Role and Regulation of Starch Phosphorylase and Starch ...

Role and Regulation of Starch Phosphorylase and Starch

Synthase IV in Starch Biosynthesis in Maize Endosperm Amyloplasts

By

Renuka M Subasinghe

A Thesis

presented to The University of Guelph

In partial fulfillment of the requirements

for the degree of Doctor of Philosophy

in Molecular and Cellular Biology

Guelph Ontario Canada

copy Renuka M Subasinghe January 2013

ABSTRACT

ROLE AND REGULATION OF PLASTIDIAL STARCH PHOSPHORYLASE AND

STARCH SYNTHASE IV IN STARCH BIOSYNTHESIS IN MAIZE

ENDOSPERM AMYLOPLASTS

Renuka M Subasinghe Advisor University of Guelph 2013 Dr Ian Tetlow

Storage starch is synthesized in sub-cellular organelles called amyloplasts

in higher plants The synthesis of the starch granule is a result of the

coordinated activity of several groups of starch biosynthetic enzymes There are

four major groups of these enzymes ADP-glucose pyrophosphorylase (AGPase)

starch synthases (SS) starch branching enzymes (SBE) and starch debranching

enzymes (SDE) Starch phosphorylase (SP) exists as both dimeric and

tetrameric forms in plastids in developing cereal endosperm and catalyses the

reversible transfer of glucosyl units from glucose-1-phosphate to the non-

reducing end of α-1-4 linked glucan chains although the precise role in the

pathway remains unclear The present study was conducted to investigate the

role and regulation of SP and SSIV in starch biosynthesis in developing maize

endosperm The results of this study showed that the tetrameric form of SP

accounts for the majority of measurable catalytic activity with the dimeric form

being barely active and the monomer catalytically inactive A catalytically active

recombinant maize SP was heterologously expressed and used as an affinity

ligand with amyloplast lysates to test protein-protein interactions in vitro

Results showed that the different multimeric status of SP influenced interactions

with other enzymes of starch synthesis Tetrameric SP interacted with SBEI and

SSIIa whilst the dimeric form of the enzyme interacted with SBEI SBEIIb All of

these interactions were enhanced when amyloplasts were pre-treated with ATP

and broken following treatment with alkaline phosphatase (APase) indicating

these interactions are regulated by protein phosphorylation In addition the

catalytic activity of SSIV was reduced following treatment with APase indicating

a role for protein phosphorylation in the regulation of SSIV activity Protein-

protein interaction experiments also suggested a weak interaction between SSIV

and SP Multimeric forms of SP regulated by protein-protein interactions and

protein phosphorylation suggested a role for SP in starch biosynthesis in maize

endosperm

iv

Acknowledgements

First and foremost I wish to express my gratitude and appreciation to my

advisor Dr Ian Tetlow for providing me the opportunity to conduct a PhD in his

laboratory at the Department of Molecular and Cellular Biology University of

Guelph and for the guidance encouragement and expert advice given through

the program

I would especially thank to Dr Michael Emes for his excellent guidance

and contribution given in his area of expertise I would like to thank Drs Robert

Mullen and Peter Pauls for serving as the members of my advisory committee I

gratefully acknowledge all members of the examination committee Dr Frederic

Marsolais (External examiner) Dr Anthony Clarke Dr Robert Mullen Dr Peter

Pauls and Dr Janet Wood

The members of the TetlowEmes research group have contributed

immensely to my personal and professional time at University of Guelph I am

especially grateful to Dr Fushan Liu for his valuable contribution and Amina

Mahmouduva for technical support given towards my research My sincere

thanks also go to Usha Zaheer Nadya Wendy Mark John Lily Ruby and all

the present and pass members in the lab for their support and friendship

I gratefully acknowledge the financial support provided by the BioCar

Initiative Project Ontario and the University of Guelph Graduate Scholarship

program

I sincerely thank to my loving mother my husband and two daughters for

their understanding sacrifice and encouragement given in my life

v

Dedicated to my Loving Family My Husband Wasantha My daughters Niki and Himi

and my mother Karuna

vi

Table of Contents

Title Page

Abstract

Acknowledgementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipiv

Dedicationv

Table of Contentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipvi

List of Figures helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxiii

List of Tables helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxviii

List of Abbreviationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxvv

Page

CHAPTER 11

1 General Introduction2

11 Starch Metabolism2

111 Molecular structure of starch3

112 Starch Biosynthesis7

1121 Starch biosynthetic enzymes8

11211 ADP-glucose pyrophosphorylase (AGPase EC 27727)8

11212 Starch synthase (SS EC 24121)13

112121 Granule bound starch synthases (GBSS)16

112122 Starch synthase I (SSI)16

112123 Starch synthase II (SSII)18

112124 Starch Synthase III (SSIIIhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20

112125 Starch synthase IV (SSIV)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21

vii

11213 Starch branching enzyme (SBEs)25

112131 Starch branching enzyme I (SBEI)25

112132 Starch branching enzyme II (SBEII)26

11214 Starch de-branching enzyme (DBE)helliphelliphelliphelliphelliphelliphelliphelliphellip27

11215 Disproportionating enzyme (D-enzyme)28

11216 Starch phosphorylase (SP)29

112161 Importance of SP in starch metabolismhelliphelliphelliphelliphelliphelliphellip30

112162 The isoforms of SP in higher plantshelliphelliphelliphelliphelliphellip30

112163 Characterization of SPhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32

112164 Biochemical characterization of SPhelliphelliphelliphelliphelliphelliphelliphelliphellip33

112165 SP and starch biosynthesis models40

112166 Evidences of interaction of SP with SSIVhelliphelliphellip41

1122 Post transitional modification of starch biosynthesis enzymes42

12 Objectives of the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43

CHAPTER 2 Biochemical Investigation of the Regulation of Plastidial

Starch Phosphorylase in Maize Endospermhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip45

21 Introductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46

22 Materials and Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip52

221 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip52

222 Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip52

2221 Amyloplast purification from maize endospermshelliphelliphelliphelliphelliphelliphellip52

viii

2222 Preparation of whole cell extractshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53

2223 Localization of SP in the plastidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54

2224 Preparation of granule bound proteinshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54

2225 Biochemical Characterization of SP in maize endospermhelliphelliphelliphelliphellip55

22251 Phosphorylation and dephosphorylation of

amyloplast lysates55

22252 Enzyme Assayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip56

222521 Starch phosphorylase glucan synthetic activity assayhelliphelliphellip56

222522 Starch phosphorylase glucan degradative activity assay56

22253 Gel Filtration Chromatography (GPC)helliphelliphelliphelliphelliphelliphelliphelliphellip57

2226 Protein analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip58

22261 Quantification of proteinshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip58

22262 Sodium dodecyl sulfate polyacrylamide gel electrophoresishellip58

22263 SP-Native affinity Zymogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip59

22264 Coomassie blue staininghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60

22265 Silver staininghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60

22266 Mobility shift detection of phosphorylated proteins

(Phos-TagTM)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60

22267 Immunological techniqueshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62

222671 Preparation of Peptides and Antiserahelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62

222672 Antibody Purificationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63

222673 Immunoblot analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64

222674 Immunoprecipitationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64

ix

23 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip66

231 Subcellular localization of SP in maize endosperm66

232 The synthetic activity of SP in developing maize endosperm69

233 Investigating the regulation of SP by protein phosphorylation71

234 Gel filtration chromatography (GPC) analysis of SP74

235 The synthetic and phosphorolytic activities of SP with

different glucan substrates82

236 Immunoprecipitation of SP85

24 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip87

3 CHAPTER 3 Using Recombinant Plastidial SP to Understand

The Regulation of Starch Biosynthesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip98

31 Introductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip99

32 Materials and Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip103

321 RNA extraction from maize endosperm and synthesis of cDNAhelliphelliphellip103

322 Quantification of nucleic acidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip103

323 Agarose gel electrophoresishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip104

324 Designing oligo-nucleotide primers and RT-PCRhelliphelliphelliphelliphelliphelliphelliphellip104

325 Ligation of complete SP cDNA sequence to the pET29a expression

vector and transformation to DH5α competent cellshelliphelliphelliphelliphelliphellip107

326 Expression of plastidial maize SP in Escherichia colihelliphelliphelliphelliphelliphelliphelliphellip108

x

327 Testing the synthetic and degradative activity of recombinant SP

using glycogen affinity native zymogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109

328 Gel filtration chromatography analysis of the recombinant SPhelliphelliphellip109

329 Immobilization of recombinant SP on S-Protein Agarose beads and

pulldown assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109

3210 Starch phosphorylase glucan synthetic activity assayhelliphelliphelliphelliphellip111

3211 Starch phosphorylase glucan degradative activity assayhelliphelliphelliphellip111

33 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip112

331 Comparison of the protein sequence of plastidial SP

of maize endosperm from the cytosolic form and other specieshellip112

332 Development of recombinant SPhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip118

3321 PCRhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip118

3322 Testing the expression level and the synthetic and

degradative activity of recombinant SP on

glycogen affinity zymogramhellip119

333 Gel Filtration Chromatography analysis of recombinant SPhelliphellip125

334 Immobilization of recombinant SP on S-Protein Agarose beadshellip127

335 The glucan synthetic and phospholytic activity of recombinant SP132

34 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip135

xi

4 CHAPTER 4 Biochemical Investigation of the Regulation of

Starch Synthase IV in Maize Endosperm146

41 Introductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip147

42 Materials and Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156

421 Analysis of the localization of SSIV in the plastidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156

422 Determination of the protein expression of SSIV

in developing endospermhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156

423 Determination of SSIV catalytic activity by zymogram analysishellip157

424 Substrate-affinity electrophoresishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip157

425 Gel Filtration Chromatography (GPC)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip158

426 Co-Immunoprecipitation of SSIVhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip158

427 Phosphorylation of SSIV using -32P-ATPhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip158

43 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip160

431 Testing the specificity of peptide specific anti-SSIV antibodieshellip160

432 Localization of SSIVhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip160

433 Determination of the expression of SSIV in developing endosperm162

434 Determination of the affinity of the SSIV in amyloplast lysates to

Different α-glucan substrateshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip163

435 Investigating the regulation of SSIV by phosphorylation using

-32P-ATPhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip166

436 Determination of the activity of ATP or APase treated

SSIV on zymogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip168

xii

437 Gel Filtration Chromatography anlysis of SSIVhelliphelliphelliphelliphellip171

438 Detection of protein-protein interactions of

SSIV by co-immunoprecipitationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip173

44 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip178

5 General Discussion185

6 List of References200

7 Appendixes218

xiii

List of Figures

CHAPTER 1

Figure 11 Structural differences between amylose and amylopectinhelliphelliphelliphelliphelliphellip5

Figure 12 Schematic representation of starch granule structure illustrating

amorphous and semi-crystalline zones of starch granule (a)helliphelliphelliphelliphelliphelliphelliphellip6

Figure 13 A summary of the role of major groups enzymes involve

in starch biosynthetic pathwayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip7

Figure 14 Domain comparison of starch synthase sequences of five

known SS isoforms in cerealhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15

CHAPTER 2

Figure 21 Schematic diagram illustrating the putative roles of plastidial

(Pho1) and cytosolic (Pho2) SP in starch metabolism in plants48

Figure 22 Immunoblots showing the subcellular localization of plastidial SP in

maize endosperm the amyloplast lysates contain soluble amyloplast

proteins the granule-bound proteins of the starch granules separated

from amyloplast the soluble protein fraction and starch granule-bound

proteins of whole cell crude extract of the endosperm and the soluble

protein fraction of the amyloplast membrane protein extracts67

Figure 23 Analysis of the localization of proteins imbedded in the granule

surface68


surface and loosely bound to the granules69

xiv

Figure 25 The activity of Pho1 was observed in developing wild-type maize

amyloplast lysates isolated 12-22 DAA using non-denaturing

affinity native zymogram containing 01 glycogen in the gel70

Figure 26 The activity of SP in amyloplast lysates at 22 DAA age in the

synthetic and phosphorolytic direction was tested on glycogen affinity

native zymogram contained 01 glycogen in the gel71

Figure 27 Determination of the different activity levels of plastidial (Pho1)

and cytosolic (Pho2) isoforms of SP following treatment with ATP and

APase72

Figure 28 Mobility shift detection of phosphorylated proteins by

Phosphate affinity SDS-PAGE using Phos-TagTM74

Figure 29 The standard curve developed to analyze the molecular weights of

the proteins eluted by gel filtration chromatography76

Figure 210A Gel filtration chromatography analysis of maize whole cell

crude extracts at 15 DAA and 35 DAA77

Figure 210BCDE Gel filtration chromatography analysis of


Figure 211 Native affinity synthetic activity SP zymogram of the amyloplast

lysates separated by GPC82

Figure 212 Immunoprecipitation of SP by peptide specific anti-SP

antibodies (30 mgmL) with 1 mL amyloplast lysates86

xv

CHAPTER 3

Figure 31 Schematic diagram of the consensus and complementary

strands showing the forward and reverse primers use to isolate

the complete cDNA sequence of the plastidial SP from maize106

Figure 32 Novagen pET29a vector used to over express plastidial SPhellip111

Figure 33 The protein sequences of the plastidial SP of maize endosperm115

Figure 34 The predicted phosphorylation sites of the plastidial maize

SP protein sequence were analyzed using NetPhos 20 Server116117

Figure 35 The PCR product of complete mRNA coding sequence (2805 bp) of

plastidial SP of maize was visualized on a 1 (wv) agarose gel contained

ethidium bromidehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119

Figure 36 Over expression of recombinant SP in Arctic express Ecoli was

analyzed by running the soluble recombinant protein on a 10 SDS gel

followed by Coomassie staining and immunoblot analyses by probing

with anti-SP specific antibodies122

Figure 37 The synthetic activity of recombinant SP in glycogen affinity

native zymogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip123

Figure 38 Testing the synthetic and degradative activity of recombinant SP

on glycogen affinity native zymogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip124

Figure 39 Gel filtration chromatography (GPC) analysis of recombinant

SP126

Figure 310 Immunoblots probed with anti-SP and anti-S-tag peptide specific

antibodies to confirm the immobilization of the recombinant GPC

fractions by S-Agarose beadshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip129

xvi

Figure 311 Immunoblots of the immobilized GPC fractions of the recombinant

SP by S-Agarose beads probed with anti-SSIIa anti-SBEI and anti-SBEIIb

peptide specific antibodieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip130

Figure 312 Immunoprecipitation of recombinant tetrameric and dimeric forms

of SP by peptide specific anti-SP antibodies bound to Protein A- Sepharose

beads131

Figure 313 Schematic diagram summarizing the protein-protein interactions

between tetrameric and dimeric forms of recombinant SP with starch

biosynthetic enzymes present in the amyloplast lysates132

Figure 314 Synthetic and degradative activities of tetrameric and dimeric

forms of recombinant SP in different glucan substrates134

CHAPTER 4

Figure 41 Amino acid sequence alignment of SSIV in different plant

Species151152

Figure 42 A schematic diagram showing major domains found within

the predicted amino acid sequence of SSIV in wheat endospermhelliphelliphelliphellip153

Figure 43 Immunoblots of amyloplst proteins probed with purified SSIV-

Specific antibodieshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip161

Figure 44 Immunodetection of SSI SSII SSIII and SSIV in stroma

and starch granules of wild-type maize amyloplasts at 22 DAAhelliphelliphellip162

Figure 45 Immunodetection of SSIV at different stages of development

in maize wild-type amyloplastshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip163

xvii

Figure 46A Determination of the relative mobility of the SSIV in amyloplast

lysates in native affinity gel electrophoresis containing varying

concentrations of amylopectin glycogen and maltoheptaose

in the gelshellip164

Figure 46B Plots of the reciprocal of the relative mobility (1Rm) of maize

SSIV against the concentration of different glucan substrateshelliphelliphelliphelliphelliphellip165

Figure 47 SSIV from amyloplast lysates was immunoprecipitated following

incubation of amyloplast lysates with -32P-ATP helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip167

Figure 48AB Zymogram analysis of SS activity in amyloplast lysates of wild-

Type maize endosperm at 22 DAA170

Figure 48C Figure 48C The activity of SS in the amyloplast lysates in the

Absence of SSIV171

Figure 49 Gel filtration chromatography analysis of SSIV

in amyloplast lysateshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip172

Figure 410A Immunoprecipitation of stromal proteins from wild-type

maize amyloplasts using peptide specific anti-SSIV antibodies

to investigate the protein-protein interactionshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip175

Figure 410B Co-Immunoprecipitation of stromal proteins from wild-type



Figure 411 Co-immunoprecipitation of ATP and APase treated stromal

proteins from wild-type maize amyloplasts using peptide specific

anti-SSIV antibodies to investigate the protein-protein interactions

of SSIV with other starch biosynthetic enzymeshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip177

xviii

List of Tables

CHAPTER 1

Table 11 The Km and Vmax values of starch phosphorylase in different

plant specieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34

CHAPTER 2

Table 21 The composition of stacking and resolving gels for

SDS-PAGEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip59

Table 22 Composition of non- denaturing 8 acrylamide gels (without SDS)

containing 01 (wv) glycogen prepared as followshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60

Table 23 The gel preparations for Phos-TagTM analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62

Table 24 The synthetic peptides sequences derived from the

N-terminal sequences of starch biosynthetic enzyme isoforms of

maize there location in full length sequence and the GenBank

accession numbershelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63

Table 25 Synthetic and phosphorolytic activities of SP in different glucan

substrates84

Table 26 Km and Vmax values of SP in amyloplast lysates in the

phosphorolytic direction85

CHAPTER 3

Table 31 The Km and Vmax values of dimeric and tetrameric forms of

recombinant SP in phosphorylitic directionhelliphelliphelliphelliphelliphelliphellip134

xix

CHAPTER 4

Table 41 Comparison of Kd values of maize SSIV with SSI SSII and

SP estimated by Coummri and Keeling (2001) in different

glucan substrateshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip166

xx

List of Abbreviations

3-PGA ndash 3-phosphoglycerate

ae ndash amylose extender

ADP ndash adenosine diphosphate

AGPase ndash ADP-glucose pyrophosphorylase

AGP-L ndash AGPase large subunit

AGP-S ndash AGPase small subunit

AP - amyloplasts

APase ndash alkaline phosphatase

ATP ndash adenosine triphosphate

BCIPNBT ndash bromo-4-chloro-3-indonyl phosphatenitro blue tetrazolium

BSA ndash bovine serum albumin

cDNA ndash complementary DNA

CE ndash crude extract

D-enzyme ndash disproportionating enzyme

DBE ndash debranching enzyme

DAA ndash days after anthesis

DMSO - dimethylsulphoxide

DP ndash degree of polymerization

DTT - dithiothreitol

EC ndash enzyme commission

Ecoli ndash Escherichia coli

EDTA ndash ethylenediaminetetraacetic acid

G-1-P ndash glucose-1-phosphate

xxi


GPC ndash gel filtration chromatography

GWD ndash glucan water dikinase

IPTG ndash isopropyl-3-D-thiogalactopyranoside

Iso ndash isoamylase

Kd ndash dissociation constant

kDa ndash kilodalton

MDs ndash malto dextrins

MOS ndash malto-oligosaccharide

MW ndash molecular weight

NAD ndash nicotinamide adenine dinucleotide

NADH - nicotinamide adenine dinucleotide (reduced form)

NCBI ndash National Center for Biotechnology Information

OD ndash optimal density

PAGE ndash polyacrylamide gel electrophoresis

PBS ndash phosphate buffered saline

PCR ndash polymerase chain reaction

Pho1 ndash plastidial starch phosphorylase

Pho2 ndash cytosolic starch phosphorylase

PI ndash phosphatase inhibitor (cocktail)

Pi ndash inorganic phosphate

PPi ndash inorganic pyrophosphate

PWD ndash phosphoglucan water dikinase

RB ndash rupturing buffer

xxii

RCF ndash relative centrifugal force

Rm ndash Relative migration

SBE ndash starch branching enzyme

SDS ndash sodium dodecyl sulfate

Ser - serine

SP ndash starch phosphorylase

SS ndash starch synthase

TEMED - tetramethylethylenediamine

Thr - threonine

TTBS - tris buffered saline solution

(vv) ndash (volumevolume)

UDP ndash uridine diphosphate

(wv) ndash (weightvolume)

Wx ndash waxy mutant

1

CHAPTER 1

2

1 General Introduction

11 Starch Metabolism

Starch is the major form of carbon reserve polysaccharide being

synthesized in plants in cellular organelles called plastids (Joen et al 2010

Tetlow et al 2006) Transient starch and storage starch are two forms of starch

available in plants The chloroplasts in photosynthetic tissues such as leaves

produce transient starch during photosynthesis and store it temporally during

the light period Transient starch is converted into sucrose in the dark and which

is translocated within the plant to supply the energy and carbon demand

required for growth and development Storage starch is a long-term carbon

store in the plant which is synthesized in non-photosynthetic plastids called

amyloplasts found in tuberous tissues (eg in potatoes) or as carbon stores in

seeds (Tetlow 2006 2011) The location of starch production in the plant is

reflective of its metabolic role Storage starch is extremely important to the

plant metabolism of higher plants as a supplier of long-term energy requirement

(Gerard et al 2001) For instance storage starch in seeds will be broken down

during germination to provide the growing seed with energy until it becomes a

photoautotrophic plant

Starch is also an important polysaccharide for humans and represents up

to 80 of daily caloric intake in the human diet Seed storage reserve

carbohydrates are produced in cereal endosperms such as in rice wheat maize

barley and sorghum make up 90 of the starch world market alone (Burrell

2003) Starch is a cheap natural and renewable raw material and has numerous

industrial applications Aside from the agri-food sector starch can be fabricated

3

into pulp and paper paints textiles cosmetics pharmaceuticals biodegradable

plastics construction materials and is also used as a source of renewable

energy in the form of ethanol (Shigechi et al 2004)

111 Molecular structure of starch

Starch exists as water insoluble glucan polymers which form into a semi-

crystalline granular structure in the plastid Starch granules are composed of

two different glucosyl polymers called amylose and amylopectin The ratio of

these polymers in a starch granule is largely genetically controlled and normally

amylopectin makes up about 75 of the starch granule mass and amylose

around 25 Glucosyl units of these polymers are connected by (14) bonds

Amylose is an unbranched or less branched polymer which is created by 100ndash

10000 glucosyl units whereas amylopectin has much larger polymer units

(degree of polymerization is 105ndash106 glucose units) with both (14) and

distinctive (16) branching glycosidic links (Fig 11) The number of glucosyl

units in (14) linked linear chains and the relative position of (16) branch

linkages are determined by the inherent properties of the starch biosynthetic

enzymes There is approximately one branch point for every 20 glucose residues

in amylopectin (Manners 1989)

Amylopectin exhibits a polymodal glucan chain distribution This allows

the condensing of shorter chained glucans and the subsequent development of

efficiently packed parallel left-handed double helices which creates crystalline

lamella of the starch granule The compact helices are approximately 6 to 75

nm in length The regular branch point clusters of amylopectin create

4

amorphous lamella which are approximately 3nm in length The compact

helices coupled with regular branch point clustering gives rise to the organized

semi-crystalline nature of the starch granule (Fig 12) (Hizukuri 1986 French

1984) Amylose is found predominantly in a single-helical or random-coil form in

the amorphous noncrystalline regions (Jane et al 1992) The unique semi-

crystaline structure of starch differs from its counterpart glycogen in archaea

bacterial and animal systems glycogen exists as a globular shaped molecule

consisting of water-soluble homogenously branched glucan polymers (Roach

2002)

5

(A)

(B)

Figure 11 Structural differences between amylose and amylopectin The starch granule consists of two forms of glucan polymers amylose and

amylopectin Amylose is a relatively low branched polymer containing (14) bonds (1A) Amylopectin is a highly branched glucan polymer and has both

(14) bonds and (16) bonds (1B) = reducing end

6


amorphous and semi-crystalline zones of starch granule (a) Enlargement of semi-crystalline growth rings illustrating the arrangement of the alternating crystalline and amorphous lamellae (b and c) (Tetlow 2006)

7

112 Starch Biosynthesis

A highly complex and organized coordination of various enzymes is

required to synthesize starch in the amyloplast The major enzymes involved in

the biosynthetic process catalyze specific reactions and are present in several

isoforms in many plants There are four major groups of enzymes are involved

in starch biosynthesis adenosine 5rsquo disphosphate glucose pyrophosphorylase

(AGPase) starch synthase (SS) starch branching enzyme (SBE) and starch

debranching enzyme (DBE) These enzymes are found in several isoforms

present in all starch synthesizing organelles (Vrinten and Nakamura 2000)

Major groups of enzymes involved in amylose and amylopectin biosynthesis

process are shown in Fig 13

Figure 13 A summary of the role of major groups enzymes involve in starch biosynthetic pathway

8

1121 Starch biosynthetic enzymes

11211 ADP-glucose pyrophosphorylase (AGPase EC 27727)

ADP-glucose is the soluble precursor and the glucosyl donor for the

different classes of starch synthases the group of enzymes which are involved

in elongation of the α-glucan chains in both transient and storage starch

biosynthesis in higher plants (Preiss 1988) ADP-Glucose is produced from

glucose-1-phosphate (G-1-P) and adenosine triphosphate (ATP) by the catalytic

activity of AGPase Therefore AGPase catalyzes the key metabolic step in the

synthesis of starch in higher plants and glycogen in bacteria by providing ADP-

glucose the substrate for all SSs (Preiss 1988) The reversible reaction of ADP-

glucose and inorganic pyrophosphate (PPi) synthesis from ATP and G-1-P by the

catalytic activity of AGPase is shown in following reaction (Fu et al 1998)

Glucose-1-phosphate + ATP ADP-glucose + PPi

AGPase is present in all starch synthesizing tissues in higher plants In

spinach leaves (Morell et al 1987 Copeland and Preiss 1981) in Arabidopsis

thaliana leaves (Lin et al 1988) and in potato tubers (Okita et al 1990

Sowokinos and Preiss 1982) AGPase is found as a heterotetrameric in

structure containing two large regulating subunits (AGP-L) and two small (AGP-

S) catalytic subunits In spinach leaves and in potato tubers the large subunits

and the small subunits are respectively 54-55 kDa and 50-51 kDa in size (Okita

et al 1990 Morell et al 1987 Sowokinos and Preiss 1982) and in the wheat

developing endosperm 58 and 55 kDa respectively (Tetlow et al 2003) The

primary sequence of the rice endosperm small subunit has 76 identity to the

9

spinach subunit and the small subunit is structurally conserved in plants (Preiss

et al 1989) Similarly these subunits are coded by at least two different genes

shrunken2 (sh2) and brittle2 (bt2) for the large and small subunits of AGPase

respectively in maize (Bae et al 1990 Bhave et al 1990) The N-terminus of

the small subunit involves its catalytic properties and the heat stability of

AGPase in potato tuber (Ballicora et al 1995) In contrast the AGPase shows

homotetrameric structure in bacterial systems like Escherichia coli and

Salmonella typhimurium which have 200 kDa and 48 kDa subunits respectively

in size (Preiss 1988)

Biochemical and genetic evidence indicate that there are two distinct

AGPases are localized in the cytosol and in the plastid (Okita 1990 Denyer et

al 1996b Tetlow et al 2003 Tiessen et al 2011) In dicots AGPase is

exclusively located in the plastid and represents 98 of the total AGPase

activity in the cell (Thorbjoslashrnsen et al 1996 Tiessen et al 2011) In contrast

the localization of AGPase is predominantly in the cytosol in cereals for example

in wheat endosperm 60-70 of the AGPase activity is cytosolic (Geigenberger

2011 Tetlow et al 2003) in maize endosperm it is 95 (Denyer et al 1996)

and in developing barley endosperm it represents 80-90 (Beckles et al 2001

Tiessen et al 2011) However the large and small subunits sizes are slightly

smaller in plastidic AGPase than in cytosolic form in the amyloplast (Beckles et

al 2001 Tetlow et al 2003)

The presence of AGPase in the cytosol of cereal endosperms implies that

the synthesized precursor ADP-glucose needs to be transported to the

amyloplast for starch synthesis A specialized nucleotide sugar transporter the

10

ADP-glucoseADP transporter encoded by brittle1 gene is located at the inner

amyloplast envelop to import ADP-glucose during storage starch biosynthesis in

cereals (Shannon et al 1998 Tetlow et al 2003) and the amino acid sequence

of the maize endosperm ADP-glucose transporter termed Bt1 has been

determined (Kirchberger et al 2007) In wheat endosperm ADP-glucose

transport into amyloplasts was shown to be dependent on the adenylates ADP

and AMP as counter-exchange substrates (Bowsher et al 2007) The authors

also found that the rate of ADP exported from the amyloplasts to be equal to the

rate of ADP-glucose utilization by starch synthases

AGPase in both photosynthetic and non-photosynthetic plant sources is

allosterically regulated by the metabolites within the carbon assimilation

pathway 3-Phosphoglycerate (3-PGA) is the first intermediate in Calvin cycle of

photosynthesis and the AGPase is allosterically regulated positively by the 3-PGA

and negatively by inorganic phosphate (Pi) in leaf chloroplasts (Neuhaus and

Stitt 1990) During the light period in photosynthetic tissues the level of 3-PGA

in chloroplast stroma increase and the Pi level decreases as it is used as a

substrate in ATP synthesis through photophosphorylation process (Buchanan et

al 2000) In non-photosynthetic tissues such as the amyloplast in cereal

endosperm (Tetlow et al 2003) and in potato tubers (Sowokinos and Preiss

1982 Tiessen et al 2003) similar regulation by 3-PGA and Pi was shown Ratios

of the allosteric effectors (3-PGAPi) are important in controlling AGPase activity

For example the plastidial AGPase activity in wheat endosperm is insensitive to

3-PGA activation compared to potato tubers (Gomez-Casati and Iglesias 2002

Tetlow et al 2003 Ballicora et al 1995 Hylton and Smith 1992)

11

The purified wheat endosperm AGPase activity is also inhibited by

adenosine diphosphate (ADP) and fructose-16-bisphosphate and the inhibition

can be reversed by adding 3-PGA and fructose-6-phosphate (Gomez-Casati and

Iglesias 2002) The regulation of plastidic form of AGPase in wheat amyloplast

in synthetic direction required 15 mM 3-PGA to achieve a 2-fold stimulation in

rate and was only inhibited approximately 40 by a 20 mM high concentrations

of Pi (Tetlow et al 2003) In contrast AGPases from photosynthetic tissues of

wheat were regulated by 3-phosphoglycerate (activator A05=001 mM) and

orthophosphate (inhibitor I05=02 mM) shows higher sensitivity of chloroplast

AGPase to 3-PGA (Gomez-Casati and Iglesias 2002) Interestingly the subunits

of the cytosolic and plastidic forms not only differ in their sizes but also in their

kinetic properties in wheat (Tetlow et al 2003) The ratio of pyrophosphorolytic

to synthetic activity indicates a preference towards the pyrophosphorolysis

direction in cytosolic form of AGPase and toward synthesis in plastidial AGPase

(Tetlow et al 2003) The inhibition of the enzyme activity by Pi on the synthetic

direction in whole cell extracts could be restored by 3-PGA whereas the

synthetic reaction in amyloplasts was more sensitive to Pi and this inhibition

was not restored by up to 15 mM 3-PGA (Tetlow et al 2003) Further pyridoxal

phosphate (pyridoxal-P) was shown as an allosteric activator of spinach leaf

AGPase (Morell et al 1988) Pyridoxal-P covalently binds to both the 54 kDa and

51 kDa subunits at or near the allosteric activator site(s) of the enzyme AGPase

shows higher affinity to pyridoxal-P than 3-PGA and binding of pyridoxal-P to

each protein is inhibited by the presence of either the allosteric activator of the

enzyme 3-PGA or the allosteric inhibitor Pi (Morell et al 1988) However the

12

maximum activation by pyridoxal-P is 6-fold and it is comparatively less

compared with 25-fold by 3-PGA (Morell et al 1988)

The activity of AGPase is also influenced through post-translational redox

modulation in several species which involves in reversible disulfide-bridge

formation between the two small catalytic subunits of the enzyme (Tiessen et al

2002 Hendriks et al 2003) The catalytic subunits of the enzyme were detected

by their mobility in non-reducing SDS gels as a dimer in oxidized form and as a

monomer in reduced form where the overall activity of the enzyme was

increased in monomeric and lower in dimeric forms (Kolbe et al 2005) The

activity of recombinant AGPase developed from potato was increased in 4-fold

by adding a reducing agent dithiothreitol (DTT) (Sowokinos and Preiss 1982)

Further AGPase from potato tubers was activated by a small protein (12 kDa)

which facilitates the reduction of other proteins called thioredoxin f and m

leading to an increase in catalytic-subunit monomerization and increased

sensitivity to activation by 3PGA (Ballicora et al 2000) In contrast AGPase

activity was partially inactivated following exposure to oxidized thioredoxin due

to formation of disulfide bonds between the N-termini of the AGPase small

subunit (ADP-S) in the potato tubers (Fu et al 1998) Tiessen et al 2002 also

showed that potato tuber AGPase is subject to redox-dependent

posttranslational regulation involving formation of an intermolecular cysteine

(Cys) bridge between the two small catalytic subunits of the heterotetrameric

holoenzyme Hendriks et al (2003) further analyzed that the intermolecular Cys

bridge between the two smaller catalytic subunits is rapidly converted from a

dimer to a monomer when isolated chloroplasts are illuminated or when sucrose

13

is supplied to leaves via the petiole in the dark and from a monomer to a dimer

when pre-illuminated leaves are darkened in pea potato and Arabidopsis

leaves This redox activation not only responds to the changes in sugars in

chloroplast but also in potato tubers (Tiessen et al 2002) However the AGPase

is regulated by a light-dependent signal in photosynthetic tissues Further

studies carried out by Tiessen et al 2003 suggested that sucrose and glucose

lead to redox activation of AGPase via two different signaling pathways involving

SNF1-related protein kinase (SnRK1) and hexokinase respectively which are

implicated in a regulatory network that controls the expression and

phosphorylation of cytosolic enzymes in response to sugars in potato tubers

(Geigenberger 2011)

11212 Starch synthase (SS EC 24121)

The starch synthases catalyze the transfer of the glucosyl moiety of ADP-

glucose to the non-reducing end of an - (14)-linked glucan primer in higher

plants Among the entire starch biosynthesis enzymes SS has the highest

number of isoforms (Fujita et al 2011) This group of enzymes is divided into

two groups first the granulendashbound starch synthases (GBSS) which are

encoded by the Waxy (Wx) gene are involved in amylose biosynthesis

(Nakamura et al 1993 Sano 1984 Echt and Schwartz 1981) The second

class of starch synthases consists of four major isoforms SSI SSII SSIII and

SSIV which are involved in amylopectin synthesis Isoforms of the major classes

of SSs are highly conserved in higher plants (Ball and Morell 2003) A region of

approximately 60kDa is highly conserved in C-terminus of all these enzymes in

14

higher plants and green algae whereas this region is distributed across the

protein sequence in prokaryotic glycogen synthases (Tetlow 2011) The KndashXndashGndash

GndashL motif is thought to be responsible for substrate (ADP-glucose) binding in

prokaryotic glycogen synthase (GSs) and in higher plant SSs (Furukawa et al

1990 1993 Busi et al 2008) and is also found only in the C-terminus of higher

plants and green algal SSs (Nichols et al 2000) where as the K-X-G-G-L

domains are distributed across the GSs protein sequence in prokaryotes

(Fukukawa et al 1990) The presence of lysine in the KndashXndashGndashGndashL domain

determines glucan primer preference (Gao et al 2004) Further the glutamate

and aspartate are found as important residues for catalytic activity and

substrate binding in maize SSs (Nichols et al 2000) SSs show considerable

variation within the N-terminus upstream of the catalytic core and this region

can vary greatly in length from 22 kDa in granule-bound starch synthase I

(GBSSI) to approximately 135 kDa in maize SSIII (Gao et al 1998) (Fig 14)

The phylogenetic and sequence analysis of plants SS (Arabidopsis thaliana

wheat and rice) and algal SS and prokaryotic GS isoforms on the basis of

predicted amino acid sequence suggests that SSIs SSIIs and GBSSIs have

distinct evolutionary origins as compared to SSIIIs and SSIVs (Leterrier et al

2008) Especially the valine residue within the highly conserved K-X-G-G-L

motif appears to have faced strong evolutionary selection in SSIII and SSIVs

and it may affect primersubstrate binding of these SSs compared to SSIs SSIIs

and GBSSIs (Leterrier et al 2008) The other prominent difference in SSIII and

SSIV from other SSs is the highly conserved G-X-G motif near the nucleotide-

binding cleft (Leterrier et al 2008)

15

Figure 14 Domain comparison of starch synthase sequences of five known SS

isoforms in cereal The C-terminal catalytic domains (in black color) includes K-X-G-G-L motif which is a putative ADPG-binding domain SSs vary in the length of the N- terminal region (shown as hatched bars) The N-terminal arm is

believed to provide isoform specificity possibly through binding to other proteins SSIII in particular has a unique N-terminal extension thought to be

involved in controlling proteinndashprotein interactions (Sequence lengths are not drawn to scale) (Source Tetlow 2011)

112121 Granule bound starch synthases (GBSS)

There are two isoforms of GBSS GBSSI and GBSSII both of which are only

found in the granule matrix of starch biosynthesizing tissues GBSSI is

responsible for elongating amylose in storage tissues and GBSSII in tissues such

as pericarp leaf stem and root (Yandeau-Nelson et al 2010 Vrinten and

Nakamura 2000) The waxy mutant results in a lack of amylose production

(Vrinten and Nakamura 2000 Vrinten et al 1999) All of the GBSSI protein in

maize endosperm is remained as granule associated (Mu-Forster et al 1996)

However the Waxy or low amylose starches are still able to form a granule and

16

maintain its semi-crystalline property suggesting that amylose is not required

for insoluble granule synthesis (Denyer et al 1999)

112122 Starch synthase I (SSI)

SSI is responsible for the synthesis of shorter glucan chains up to ten or less

than ten glucosyl units in maize endosperm (Commuri and Keeling 2001) SSI

synthesizes shorter glucan chains with the degree of polymerization (DP) less or

equal to 10 (DPle10) in transient starch synthesis in leaves (Delvalle et al

2005) The soluble SSI in maize is 76kDa in size (Mu et al 1994) The degree of

association of SSI in the starch granule is significant representing 85 of total

SSI content in maize endosperm (Mu-Forster et al 1996) Further the affinity of

SSI for amylopectin (Kd= 02 mgmL) was higher compared to starch (Kd= 049

mgmL) glycogen (Kd= 10 mgmL) and amylose (Kd= 06 mgmL) (Commuri

and Keeling 2001)

The amino acid sequence of SSI in maize shares 757 sequence identity

to rice SSI (Knight et al 1998) In japonica rice lacking SSII (Nakamura et al

2005) SSI accounts 70 of the total SSs activity (Fujita et al 2006) However

the relative contribution of SS isoforms is different in different species (eg

SSIII contributes gt70 of total SS activity in potato) (Abel et al 1996) The

accumulation of SSI total transcripts was higher at 5ndash10 days-post-anthesis

(DPA) than at 15ndash25 DPA in developing wheat endosperm (Peng et al 2001)

During the endosperm development the relative abundance of SSI did not vary

in starch granules whereas SSI concentration in the endosperm soluble

fractions was highest from 10-15 DPA and below detection levels at 5 DPA The

17

wheat endosperm SSI further exhibited similar concentration per kernel from

15-25 DPA in endosperm soluble fractions but at considerably higher

concentrations in starch granules as compared to endosperm soluble fractions

(Peng et al 2001) SSI from japonica rice produces chains with a degree of

polymerization (DP) 8-12 from short and DP 6-7 chains emerging from the

branch point in the A and B1 chains of the amylopectin (Fujita et al 2006)

Further SSI mutant showed decreased number of DP 8-12 glucan chains and

increased number of both DP 6-7 and DP 16-19 chains in endosperm

amylopectin in japonica rice (Fujita et al 2006) However SSI mutants in

transgenic potato plants displayed no visible phenotypic changes in starch

structure (Kossman et al 1999) The overlapping function of SSI and SSIII were

revealed by creating double-recessive homozygous mutants from SSI null

mutants with SSIII null mutants in japonica rice (Fujita et al 2011) The seeds

from these mutants remained sterile and the heterozygous mutants produced

fertile opaque seeds further confirmed that SSI or SSIII is required for starch

biosynthesis in rice (Fujita et al 2011)

112123 Starch synthase II (SSII)

Two SSII isoforms are present (SSIIa and SSIIb) in higher plants SSIIa

predominates in cereal endosperm while SSIIb is mostly confined to vegetative

and photosynthetic tissues (Morell et al 2003) SSII is also partitioned in both

the starch granule bound protein fraction and in the soluble protein fraction in

the plastid (Li et al 1999) SSIIa mRNA level showed a higher accumulation

during the period of starch accumulation in developing maize endosperm (Harn

18

et al 1998) SSIIa plays a role in medium chain length extension and appears to

be involved in elongating glucan chains produced by SSI leading to the

production of medium length chains of DP=12-25 (Zhang et al 2004 Morell et

al 2003) The sex6 mutant of barley lacking SSII activity in the endosperm has

a shrunken endosperm phenotype and reduced starch content due to substantial

decrease in amylopectin content However the amylose content was increased

up to 71 and 625 compared with 25 in the wild-type (Morell et al 2003)

Moreover these mutants have altered chain-length distribution whereas the

amount of shorter glucan chains (DP= 6-11) increased from 2415 (in wild-

type) to 3818 and 3896 and the medium length glucan chains (DP= 12-

30) decreased from 6912 (in wild-type) to 5414 and 5342 in M292 and

M342 mutants respectively (Morell et al 2003) Interestingly the lack of SSII

causes a reduction in the levels of the branching enzymes SBEIIa SBEIIb and

SSI in the starch granule but not the amount of these enzymes in the soluble

fraction in barley amyloplasts (Morell et al 2003) This suggests that either SSII

mutation prevents binding of these proteins to the starch granules or they are

prevented from forming protein complexes in the amyloplast stroma and get

trapped in the granule (Morell et al 2003) The effects on chain length

distribution of ss2 mutants observed in barley are similar to sugary2 (su-2)

mutants of maize endosperm lacking SSIIa (Zhang et al 2004) indicating a

common function for SSII in starch granule assembly The su-2 mutants exhibit a

significant increase in DP= 6-11 shorter chains and a decrease in DP=13-20

medium length chains (Zhang et al 2004) In the Arabidopsis thaliana mutant

ss2 (Atss2) the growth rate or the starch quantity were not affected but

19

increased the amyloseamylopectin ratio increased total amylose (43 of total

amylose) and DP=12-28 medium length glucan chains were significantly

decreased as similar as in the endosperms of many cereals (Zhang et al 2008)

In addition the total SSs activity was recorded as 212 plusmn 87 nmol

productminmg proteins in wild-type leaf extract was increased up to 237 plusmn

87 in Atss2 (Zhang et al 2008) These results suggested that the loss of SSII

activity can be restored by any other conserved SS specifically SSI GBSSI or

SSIII or SSIV in transient starch biosynthesis (Zhang et al 2008)

112124 Starch Synthase III (SSIII)

The SSIII was found as 1392 kDa size in potato (Abel et al 1996) and gt200

kDa in maize endosperm (Cao et al 1999) and is expressed throughout the

developmental stages of these plants The calculated molecular masses of the

isoforms found in rice endosperm (OsSSIII-1) and leaves (OsSSIII-2) were 138

kDa and 201 kDa respectively (Dian et al 2005)

SSIII is coded by the DUI gene in maize endosperm (Cao et al 1999)

The du1 mutations alter starch structure indicates that DU1 provides a specific

function(s) that cannot be compensated for by the remaining soluble SS activity

(Abel et al 1996) The ss3 mutant showed a significant decrease in total SS

activity by 13-29 compared to 100 of SS activity in the wild-type without

any significant effect on the plant phenotype amylose content or the tuber yield

in potato (Abel et al 1996) The granule morphology was changed in ss3 single

mutants producing small granule structures (Abel et al 1996)

20

SSIII elongates comparatively longer glucan chains than SSII producing

DP= 25-40 or greater glucan chains in amylopectin (Tomlinson and Denyer

2003 Edwards et al 1999 Abel et al 1996) The frequency distribution of the

linear glucan chain in ss2 and ss3 single mutant lines showed strong

enrichments at DP= 6-9 and ss2ss3 double mutant lines showed strong

enrichments at both DP= 7ndash8 and DP= 12ndash13 (Edwards et al 2002) The

frequency distribution of the linear glucan chain was determined in transient

starch from Arabidopsis thaliana Atss3-1 Atss3-2 mutant lines (Zhang et al

2005) The frequency of shorter chains of DP= 5-10 and DP= 26-37 are

respectively increased but chains of DP= 14-20 and DP= 43-46 are respectively

decreased suggesting that SSIII is involved in producing comparatively longer

glucan chains compared with SSI and SSII (Zhang et al 2005 Edwards et al

2002) SSIII appears to be a vital enzyme in transient starch biosynthesis

starch granule initiation requires the presence of either SSIII or SSIV in

Arabidopsis leaves (Szydlowski et al 2009)

112125 Starch synthase IV (SSIV)

SSIV is exclusively present in the stroma of the plastids (Leterrier et al 2008

Roldan et al 2007) The role of SSIV in chain length distribution is not clear but

it may play a selective role in priming starch granule formation (Roldan et al

2007) SSIII and SSIV in rice have two isoforms in each enzyme OsSSIII1

OsSSIII2 and OsSSIV1 OsSSIV2 (Dian et al 2005) The SSIII2 and SSIV1

genes are mainly expressed in rice endosperm whereas the other two isoforms

were expressed mainly in leaves The cDNA sequence of wheat SSIV

21

preferentially expressed in leaves is most similar to rice SSIV2 which share a

similar exon-intron arrangement (Leterrier et al 2008) suggesting that the SSIV

present in leaves and endosperms may have slight variation in amino acid

sequences (eg as similarly observed in SBEIIa and SBEIIb)

The SSIV protein in Arabidopsis thaliana is 11299 kDa in size (Roldan et

al 2007) showing 71 582 568 and 583 sequence identity to Vigna

unguiculata (accession number AJ006752) wheat (accession number

AY044844) rice (SSIVa accession number AY373257) and rice (SSIVb

accession number AY373258) respectively (Roldan et al 2007) Two

independent mutant alleles of SSIV in Arabidopsis thaliana Atss4-1 [from

Columbia-0 (Col-0) ecotype] and Atss4-2 [from Wassilewskija (WS) ecotype]

showed no decrease in total soluble SS activity but lower growth rates were

recorded in the mutant plants grown under a 16-h day8-h night photo period

approximately as 100 mgFW (fresh weight) and 50 mgFW of the plant

compared with their respective wild types which conserved 550 mgFW and 275

mgFW of growth rates (Roldan et al 2007) However the fruit size number of

seeds per silique and germination ratios were not altered in the mutant lines

but the starch contents in the leaves were significantly reduced in both mutants

by 35 for the Atss4-1 and 40 for the Atss4-2 line with respect to their wild

types at the end of the illuminated period Although the total SS activity was

not affected the total activity of both cytosolic and plastidial forms of starch

phosphorylase (SP) was increased by 14ndash2-fold in both Atss4-1 and Atss4-2

mutants which may be due to a metabolic alteration that triggers the induction

22

of SP isoform gene expressions in ss4 mutants (Roldan et al 2007) The exact

reason for the increase of SP in ss4 mutant is not known

In Arabidopsis thaliana leaves amyloseamylopectin ratio was not

changed in Atss4 mutants (Roldan et al 2007) The chain length distribution

pattern was determined in Atss4 mutants and their respective wild types and

indicated that the Atss4 mutation had minor effects on the structure of

amylopectin and only a slight reduction in the number of shorter chains of DP=

7ndash10 were observed The microscopic analysis of starch granules collected at 4

and 12 h during the light phase showed a greater difference in size between Col-

0 and Atss4-1 starch granules the surface area was increased by 10 times at

the end of the day in Atss4-1 mutant plants A single starch granule was

contained in the mutant whereas in wild-type showed 4ndash5 starch granules per

chloroplast concluding that the mutation at the AtSS4 locus affects both the

number and size of starch granules synthesized in the chloroplast These

observations further suggested that the SSIV may be involved in the priming of

the starch granule (Roldan et al 2007) However this hypothesis was not yet

confirmed in any plant species The essential mechanism of starch granule

initiation is largely unknown

SSIV in wheat endosperm was found as 1031 kDa protein which is 87

homologous to the OsSSIVb in rice endosperm [Genbank AAQ82623] (Leterrier

et al 2008) Like all other SSs the N-terminus of wheat SSIV is unique the

SSIV-specific region from amino acids 1ndash405 contains two coiled-coil domains

and a 14-3-3-protein recognition site (Leterrier et al 2008) The coiled-coiled

domains are commonly involved in crucial interactions such as transcriptional

23

control (Mason et al 2004) and 14-3-3 proteins are commonly linked to binding

with various signaling proteins such as kinases and phosphatases (Comparot et

al 2003) The mRNA expression of SSIV was highest in non-endosperm tissues

such as in leaf embryo and roots in wheat and the level of expression in the

endosperm was comparatively lower and the expression was independent from

the regulation of the circadian clock Therefore the transcript accumulation

levels did not coincide with the period of high carbon flux to starch in the wheat

endosperm (Leterrier et al 2008)

To elucidate the function of SSIV in the priming process of starch granule

formation different combinations of homologous double SSs mutations in ss4

mutant backgrounds were developed in Arabidopsis thaliana ss1ss4 ss2ss4

and ss3ss4 (Szydlowski et al 2009) Decreased levels of starch accumulated in

ss1ss4 and ss2ss4 double mutants were equal with the sum of the decreases

starch levels in their respective single mutant lines At the end of 12h light

period the starch accumulation in the respective ss3 and ss4 single mutants

were recorded as 122 (Zhang et al 2005) and 62 (Rolden et al 2007)

respectively compared to their wild types However the ss3ss4 double mutant

did not accumulate any measurable amounts of starch despite the dark or light

conditions (Szydlowski et al 2009) Therefore the presence of either SSIII or

SSIV is a crucial requirement in transient starch biosynthesis (Szydlowski et al

2009) Further although the ss3ss4 double mutants did not affect on other

starch metabolism enzymes (such as phosphoglucomutase AGPase and starch

branching enzymes (SBE) they showed a significantly increased SP activity

(more than 8 fold in wild-type) (Szydlowski et al 2009) which may be due to

24

an alternative route of starch biosynthesis available using hexose phosphates via

a starch phosphorylase (SP)-mediated pathway (Fettke et al 2010) (see the

section 11216 for the details)

SSIV is a newly identified SS isoform existing in the plastids However

the exact function of SSIV in storage starch biosynthesis has yet to be identified

The expression of SSIV during the endosperm development is high at the later

stage of the grain filling (Dian et al 2005) The coordination and interactions of

the various enzyme classes are explained through the operation of protein-

protein interactions (see the section 1122) Chapter 4 of this thesis present

an investigation of the catalytic activity and regulation of SSIV by protein-

protein interaction with other starch biosynthetic enzymes in maize amyloplasts

11213 Starch branching enzyme (SBEs EC 24118)

The branching structural arrangement of amylopectin is generated by

starch branching enzymes (SBEs) These enzymes generate the -(16)

linkages through cleavage of internal -(14) glycosidic bonds The reducing

ends are then transferred to the C6 hydroxyls forming a new branch point In

common with the other classes of starch biosynthetic enzymes SBEs have

multiple isoforms (eg SBEI SBEIIa and SBEIIb) some of which are plant

tissue andor developmental specific in their expression patterns (Regina et al

2005 Gao et al 1997)

25

112131 Starch branching enzyme I (SBEI)

SBEI and the SBEIIrsquos differ in the length of the glucan chain they transfer

in vitro and show different substrate specificities SBEI exhibits a higher rate of

branching with amylose and transfers longer chains compared to SBEII which

has a higher affinity towards amylopectin (Guan and Preiss 1993 Takeda et al

1993) The amylopectin chain-length profile of the maize SBEI mutant (sbe1)

was not affected compared with wild-type (Blauth et al 2002) However SBEI

has a lower Km for amylose and tends to produce shorter constituent chains

compared to SBEIIa or SBEIIb when reacted with amylose in vitro (Gao et al

1996) In maize SBEI is expressed moderately during middle stages of kernel

development (12ndash20 DAA) strongly during the later stages of kernel

development (22ndash43 DAA) and is moderately expressed in vegetative tissues

(Kim et al 1998) When compared to the total SBE activity in mutants of SBEI

SBEIIa and SBEIIb in maize a loss of enzyme activity have been identified for

only SBEIIa and SBEIIb (Blauth et al 2002) showed that the lack of SBEI was

compensated by other two SBE isoforms Alternatively SBEI does not have a

significant role in determining starch quantity or quality in leaves or endosperm

(Blauth et al 2002) SBEI is highly conserved in plants and has been shown to

interact with other starch biosynthetic enzymes (Liu et al 2009 Tetlow et al

2004) indicating that SBE plays some function in regulating the starch

biosynthetic process

26

112132 Starch branching enzyme II (SBEII)

In monocots two SBEII gene products (SBEIIa and SBEIIb) are closely related

(Rahman et al 2001) However in wheat endosperm their expression patterns

are considerably different where SBEIIa is expressed at a higher level than

SBEIIb (Regina et al 2005) SBEII mutations show a more pronounced

phenotypic change compared to SBEI A mutation of the gene encoding SBEIIb

in maize produces a high-amylose starch phenotype known as the amylose

extender (ae-) (Banks et al 1974 Yu et al 1998) Mutations in SBEIIb in maize

(ae mutant) produce resistant starch genotype which characteristically produces

less branched and longer glucan chains in amylopectin (Nishi et al 2001

Klucinec and Thompson 2002)

Phenotypic changes in SBEIIa mutations are dependent on the source of

starch In maize there was a visible change in leaf starch in SBEIIa mutants

however no significant changes occurred in storage starches of maize kernels

(Blauth et al 2001) The catalytic activity of SBEIIa and SBEIIb is regulated by

protein phosphorylation in wheat endosperm (Tetlow et al 2004) and show a

high expression of SBEIIa compared to SBEIIa in developinf wheat endosperm

(Morell et al 1997 Regina et al 2005) In contrast in maize endosperm

SBEIIb is the predominant form being expressed at approximately 50 times the

level of the SBEIIa form (Gao et al 1997) it is the most abundant protein in the

maize endosperm amylopast stroma (Mu et al 2001)

27

11214 Starch de-branching enzyme (DBE EC 32141 and EC

32168)

Starch debranching enzymes play an important role in the development of

crystalline amylopectin There are two types of DBEs The isoamylase-type (ISO)

hydrolyzes -(16) linkages in amylopectin and pullulanase-type (PUL)

hydrolyzes -(16) linkages in amylopectin and pullulan a fungal polymer of

malto-triose There are three isoamylase-type DBE isoforms (ISO1 ISO2 and

ISO3) Rice and maize mutants lacking ISO1 (sugary1) demonstrate an increase

in the disordered water-soluble highly and randomly branched polysaccharide

called phytoglycogen (Nakamura 2002 James et al 1995) Although the

respective substrates of isoamylase and pullulanase type DBEs are known their

specific roles in starch biosynthesis are not clear However there are two

existing models for their function The glucan trimming model proposes that

DBEs remove any branches that would inhibit crystallization of the developing

granule (Ball et al 1996 Myers et al 2000) Another model suggests that DBEs

clear away any soluble glucan not attached to the granule (Zeeman et al 1998)

The theory is based on the concept that SSrsquos and SBErsquos will continue to

synthesize glucan polymers if sufficient substrate is present therefore causing

phytoglycogen accumulation Although the latter model would explain the

increase of phytoglycogen in DBE mutants it is possible these models are not

mutually exclusive

ISO1 and ISO2 form a hetero-oligomeric complex to form a functional

enzyme (Hussain et al 2003) This complex is approximately 400 kDa in size

and is also found with a 300 kDa complex containing ISO1 but not ISO2 in

28

maize Loss of ISO1 prevents formation of the complexes indicating that ISO1

is required for the complex assembly (Kubo et al 2010) ISO3 thought to be

involved in starch degradation (Dinges et al 2003) In Arabidopsis leaves ISO3

is catalytically active on water-soluble polysaccharides that have been produced

by β-amylase and starch phosphorylase (Wattebled et al 2005)

11215 Disproportionating enzyme (D-enzyme E C 24125)

D-enzyme catalyzes the hydrolysis of -(14) linkages of unbranched

malto-oligosacharides and subsequent transfer of the glucan released at the

non-reducing end to a non-reducing end of the acceptor molecule to form a new

-(14) linkage D-enzyme mutation in Arabidopsis show reduced rates of

nocturnal starch degradation indicating that D-enzyme plays a part in the

pathway of chloroplast starch degradation (Critchley et al 2001) Some

research evidence suggested that the D-enzymes work in conjunction with SP

contributing to starch synthesis via the phosphorolytic SP reaction (Takaha et al

1998) According to this model the short-chain MOS liberated in the trimming

reaction by DBEs are converted to longer-chain glucans by D-enzyme which are

the substrates for phosphorolysis by SP liberating G-1-P used to synthesize

ADP-glucose by plastidial AGPase (Takaha et al 1998) In addition in

Chlamydomonas reinhardtii the phosphorolytic SP reaction is stimulated by the

presence of D-enzyme (Colleoni et al 1999)

29

11216 Starch phosphorylase (SP EC 2411)

Starch phosphorylase exists in both tetrameric and dimeric states and

catalyses the reversible transfer of glucosyl units from glucose-1-phosphate (G-

1-P) to the non-reducing end of α-1-4 linked glucan chains as shown in the

following equation

G-1-P + (14-α-D-Glucose)n (14-α-D-Glucose)n+1 + Pi

112161 Importance of SP in starch metabolism

SP has often been regarded as a glucan degradative enzyme (Preiss

1982 Preiss 1984) The α-glucan phosphorylase (EC 2411) found in animals

fungi and prokaryotes plays a major role in glucan catabolism (Preiss 1984)

and the amino acid sequence of the enzyme is found to be highly conserved

among prokaryotes and eukaryotes (Newgard et al 1989) Genetic analyses in

Chlamydomonas showed that the mutation of plastidial SP affected starch

accumulation (Dauvilleacutee et al 2006) In addition the mutation of plastidial α-

glucan phosphorylase could not change the total accumulation of starch or the

starch structure during the day or its remobilization at night when the

phosphorylase gene activity was eliminated by T-DNA insertion in Arabidopsis

thaliana leaves where transient starch is synthesized (Zeeman et al 2004) In

contrast research evidence demonstrated that the SP has a certain effect on the

storage starch biosynthesis that the development of plastidial SP activity

coincides with starch accumulation in developing cereal endosperms in rice

(Satoh et al 2008) in wheat (Schupp and Ziegler 2004 Tickle et al 2009) and

30

in maize (Yu et al 2001) Above evidence further suggests that the plastidial

forms of SP are involved in starch synthesis rather than the degradation in

higher plants

112162 The isoforms of SP in higher plants

Two major isoforms of SP are present in plants and differ in their

intracellular localization and are designated as plastidic (Pho1) and cytosolic

(Pho2) isoforms (Nakano and Fukui 1986) In developing rice endosperm

plastidial Pho1 accounts for about 96 of the total phosphorylase activity and it

is restricted to the stroma (Satoh et al 2008) The predicted protein sequence

alignment of Pho1 and Pho2 isoforms show a significant 50 amino acid extension

in the N-terminus of Pho1 which represent the transit peptide (Nakano and

Fukui 1986) In this thesis the term SP is generally used for the plastidial form

The plastidial form of SP (112 kDa in maize Mu et al 2001) is known to

be the second most abundant protein in the maize amyloplast stroma next to

SBEIIb (Yu et al 2001) Peptide sequences of plastidial SP in maize showed

higher identities to potato sweet potato and spinach and the N-terminus

sequence was unique in maize amyloplast it can not be aligned with any other

N-terminus sequences of Pho1 available in the gene bank (Yu et al 2001)

Excluding the N-terminus difference between Pho1 and Pho2 a unique 78-amino

acid insertion in the middle of the Pho1 sequence is a prominent characteristic of

the plastidial isoform in higher plants (Yu et al 2001) In potato Pho1 and Pho2

showed 81 - 84 amino acid sequence similarity over most part of the

sequence with the exception of N-terminal transit peptide and the large L-78

31

insertion located between the N and C terminal domains (Albrecht et al 1998)

Significant variation is found in the molecular mass of the Pho1 and Pho2 in

wheat endosperm as 100 kDa and 90 kDa respectively (Albrecht et al 1998)

The peptide sequence ILDNADLPASVAELFVK is a common sequence fragment

found in the L-78 region in maize and potato (Yu et al 2001 Albrecht et al

1998) In addition the sequence comparison among SP from potato tuber

rabbit muscle and Escherichia coli revealed the presence of the characteristic

78-residue insertion only in the middle of the polypeptide chain of the potato

enzyme (Nakano and Fukui 1986) (Fig 33 in Chapter 3) suggesting the L-78

region is specific to plants The proposed function of the L-78 insertion is

thought to be the obstruction of the binding of Pho1 to large highly branched

polysaccharides (Albrecht et al 1998) This idea was further confirmed by the

observation that the L-78 insertion in sweet potato (Ipomea batatas) blocked

the starch-binding site in Pho1 molecule showing low affinity towards starch

(Young et al 2006) Several serine phosphorylation sites were also found in the

L-78 insertion suggested that the regulation of Pho1 is phosphorylation

dependent (Young et al 2006) This research group was able to purify a 338

kDa protein kinase activity from sweet potato roots using liquid chromatography

methods and which actively phosphorylates the L-78 insertion (Young et al

2006) Interestingly this phosphorylation modification was not found in Pho2

isoform or after L-78 insertion was proteolytically removed from Pho1 (Young et

al 2006)

32

112163 Characterization of SP

All phosphorylases exist as dimers or tetramers of identical subunits and

have similar kinetic and structural properties but their regulatory mechanisms

may vary depending on the source of the enzyme (Dauvilleacutee et al 2006

Weinhaumlusel et al 1997 Brisson et al 1989) or its multimeric state (see later)

The α-glucan phosphorylase found in bacterial forms has a homodimeric

molecular structure (Dauvilleacutee et al 2006 Weinhaumlusel et al 1997) Gel filtration

chromatography studies revealed that the native enzyme consisted of two

identical subunits in maize (Mu et al 2001) which coincides with findings of

Tanabe et al (1987) on availability of dimeric form (203 kDa) of α-glycogen

phopsphorylase in yeast The purified form of SP from maize endosperm was

thermally labile above 50degC where optimum enzyme activity is at pH 60 in the

synthetic direction and pH 55 in the phosphorolytic or degradative direction at

40degC (Mu et al 2001)

112164 Biochemical characterization of SP

According to their affinities for glucan substrates SPs are further

classified as low affinity (SP-L) and high affinity (SP-H) isoforms respectively in

potato tuber and leaf (Mori et al 1993) When the L-78 insertion in SP-L was

replaced by high affinity SP-H sequence the SP-L showed less affinity to

glycogen compared to SP-H form (Km=10400 and Km=10 μgmL) (Mori et al

1993) The L-78 insertion-replaced chimeric enzyme was five times less active

than the SP-L isoform but still showed low affinity to glycogen than in SP-L

(Km= 24 μgmL) However when the glycogen was replaced by amylopectin

33

and amylose (DP=30) the affinity increased in SP-L (Km= 82 and Km=76

μgmL respectively) in SP-H form (Km=36 and Km=87 μgmL respectively)

and in chimeric form (Km=53 and Km=2 μgml respectively) Among all the

isoforms the SP-H form has the highest affinity to amylopectin suggesting that

the L-78 region has greater affinity towards low molecular weight substrates

(Mori et al 1993) In addition two isoforms named Pho1a and Pho1b were

identified in potato (Sonnewald et al 1995) The homodimeric form of Pho1a

isoform was immunochemically detectable only in tuber extracts where both

Pho1a and heterodimeric Pho1b were present in leaf extracts in potato (Albrecht

et al 1998) Wheat has three forms of SP (designated as P1 P2 P3) which are

distinguished in non-denaturing separation gels containing glycogen (Schupp

2004) The activity form P3 is plastidic in where as P1 and P2 are cytosolic and

found mainly in younger leaves (Schupp 2004) However mature leaves only

contain the plastidic form which was also strongly evident in the endosperm of

the developing seeds Cytosolic forms are more prominent in germinating seeds

(Schupp 2004) suggestive of the involvement of cytosolic SP forms in the

utilization of α-glucans resulting from starch degradation

The plastidial and cytosolic SP show different affinity towards high and low

molecular glucan polymers in synthetic direction (Table 11) Plastidial SP

prefers amylopectin than the glycogen potato tuber (Liddle et al 1961) spinach

leaf (Shimomura et al 1982) and sweet corn (Lee and Braun 1973) and maize

(Yu et al 2001) In maize endosperm the Km value for amylopectin in the

synthetic direction of the SP reaction was 34-fold lower and the Kd value was

40-fold lower than of glycogen (Yu et al 2001) The kinetic analysis indicated

34

that the Km value for amylopectin was eight-fold lower than that of glycogen

and the phosphorolytic reaction was favored over the synthetic reaction when

malto-oligosaccharides (DP= 4 to 7 units) were used as substrates (Mu et al

2001)

Table 11 The Km and Vmax values of starch phosphorylase in different plant species SP-L =plastidial form of SP SP-H= cytosolic form of SP (s) = synthetic

direction (p) = phosphorolytic direction

Plant Tissue

Substrate

Vmax

(umolminmg)

Km

(mgml)

Reference

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

058 (s)

063 (s)

013 (s)

045 (s)

Yu et al

2001

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

Maltoheptaose

73 (s) 111 (p)

716 (s) 1180(p)

78 (s) 1993 (p)

0017 (s) 0028(p)

025 (s) 094(p)

008 (s) 01 (p)

Mu et al

2001

Sweet potato

tuber crude

extract

Starch

G-1-P

Pi

0077 (s)

0115 (p)

1052 (s) 1498(p)

Young et al

2006

Potato tubers

Recombinant

proteins of

SP-L and SP-

H types

Maltopentaose

SP- L type

SP- H type

Glycogen

SP- L

SP- H

Amylopectin

SP- L

SP- H

Amylose

DP=30

SP- L

SP- H

396 (s) 165 (p)

961 (s) 368 (p)

83 (p)

94 (p)

79 (p)

83 (p)

139 (P)

182 (P)

013 (s)

112 (s)

10400 (p)

98 (p)

82 (p)

36 (p)

76 (P)

87 (P)

Mori et al

1993

35

ADP-glucose the major precursor for starch biosynthesis has been known

for long time as an inhibitor of activity of SP in the synthetic direction (Matheson

and Richardson 1978) ADP-glucose (at 4 mM) reduced the synthetic activity of

plastidial SP and G-1-P (at 10 mM) reduced the activity of cytosolic SP by 18

to 22 respectively in pea seeds (Matheson and Richardson 1978) Low

concentration of G-1-P and high PiG-1-P ratio increase the degradation activity

by glycogen phosphorylase in vivo (Schupp and Ziegler 2004 Newgard et al

1989) suggesting SP degradative activity is increased by inorganic phosphate

(Pi) In addition in developing barley endosperm cytosolic Pi concentration was

very higher (over 23 folds) than G-1-P where cytosolic form of SP required

higher level of Pi (Tiessen et al 2011) However according to the findings of

Hwang et al 2010 incorporation of [14C]-G-1-P into starch was only partially

affected by Pi Even under physiological G-1-P substrate levels (02 mM)

plastidial SP from rice was still able to carry out the biosynthetic reaction

although at low rates in the presence of 50-fold excess of Pi in vitro Hence

under conditions that would favor the degradation of starch plastidial SP

preferentially carries out biosynthesis

The animal orthologue of SP glycogen phosphorylase consists of two

identical subunits each of which have a highly conserved C-terminal region

incorporating a pyridoxal phosphate molecule which is essential for activity and

a site effecting non-catalytic glucan binding (Newgard et al 1989) The activities

of animal glycogen phosphorylases in releasing glucose for dissimilative

metabolism are highly regulated by allosteric effectors and covalent

modifications (Johnson 1992 Newgard et al 1989) All known α-glucan

36

phosphorylases require pyridoxal 5-phosphate for activity as a cofactor (Yanase

et al 2006) The maize shrunken-4 mutant is found to be lacking SP activity in

the endosperm and the mutants had reduced the starch content and the soluble

protein content by two-third than in the wild type kernel (Tsai and Nelson

1969) The activities AGPase and SS are also reduced in the shrunken-4 mutant

while reducing the total amount of pyridoxal-5-phosphate in the endosperm by

8-fold than in the wild type endosperm (Tsai and Nelson 1969) This reduction

was identified as the lack of SP cofactor pyridoxal-5-phosphate in the shrunken-

4 mutant in the maize (Tsai and Nelson 1969) However external addition of

pyridoxal-5-phosphate to the assay system for SP activity of the mutant did not

affect the activity (Yu et al 2001) Thioreactive agents such as diethyl

pyrocarbonate phenylglyoxal have also been identified as some of the chemical

inhibitors of SP (Mu et al 2001)

The pho1 mutants developed in rice endosperm have helped to elucidate

the in vitro role of SP on the other major starch biosynthetic enzyme isoforms

(Satoh et al 2008) Induced mutagenesis of SP by N-methyl-nitrosourea

treatment led to the creation of a series of mutants with a considerable

reduction in starch contents from the seed morphologies varies from white-core

pseudonormal to shrunken in rice particularly at different temperatures (varied

from 20oC to 30oC) (Satoh et al 2008) The white-core phenotypes made

approximately 18 and 20 mg of grain weight in pseudonormal approximately

18 and 19 mg and in shrunken made 10 and 8 mg of grain weight where the

wild type approximately made 22 mg both at 30oC and 20oC temperatures

respectively Scanning electron microscopy showed that the sizes of the starch

37

granules were decreased (shrunken phenotype had the smallest granules than in

the wild type) in the mutant lines and some granules were more spherical than

the irregular polyhedron-shaped granules typical of wild-type starch grains

High-resolution capillary electrophoresis technique was used to measure the

chain length distribution of the amylopectin in the endosperm The mutants

created a higher proportion of DP=11 shorter glucan chains with a decrease in

the proportion of intermediate chains with a DP= 13-21 Even though the seed

weight was varied within the white-core pseudonormal and shrunken

phenotypes of the mutants they have demonstrated a similar change in chain

length distribution in the amylopectin In contrast this study also showed that

the Pho1 mutants did not have any effects on the measurable activity levels of

the other major starch biosynthetic enzymes such as AGPase DBE isozymes

(isoamylase and pullulanase) SBE isoforms (SBEI SBEIIa and SBEIIb) and SS

isoforms (SSI and SSIIIa) (Satoh et al 2008) Based on these results the

authors suggested that the SP could operate at two distinct phases of starch

biosynthesis one phase consisting of starch initiation and a second phase is in

starch elongation (Satoh et al 2008) The in vitro analysis of chain length

elongation properties of recombinant SP and SSIIa from rice were compared on

MOS of DP=4 DP=6 or DP=7 glucan primers Despite the type of primer used

in the reaction the two enzymes showed different product distributions to each

other (Satoh et al 2008) SP produced a broad distribution of MOS products of

increasing size mostly DP= 6-11 SSIIa showed a much narrower distribution

(DP= 6-7) of MOS products The results clearly indicated that SP can synthesize

much longer linear glucans (DP= 16) than SSIIa (DP= 7-9) (Satoh et al 2008)

38

In addition the catalytic activity of SP from rice is significantly higher (75

mmoles G-1-Pmg proteinmin) toward MOS than SSIIa is (24 nmoles

ADPglucosemg proteinmin) Therefore these results support a role for SP in

extending small MOS whereas rice SSIIa is unlikely to be involved in this

process The authors further suggested that these longer linear glucan chains

which are produced by SP could presumably be the linear substrates for SBE to

form branched glucans in the starch initiation process (Satoh et al 2008)

Functional interactions between SP and SBE isoforms were observed in

vitro and showed that purified SP from rice endosperm synthesized glucans

from G-1-P in the presence of different isoforms of SBE even without any

exogenous glucan primer (Nakamura et al 2012) Glucan production was higher

by SP when SBEI was present compared to SBEIIa or SBEIIb and produced

glucan polymers with DP =11 7 and 6 respectively (Nakamura et al 2012)

Activities of SP and SBE were depended on the mutual availability SP and SBE

and showed mutual capacities for chain elongation and chain branching

(Nakamura et al 2012)

The isoforms of the major enzymes involved in starch biosynthesis are

regulated by protein phosphorylation and protein-protein interactions (Liu et al

2009 Hennen-Bierwagen et al 2008 Tetlow et al 2008 Tetlow et al 2004)

Plastidial SP in wheat endosperm is also involved in formation of active protein

complexes with the SBEI and SBEIIb particularly in wheat amyloplast stroma in

a phosphorylation-dependent manner (Tetlow et al 2004) Novel complexes of

starch synthesis enzymes assembled in the amylose extender (ae-) mutant

(lacking SBEIIb) of maize (Liu et al 2009) The complex formed by SSI SSII

39

with SBEIIb in wild-type was replaced by forming SBE1 combined with SP in the

ae- mutant (Liu et al 2009) Genetic analyses further revealed that the loss of

SBEIIb in ae mutant could cause a significant increase in the SBEI SBEIIa

SSIII and SP in the starch granule (Liu et al 2009 Grimaud et al 2008)

112165 SP and starch biosynthesis models

Based on recent genetic and biochemical evidence some researchers

suggested that SP may play a role in the initiation of starch biosynthesis (Satoh

et al 2008 Leterrier et al 2008 Roldan et al 2007) Tickle et al (2009)

recently suggested a model in which SP plays a role in starch synthesis via two

pathways First SP degrades the soluble malto-oligosaccharides (MOS) which

are made from starch via the action of DBE into G-1-P in the amyloplast

stroma This G-1-P can then be converted to ADP-glucose by AGPase and to

recycled back into starch The second mechanism suggests that SP can directly

act on the surface of the starch granule where it could phosphorolytically

modify the structure of starch to produce G-1-P (Tickle et al 2009) Recent

mutant analysis in Arabidopsis suggests plastidial SP is not required in starch

degradation in chloroplasts (Zeeman et al 2004) The leaves of mature SP

mutant plants had small white lesions on the tips or margins of fully expanded

leaves It was suggested that SP may play a role in creating tolerance to abiotic

stresses in leaves by providing an alternate route for starch degradation

(Zeeman et al 2004)

40

The existence of a complementary path of forming reserve starch was

discussed in potato by analyzing the effect of the G-1-P-dependent intracellular

carbon flux (Fettke et al 2010) The tuber discs of wild-type and various

transgenic potato lines expressing an antisense construct directed against the

plastidial SP isofoms were incubated with 14C-lablled G-1-P G-6-P sucrose and

maltose Highest amount of starch was measured in G-1-P substrate compared

to G-6-P sucrose and maltose indicating that the path of starch biosynthesis is

functional that is selectively initiated by the uptake of the anomeric glucose

phosphate ester (Fettke et al 2010) The initiation of this path is separated

against external glucose 6-phosphate Rice SP mutants grown at 300C produced

about 6 of the shrunken phenotypes (compared to 100 in wild-type) the

starch content was similar in the wild-type and the percentages of shrunken

phenotype was increased in SP mutant plants when the temperature was

decreased to 250C and 200C by 35-39 and 66 respectively with a severe

reduction in starch accumulation It was suggested that SP may play an

important role in starch biosynthesis during fluctuating andor adverse

temperature conditions in rice (Satoh et al 2008)

112166 Evidence of interaction of SP with SSIV

Research evidence suggested potential interactions between the SP and

SSIV enzymes In Arabidopsis thaliana leaves the activity of SP increased in ss4

mutants by 14 -2 fold compared to the wild-type without changing starch

structure or the amyloseamylopectin ratio and the concentrations of soluble

sugars remain unchanged (Roldan et al 2007) However granule size was

41

increased in ss4 mutants with a reduction in the granule number to 2-3 granules

per amyloplast compared to the 4-5 granules in wild-type (Roldan et al 2007)

Interestingly the double mutant of ss4 and sp produced granule size of at least

4 times higher than starch granules originating from the wild-type plants

(Planchot et al 2008)

1122 Post translational modification of starch biosynthesis enzymes

Protein phosphorylation allosteric and redox modification are the major

post translational modifications which take place in order to control the activity

of enzymes Phosphorylation of major starch biosynthetic enzymes was recently

discovered by Tetlow et al (2004) who investigated the role of protein

phosphorylation as a mechanism of regulation of the starch synthesis in

developing wheat endosperm After incubating intact plastids from wheat with -

[32P]-ATP it was found that three isoforms of SBErsquos (SBEI SBEIIa and SBEIIb)

were phosphorylated on serine residues (Tetlow et al 2004) The activity of

SBEIIb in amyloplasts and SBEIIa in chloroplasts was stimulated by

phosphorylation whereas dephosphorylation using alkaline phosphatase reduced

catalytic activity (Tetlow et al 2004)

There is increasing evidence that starch synthesis does not consist of

several isolated and simple reactions as indicated in Figure 13 The interaction

and coordination of starch biosynthetic enzymes appears to be a general feature

of starch biosynthesis in plants Starch biosynthetic enzymes form heteromeric

protein complexes that are probably involved in starch synthesis (Hennen-

Bierwagen et al 2008 Tetlow et al 2008 Tetlow et al 2004) Co-

42

immunoprecipitation experiments revealed that SP SBEIIb and SBEI form a

protein complex of three enzymes when only these enzymes are phosphorylated

within the soluble protein fraction in wheat amyloplasts lysates (Tetlow et al

2004) Dephosphorylation with alkaline phosphatase disassembled the complex

formed (Tetlow et al 2004) suggesting that the protein-protein interactions are

likely to be phosphorylation-dependent In developing endosperm of barley the

sex6 mutant lacking SSIIa resulted a reduction in amylopectin synthesis to less

than 20 of the wild-type levels and production of high amylose starches

(Morell et al 2003) A pleiotropic effect of the SSIIa mutation abolished the

binding of SSI SBElla and SBEIIb to the starch granules while not significantly

altering their expression levels in the soluble fraction (Morell et al 2003) In

wheat endosperm physical interactions between SSrsquos and SBErsquos were detected

and two distinct complexes identified (Tetlow et al 2008) The authors found

one complex consisting of SSI SSII and SBEIIa and another complex with SSI

SSII SBEIIb Furthermore both of these complexes are phosphorylated and in

vitro dephosphorylation with alkaline phosphatase resulted in disassociation of

the proteins In maize amyloplasts a multi-subunit complex containing SSIIa

SSIII SBEIIa and SBEIIb was detected using gel permeation chromatography

(Hennen-Bierwagen et al 2008) The authors also located another complex

consisting of starch synthesizing enzymes SSIIa SBEIIa and SBEIIb In the ae-

mutant lacking SBEIIb a novel protein complex was found in which SBEIIb was

replaced by SBE1 and SP (Liu et al 2009) Analyses further revealed that

eliminating SBEIIb in ae- mutant caused significant increases in the abundance

of SBEI SBEIIa SSIII and SP in the granule (these proteins are not found in

43

the granule in the granules of wild-type maize) without affecting SSI or SSIIa

(Grimaud et al 2008) Staining the internal granule-associated proteins using a

phospho-protein specific dye revealed phosphorylation of at least three proteins

GBSS SBEIIb and SP (Grimaud et al 2008) This evidence added weight to the

hypothesis that starch synthesizing enzymes exists as hetero complexes in

developing cereal endosperm and these proteins eventually become granule-

associated via as yet unknown mechanisms

12 Objectives of the study

As the research evidence indicates SP may have the potential to be

involved in starch synthesis possibly involving the formation of protein

complexes with other enzymes Therefore the first aim of this research project

was

To determine whether the SP is involved in starch biosynthesis in maize

endosperm by interacting with starch biosynthetic enzymes and forming

protein complexes

The second objective was to understand the involvement of SP in starch

synthesis in maize and explore possible interactions with SSIV

The third objective was to investigate if the SP-involved protein-protein

interactions are regulated by protein phosphorylation

The results in this thesis discuss the possible interaction of SSIV and SP

and the mechanisms of their regulation through phosphorylation in wild type

developing maize endosperm using the amyloplast lysates and partially purified

44

recombinant SP This research aims to provide further insight into our growing

understanding of coordinated activity of different enzymes associated in starch

synthesis through protein-protein interactions and complex formation in

developing maize endosperm The results in the thesis outline the biochemical

characterization of SP and SSIV in developing maize endosperm and explore

possible protein-protein interactions of SP and other starch biosynthetic

enzymes The protein complexes in amyloplasts could influence the quality as

well as the quantity of starch in developing endosperm through the modulation

of the granule structure Understanding of the basis of these modulations in

starch is therefore essential Starch produced in plastids provides up to 80 of

the food calorie requirement of humans with various potential applications in

nonndashfood industries Application of starch in food and non-food industries is

depends on different structural and functional properties of starch which can be

modified with the knowledge of its genetic manipulations This research

expected to enhance our understanding of the basics of starch biosynthesis to

develop models of starch structure assembly

45

CHAPTER 2

46

Biochemical Investigation of the Regulation of Plastidial Starch

Phosphorylase in Maize Endosperm

21 Introduction

Starch phosphorylase (SP) is a tetrameric orand dimeric enzyme which

catalyses the addition of glucosyl units from glucose-1-phosphate (G-1-P) to the

non-reducing end of α-1-4 linked glucan chains liberating inorganic phosphate

(Pi) in forward reaction and produces G-1-P while degrading glycosyl units in

reverse reaction SP is potentially involved in both starch synthesis and

degradation as shown in the following equation


Two isoforms of SP are found in higher plants designated by their sub-

cellular localization the plastidial (Pho1) and the cytosolic (Pho2) (Zeeman et al

2004 Steup et al 19881981 Nakano and Fukui 1986) The plastidial form

(Pho1) in maize endosperm is designated as SP in this thesis

211 Cytosolic form of SP (Pho2)

The extraplastidic (Pho2) starch phosphorylases do not contain L-78

amino acid insertion as in plastidial form (Pho1) and they are much more

effective in degrading processes (Zeeman et al 2004 Steup et al 1988) Pho2

preferentially degrades branched starch molecules and can even attack starch

47

granules in vitro (Steup et al 1988) However in starch-accumulating tissues

like developing seeds and leaves which maintain intact amyloplasts or

chloroplasts cytosolic Pho2 has no direct access to the starch inside the plastid

Cytosolic SP may be involved in regulating the cytosolic G-1-P level by

glucosylating and trimming a heteropolysaccharides found in the cytosol

produced mainly from maltose (a product of starch breakdown inside the

plastid) which is translocated to the cytosol through MEX1 transporter located in

the plastidic membrane (Yang and Steup 1990 Steup et al 1991 Buchner et al

1996 Pyke 2009 Rathore et al 2009) The production of metabolites such as

maltose and glucose which are exported to cytosol are involved in glycan

metabolism by the action of cytosolic phosphorylase (Pho2) disproportionating

enzyme cytosolic transglucosidase and Pho2 produces G-1-P in the cytosol

(Pyke 2009 Zeeman et al 2004) Fig 21 illustrates the putative roles of

plastidial (Pho1) and cytosolic (Pho2) SP in starch metabolism in plants

212 Plastdial SP (Pho1)

The plastidial isoform of SP Pho1 is present throughout endosperm

development in cereals (Schupp and Ziegler 2004 Satoh et al 2008 Tickle et

al 2009) The Pho1 also contributes the highest proportion of the total SP

activity in the endosperm and remains active throughout the endosperm

development in rice endosperm (Satoh et al 2008) Also the mutation in Pho1

in rice endosperm produces a shrunken phenotype endosperm with reduced

starch content and altered starch granule structure in rice (Satoh et al 2008)

The shrunken 4 mutants lacking plastidial SP activity in maize endosperm

48

produce endosperms with reduced starch contents (Tsai and Nelson 1969) and

the fact that Pho1 does not appear to influence starch degradation in

Arabidopsis thaliana (Zeeman et al 2004) suggests plastidial SP may play a role

in the storage starch biosynthesis or play a subsidiary role in to the α-

amylolytic pathway in starch in starch degradation

Figure 21 Schematic diagram illustrating the putative roles of plastidial (Pho1) and cytosolic (Pho2) SP in starch metabolism in plants The dashed lines indicate

that there may be intermediate steps in the pathways ADGP=ADP glucose pyrophosphorylase SS= starch synthases SBE= starch branching enzymes DBE= debranching enzymes DPE1DPE2= Disproportionating enzymes GWD=

glucan water dikinase PWD=phospho-glucan water dikinase Glc-1-P= glucose-1-phosphate GT= glucose transporter MEX1= maltose transporter TPT= triose

phosphate transporter (Modified from Rathore et al 2009)

49

The biochemical characteristics of plastidial SP such as the lower affinity

towards the high molecular starch and the higher affinity towards the low

molecular weight linear malto-oligosaccharides (MOS) in sweet potato tubers

(Young et al 2006) suggested the possibility that SP acts on elongating the

shorter glucan chains and might be also involved in the process of granule

initiation The 78 amino acid insertion (L-78) in the middle of the sequence in

Pho1 but not in cytosolic Pho2 is a prominent molecular characteristic in all the

plant species investigated This insertion prevents the binding of SP to large

highly branched polysaccharides in sweet potato tubers (Young et al 2006) In

contrast in cereals SP showed higher affinities towards to amylopectin than

glycogen in synthetic direction and to MOS in phosphorylitic direction (Mori et al

1993 Mu et al 2001 Schupp and Ziegler 2004)

The plastidial form of SP in maize endosperm amyloplasts is 112 kDa in

size and known to be the second most abundant enzyme presence next to the

SBEIIb (Yu et al 2001) In addition to the presence of the L-78 insertion in the

middle of the maize SP protein sequence the N-terminus of maize amyloplast

SP does not align with any other N-terminus sequences of SP available in the

gene bank (Yu et al 2001) Due to the variability in the N- terminus of the

enzyme SP in maize and other plastidial SP forms may have different regulatory

mechanism for example the N-terminus of the protein generally contain signal

recognition peptides targeting peptides and important in enzyme regulation

(Fig 22)

The first evidence for the post translational regulation of SP described the

phosphorylation of SP and its involvement in phosphorylation-dependent

50

protein-protein interactions in wheat amyloplast stroma with SBEI and SBEIIb

(Tetlow et al 2004) In the maize ae1 mutant amyloplasts lacking SBEIIb

novel protein complexes are found with SP these include SSI SSIIa SBEI and

SBEIIa (Liu et al 2009) The ae2 mutant contains an inactive form of SBEIIb

found to be associated in complex formation with SSI SSIIa and SBEI both in

the stroma and the granule (Liu et al 2012) Interestingly the SP is not involved

in complex formation in ae2 mutant as seen in ae1 mutant (Liu et al 2012)

Indirect evidence implicates interactions between SP and SSIV in

mutants of Arabidopsis The activity of both Pho1 and Pho2 increased in SSIV

mutants (Atssiv1 and Atssiv2) by 14 -2 fold compared with the wild-type in

Arabidopsis thaliana leaves where transient starch is synthesized (Roldan et al

2007) However there was no significant influence on starch structure or the

amyloseamylopectin ratio in these mutants and the concentrations of soluble

sugars remain unchanged (Roldan et al 2007) A double mutant produced by

the insertion of an heterologous T-DNA within the nucleic sequence of an intron

or an exon lacking both Pho 1 and SSIV activity produced 1-2 granules per

plastid (3-4 granules per plastid in wild-type) but increased the granule size by

at least four times higher than the starch granules originating from the their

single mutants plants in Arabidopsis (Planchot et al 2008 patent EP1882742)

However no evidence is currently available to show any direct relationship

between SP and SSIV in storage starch synthesizing tissues

The active Pho1 enzyme exists as an assembly of dimeric or tetrameric

subunits in maize and different multimeric forms of SP in maize might be

involved in the formation of different protein complexes (Liu et al 2009 Mu et

51

al 2001) Previous studies confirmed that SP activity can be modulated by the

substrates ratio of G-1-PPi (Schupp and Ziegler 2004 Mu et al 2001) and

ADP-glucose (Matheson and Richardson 1978) Comparatively less information

is available on SP regulation by protein phosphorylation in storage starch

synthesizing tissues Unlike the SP mutant lines developed in rice (Satoh et al

2008) and Arabidopsis (Roldan et al 2007 Planchot et al 2008) there are no

genetically developed mutants lines available in maize The shrunken-4 mutant

has reduced SP activity but this is probably due to alterations in levels of

pridoxal-5-phosphate the essential cofactor for SP activity in the endosperm

(Tsai and Nelson 1969)

The objectives of this study were to characterize and investigate the role

and regulation of Pho1 in maize wild-type amyloplasts by protein

phosphorylation and protein-protein interactions Moreover the possible

involvement of SP in granule initiation was tested specifically by testing the

possibility of interactions between SP and SSIV in the amyloplast

52

22 Materials and Methods

221 Materials

2211 Plant materials

The wild type maize (C G 102) (Zea mays) was used in all experiments

The cobs were collected at different growth stages (5-35 days after anthesis)

from wild type maize plants grown under the normal field conditions Cobs were

kept at +40C cold room until use for amyloplast extractions The kernels were

also collected and frozen at -800C for future use for whole cell (crude) extracts

2212 Chemicals

All chemicals were obtained from Sigma Aldrich unless otherwise stated

222 Methods

2221 Amyloplast purification from maize endosperms

Endosperms harvested at 22 days after anthesis (DAA) from the wild-type

of maize plants were mainly used to purify the amyloplasts in the experiments

unless otherwise stated This stage of endosperm development was found to be

the major grain-filling period (Liu et al 2009) Amyloplasts are purified to

remove any contaminating proteins that may be found in maize whole cell

lysates Maize amyloplast extraction was performed as described by Liu et al

2009

Approximately 100g of the endosperms were taken from the developing

kernels with a spatula and gently chopped with a razor blade in 40-50 mL of ice-

cold amyloplast extraction buffer (50 mM N-(2-hydroxyethyl) piperazine-Nrsquo-

53

ethanesulphonic acid (HEPES)KOH pH 75 containing 08 M sorbitol 1 mM

KCl 2 mM MgCl2 and 1 mM Na2-EDTA) on a petri dish on ice until firmly

chopped in to creamy solution The resulting whole cell extract was then filtered

through four layers of Miracloth (CalBiochem catalogue no 475855) wetted in

the same buffer Then the filtrate was then carefully layered onto 15 mL of 3

(wv) Histodenz (Nycodenz Sigma catalogue no D2158) in amyloplast

extraction buffer followed by centrifugation at 100xg at 40C for 20 min and the

supernatant was carefully removed The pellet with intact amyloplasts was

ruptured with 1 mL of ice-cold rupturing buffer containing 100mM N-tris

(hydroxymethyl) methyl glycine (Tricine)KOH pH 78 1 mM dithiothreitol

(DTT) 5 mM MgCl2 and a protease inhibitor cocktail (5μl per 1 mL buffer) (see

Appendix 09 for details) Then the mix was transferred into micro-centrifuge

tubes and centrifuged at 13000xg at 40C for 5 min to remove starch The

soluble fractions were frozen in liquid nitrogen and stored at -800C until further

use The amyloplast lysates were ultra-centrifuged at 100000xg for 15 min

before use to remove plastidial membranes

2222 Preparation of whole cell extracts

Whole cell extracts were prepared as described previously by (Tetlow et

al 2003) Approximately 10 g of endosperm tissue was quickly frozen in liquid

nitrogen and immediately ground into a fine powder adding liquid nitrogen on

ice using a chilled mortar and pestle The frozen powder was mixed with ice-cold

rupturing buffer (same rupturing buffer used in amyloplast purification) and a

protease inhibitor cocktail (5 μL per 1 mL buffer) (see Appendix 09 for details)

54

The mixture was further mixed and allowed to stand on ice for 5 min followed by

centrifugation at 13000xg for 5 min at 40C The supernatant was subjected to

ultracentrifugation at 100000x g for 15 min in a Beckman Coulter Optima-Maxndash

XP ultracentrifuge to remove membranes and particulate material The

supernatant obtained following the ultracentrifugation was used for experiments

2223 Localization of SP in the plastid

To investigate the proportional of SP and other starch biosynthetic

proteins in the stroma-granule interface where the proteins are imbedded on

granule surface the remaining pellet (approximately 1 g of fresh weight) from

the isolation of amyloplast lysates (as described in section 2221) was

subjected to a series of washings (for up to 10 times) with rupturing buffer (03

mLwashing stage) used in amyloplast extraction The supernatant was collected

after centrifugation at 13000xg for 5 min and the proteins were separated on

the SDS gels and the proteins are visualized by silver staining and identified by

immunoblotting

2224 Preparation of granule bound proteins

The granule bound protein was isolated as the method described by

(Tetlow et al 2004) After rupturing of the amyloplasts and the separation of

soluble protein fractions by centrifugation (as described in section 2221) the

remaining pellets (approximately 1g) were resuspended in 1 mL of cold aqueous

washing buffer [50 mM Tris (hydroxymethyl) aminomethane (TRIS)-acetate pH

75 1 mM Na2 -EDTA and 1 mM DTT] and centrifuged at 13000 rpm for 1 min

55

at 40C This washing step was repeated 8 times The pellet was then washed

three times with 1 mL acetone each time followed by three washes with 2

(wv) SDS (1 mL each time) Starch granule bound proteins were extracted by

boiling the washed starch in 2XSDS loading buffer [625 mM TRIS-HCl pH 68

2 (wv) SDS 10 (wv) glycerol 5 (vv) β-mercaptoethanol 0001 (wv)

bromophenol blue] for 5 min at 900C The boiled samples were cooled and

centrifuged at 13 000xg for 5 min and supernatants separated by SDS-PAGE

2225 Biochemical characterization of SP in maize endosperm

22251 Phosphorylation and dephosphorylation of amyloplast lysates

The amyloplast lysatescrude extracts were incubated with 1 mM ATP to

stimulate protein phosphorylation by protein kinases present in the endosperm

To prevent in vitro dephosphorylation the lysates were also incubated with

phosphatase inhibitor cocktail (10 μl1ml lysates) in a separate tube as a

control Another treatment involved the incubation of maize amyloplast lysates

with alkaline phosphatase conjugated to agarose beads (APase insoluble form

suspension in (NH4)SO4 final conc 25 unitsmL) to promote non-specific

dephosphorylation Untreated amyloplast lysates were used as the control in all

phosphorylation experiments All samples had gt1 mM MgCl2 Rupturing buffer

was added to balance the total end-volumes of the treatments Phosphatase

inhibitor (PI) was added to inhibit the endogenous alkaline phosphatases in the

sample as a control (see appendix 09 section 1 for the details about PI)

56

22252 Enzyme assays

222521 Starch phosphorylase glucan synthetic activity assay

The synthetic activity of SP was assayed in vitro by using amylopectin

glycogen and maltoheptaose as the substrates 80 μL of glucan substrates

(25 [wv] prepared in 100 mM MES-NaOH [pH 60] only amylopectin was

gelatinized before adding to the mixture) and 20 μL [U14C]-G-1-P (GE Health

care catalogue No CF0113 10 mM stock 01μCi prepared in 100 mM MES

[pH 60]) were added to a clean 15 mL micro centrifuge tube [U-14

C]-G1P was

used The reaction was initiated by adding 100 μL extract in 10 second intervals

and terminated after incubated for 30 minutes at 37degC by the addition of 1 mL

stop solution (75 [vv] methanol 1 [wv] KCl) Samples were then

centrifuged at 10000g for 5 minutes The supernatant was removed and the

remaining pellet was resuspended in 300 μL H20 before the addition of 1 mL

stop solution Samples were then centrifuged for a further 5 minutes at

10000xg for 5 min and the supernatant was removed The pellet was

resuspended in 300 μL H20 and added to 37 mL Ecoscinttrade scintillation cocktail

and radioactivity was measured in a liquid scintillation analyzer (Bekman

Coulter-USA ls-6500 Multi-purpose scintillation counter) Amount of [U-14

C]-G-

1-P incorporated into glucan was calculated

222522 Starch phosphorylase glucan degradative activity assay

SP phosphorolytic activity was determined based on the procedure

described by (Tickle et al 2009) The G-1-P formed in the phosphorolysis

57

direction was converted to glucose-6-phosphate (G-6-P) by

phosphoglucomutase and then the G-6-P converted to 6-phopsphogluconate by

glucose-6-phosphate dehydrogenase The amount of NADH was released at this

step was analyzed at 340nm the amount of NADH was equal to the amount of

G-1-P produced in the reaction In the procedure for one reaction (1 mL final

volume) final concentration of 20 mM HEPES (pH 70) was added to a 1 mL

plastic cuvette with final concentrations of 5 mM MgCl2 025 mM NAD 0024

mM glucose-16-bisphosphate and 1 [wv] substrate (glycogen amylopectin

and maltoheptose) (all solutions were prepared in 50 mM HEPES [pH 70]) 37

μL phosphoglucomutase (05 unitsμL-1

) 100 μL of amyloplast lysates (095

mgmL concentration) and 16 μL glucose-6-phosphate dehydrogenase (032

unitsμL-1

) This reaction was initiated by the addition of 45 mM Na2HPO

4 as the

source of Pi

22253 Gel filtration chromatography (GPC)

Extracts of soluble proteins from maize amyloplasts and whole cell

extracts (500 μL loading volume) were separated through a Superdex 200

10300GL gel permeation column (equilibrated with two column volumes of the

rupturing buffer) on an AKTA- FPLC system (Amersham Pharmacia Biotech

model No 01068808) The column was calibrated using commercial protein

standards from 137 kDa to 669 kDa (GE Healthcare Gel Filtration Calibration

Kits low molecular and high molecular weight) The column was pre-equilibrated

with two column volumes of running buffer containing 10 mM HEPES-NaOH pH

58

75 100 mM NaCl 1 mM DTT and 05 mM PMSF at a flow rate of 025 ml

min_1 05 ml fractions were collected

2226 Protein analysis

22261 Quantification of proteins

Protein content was determined using the Bio-Rad protein assay (Bio-Rad

Laboratories Canada) according to the manufacturerrsquos instructions and using

bovine serum albumin as the standard

22262 Sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE)

SDS-PAGE was performed using a Mini-Protean III Vertical Electrophoresis

System (Bio-Rad) according to the manufacturerrsquos instructions Proteins were

separated on SDS-PAGE on 10 acrylamide gels The compositions of 3

stacking gel and the separation gel was shown in Table 21 Prior to

electrophoresis proteins were mixed with SDS sample buffer (625 mM Tris-HCl

pH 68 2 [wv] SDS 10 [wv] glycerol 5 [vv] b-mercaptoethanol

0001 [wv] bromophenol blue) and boiled for 5 min at 900C The gel was run

using 025 M Tris (pH 72) 192 mM Glycine 04 SDS running buffer at 120V

for 15hr

59

Table 21 The composition of stacking and resolving gels for SDS-PAGE

Stock solution SDS-PAGE (10 mL) Stacking gel Resolving gel

(5 acrylamide) (10 acrylamide)

ProtoGelreg

Acrylamidebisacrylamide 168 34 (30[wv])

05M Tris (pH 68) 128 - 15M Tris (pH 88) - 26 10 (wv) SDS 01 01

10 (wv) ammonium persulfate 0112 01 Distilled water 7 38

TEMED 0008 001

22263 SP-Native affinity zymogram

Zmogram analysis was used to identify the activity of the proteins as

described by (Tickle et al 2009) The extracts were separated by substrate-

affinity (glycogen amylopectin and maltoheptaose) non-denaturing PAGE The

non-denaturing gels were prepared as 8 (wv) polyacrylamide gels containing

glycogen (01 wv) (Table 22) The composition of the stacking gel and the

resolving gel are shown in Table 22 Following electrophoresis the gels were

incubated for 16 hours at 28degC in substrate buffer containing (01 [wv]

glycogen 20 mM G-1-P made up in 100 mM sodium citrate [pH 65]) to test the

synthetic activity Phosphorylitic activity was tested by incubating the gel

containing (01 [wv] glycogen 20 mM Na2HPO4 made up in 100 mM sodium

citrate [pH 65]) and incubated under same conditions as used in synthetic

activity gels Gels were then rinsed briefly in sodium citrate (100 mM pH 65)

before covering the gel for up to 1 minute in Lugol solution (02 [vw] iodine

2 [vw] potassium iodide) Gels were subsequently rinsed in distilled water

and photographed immediately

60


containing 01 (wv) glycogen prepared as follows

Stock solution Resolving gel (10 mL) Stacking gel (5 mL) H2O 47 (mL) 355 (mL)

30Acrylamide 26 (mL) 084 (mL) 15M Tris pH 88 26 (mL) -

1M 5M Tris pH 68 - 064 (mL) 10 APS 01 0056 01 glycogen 10 (mg) -

TEMED 10 μL 4 μL

22264 Coomassie blue staining

Polyacrylamide gels were stained in Coomassie Blue stain (42 [vv]

methanol 18 [vv] acetic acid 01 [wv] Coomassie Brilliant Blue R 250) for

1hr and destained overnight in 42 [vv] methanol 18 [vv] acetic acid

Then the gel was washed in distilled water

22265 Silver staining

Following the electrophoresis the polyacrylamide gel was kept in 50 mL

fixing solution (50 Methanol [vv] 5 acetic acid [vv]) for 20min on a

shaker and washed the gel in washing buffer (50 Methanol [vv]) for 10min

and with distilled water at least for 1hr Then the gel was transferred to

sensitizing buffer (002 Na2S2O3 [wv]) for 1min and washed the gel twice in

distilled water for 2 min each time The gel was stained in ice-cold silver nitrate

buffer (01 AgNO3 [wv]) for 20 min and washed the gel in distilled water for 2

min each time Developed the gel in developing solution (2 Na2CO3 [wv]

004 formalin [vv]) for 5-7 min until the proteins bands were visualized

61

Staining was stopped by adding the stop solution (5 acetic acid [vv]) for 5

min and transferred to distilled water


(Phosphate affinity SDS-PAGE using Acrylamide-pendant Phos-TagTM

The Phos-Tag affinity ligand (10 μmolL Phos-tagTM AAL) was used to

detect phosphorylated proteins using the SDS-PAGE gels A dinuclear metal

complex (Mn2+) acts as a selective phosphate-binding tag molecule and the

Phos-Tag binds to the phosphate group of the phosphorylated protein and

retards the movement of the phospho protein in the SDS-PAGE gel The

phosphorylated and nonphosphorylated proteins were detected by immunoblot

analysis The composition of the gel prepared (see in Table 23) and the

experimental procedure is described as below The gel was run at 10 mA for 14

hours at room temperature

Solutions

1 Stock solution of 50 mmolL Phos-tagTM AAL Solution containing 3 (vv)

MeOH (Phos-tagTM AAL-107 10 mg was mixed with methanol 010 mL and

distilled water 32 mL) This oily product was stored in dark at 40C until use

2 10 mmolL MnCl2 Solution was prepared by dissolving 010 g MnCl2 (H2O)4

(FW 198) in 50 mL of distilled water

62

Table 23 The Gel preparations for Phos-TagTM analysis

Stock solution Resolving Gel (10 mL) Stacking Gel (10 mL)

10 (wv) acrylamide and (5 (wv) acrylamide) 50 μmolL Phos-tag TM AAL)

30 (wv) Acrylamide Solution 40 mL 150 mL

15 molL TrisHCl Solution pH 88 2 25 mL 250 mL (pH 68) 5 mmolL Phos-tag AAL Solution 01 mL - 10 mmolL MnCl2 Solution 01 mL -

10 (wv) SDS Solution 01 mL 010 mL 10 (wv) Diammonium Peroxydisulfate 01μL 010 μL

Distilled Water 31 mL 50 mL TEMED (tetramethylethylenediamine) 10 μL 80 μL

22267 Immunological techniques

222671 Preparation of peptides and antisera

Polyclonal antibodies were raised in rabbits against the synthetic peptides

derived from the sequence of maize SP (YSYDELMGSLEGNEGYGRADYFLV)

corresponding to residues 917ndash943 of the full length sequence (GenBank

accession no AAS33176) Synthetic polypeptides were raised to the polyclonal

rabbit antisera targeted to maize SSI SSIIa SBEI SBEIIa SBEIIb Iso-1 and

Iso-2 The specific peptide sequences used for the various antibodies were as

follows (Table 24)

63

Table 24 The synthetic peptides sequences derived from the primary amino acid sequences of starch biosynthetic enzyme isoforms of maize their location

in the full length sequence and the GenBank accession numbers

Enzyme Peptide Location GeveBank Accession Isoform Sequence in Full Length Number Sequence

SSI AEPTGEPASTPPPVPD 72-87 AAB99957 SSIIa GKDAPPERSGDAARLPRARRN 69-89 AAD13341

SSIV ANHRNRASIQRDRASASI 55-72 AAC197339 SBEI KGWKFARQPSDQDTK 809-823 AAC36471

SBEIIa FRGHLDYRYSEYKRLR 142-157 AAB67316 SBEIIb PRGPQRLPSGKFIPGN 641-656 AAC33764 Iso-1 FTKHNSSKTKHPGTYIAC-NH2 269-286 AAA91298

Iso-2 ARSYRYRFRTDDDGVV 37-52 NP001105666 GBSSI QDLSWKGPAKNWENV 442-456 ABW95928

222672 Antibody purification

The peptide affinity columns were used to purify the various crude

antisera The columns were prepared as follows To make a 1 mL column the

respective synthetic peptide (2 mg) was dissolved in 1 mL of TRIS-HCl pH 85

(50 mM TRIS-HCl 5 mM EDTA) 2 mL sulpholink resin slurry (Pierce) was

washed in 1 mL TRIS-HCl pH 85 for six times The dissolved peptide was added

to 1 mL washed resin in a falcon tube and incubated on a rotor for 15min in

room temperature and for additional 30 min without rotating and added to the

column and column was washed with 3 mL TRIS-HCl pH 85 and then blocked

with 1 mL of 50 mM cysteine in the same washing buffer 5 mL antisera

containing the polyclonal maize antibodies were applied to the column and mix

on a rotator for overnight at 4oC with 3 mL of PBS 001 Na azide [wv Then

64

the column was washed with 10 ml RIPA [50 mM TRIS-HCl pH 75 150 mM

NaCl 1 (wv) nonyl phenoxylpolyethoxyl ethanol (NP-40) 05 (wv) Na-

deoxycholate 01 (wv) sodium dodecyl sulphate (SDS)] The column was

further washed with 10 ml sarcosyl buffer [NETN (20 mM TRIS-HCl pH 80 1 M

NaCl 1 mM Na2-EDTA and 05 (wv) NP-40)] followed by washing again with

10 ml of 10 mM TRIS-HCl pH 78 The antibody bound to the column was eluted

with 05 mL of 100 mM glycine pH 25 to a tube contained 05 mL 1M TRIS-HCl

pH 78 and the protein contents were measures The column was neutralized by

adding 10 mL of 10 mM TRIS-HCl pH 78005 [wv] sodium azide

222673 Immunoblot analysis

After electrophoresis the proteins in polyacrylamide gels were

transferred to nitrocellulose membranes (Pall Life Sciences) using a Mini Trans-

Blotreg

Electrophoretic Transfer Cell (Bio-Rad) according to the manufacturerrsquos

instructions The transfer buffer contains 10 running buffer 20 methanol

and 70 water Then the membrane was blocked with 15 bovine serum

albumen (BSA) in 1XTBA buffer and incubated overnight in diluted antibodies

using the methods described by (Tetlow et al 2004) The anti-maize antisera

were used in immunoblot analyses were diluted in 15 BSA in 1XTBA buffer as

follows 11000 for SSI SSIIa SSIIb SBEI SBEIIb and 1500 for SP SSII and

SSIV The bound antibodies were detected with alkaline phosphatase-conjugated

anti-rabbit IgG using a 5-bromo-4-chloro-3-indolyl phosphatenitro blue

tetrazolium liquid substrate developing solution (BCIPNBT)

65

222674 Immunoprecipitation

Immunoprecipitation was performed with amyloplast lysates using

methods previously described by (Tetlow et al 2004) The SP SSIIa and SBEIIb

antibodies were added at 30 mgmL concentration and the SSIV antibodies at 60

mgmL to 10 mg of amyloplast lysates and incubated for 1hr on a rotator at

room temperature Proteins were immunoprecipitated by adding 40 μL of 50

(wv) Protein A-Sepharose slurry (60 μL of slurry for SSIV) The Protein A-

Sepharose slurry was made by adding the phosphate buffer saline (137 mM

NaCl 10 mM Na2HPO4 27 mM KCl 18 mM KH2PO4) to the Protein A-Sepharose

beads and incubated for 1hr at room temperature Protein A-Sepharoseprotein

complex was centrifuged at 100 g for 2 min at 40C in a refrigerated micro

centrifuge and the supernatant was collected and denatured with the sample

running buffer containing SDS to use as an indicator of the immunoprecipitation

efficiency The remaining pellet Protein A-Sepharoseprotein complex was

washed eight times each with 1 mL phosphate buffered saline (PBS) followed by

three similar washes with 10 mM HEPESKOH pH 75 buffer (at 100 g 1 min

centrifugation) The immunoprecipitation pellet was boiled in 2X SDS loading

buffer for 8 min Co-immunoprecipitation was tested by probing with specific

anti-peptide antibodies of major starch biosynthetic enzymes

66

23 Results

231 Subcellular localization of SP in maize endosperm

To determine the subcellular localization of SP the amyloplast lysates the

granule-bound proteins of the starch granules separated from amyloplast the

whole cell crude extracts of the endosperm and the amyloplast membrane

protein were extracted from 22 DAA wild-type maize plants Immunoblot

analysis using peptide specific anti-Pho1 antibodies showed that there is no SP

in granules and in amyloplast membranes (Fig 22) The SP is mainly found in

the amyloplast lysates The proportional existence of the SP in the interface of

the soluble fraction and the granule as the granule surface imbedded protein

was tested by collecting the extracts as the supernatants after repeatedly

washing the granules nine times with rupturing buffer Fig 23A showed the

protein profile of the extracts collected after each washing (silver stained SDS-

PAGE gel) The proteins which were separated on SDS gels were identified by

probing the immunoblots with anti-peptide specific antibodies of starch

biosynthetic proteins SP SSI SSIIa SSIII SSIV SBEI SBEIIa and SBEIIb (Fig

23B) Most of the SP was found in wash 1 and 2 and slightly in was 3 4 and 5

There was no band detectable from wash 6-9 and the protein profile of SP was

similar with SSI and SBEIIb (wash 1 to 3) and SBEI (wash 1 to 4) SSIII and

SSIV were found in only the first wash whereas SSII was found clearly from

wash 1 to 7 (Fig 23B) To determine the granule bound SP 005 mg (wet

weight) of starch was taken out after every centrifugation stage during granule

washing and it was boiled with 200 μL of 2XSDS Immunoblots were probed with

67

anti-SP and anti-SSIIa specific antibodies (Fig 24) SP was not found in the

granules as a granule-bound protein while SSIIa which was found in the granule

and could not be removed by the washing treatment (Fig 24)

Figure 22 Immunoblots showing the subcellular localization of plastidial SP in maize endosperm the amyloplast lysates contain soluble amyloplast proteins

the granule-bound proteins of the starch granules separated from amyloplast the soluble protein fraction and starch granule-bound proteins of whole cell crude extract of the endosperm and the soluble protein fraction of the

amyloplast membrane protein extracts (A) Leaf crude extracts were probed with anti-SP antibodies are shown in (B) All samples were extracted from 22

DAA wild-type maize plants The blots were developed in two different experiments and both were probed with pastidial peptide specific anti-SP antibodies after equal amounts (25 microg) of proteins were run on SDS-PAGE

Arrows indicate the location of SP

(A) (B)

68


surface Approximately 1 g of fresh weight of starch granules from the amyloplast was subjected to a series of washings with the 03 mL of 100mM

rupturing buffer for 9 times The supernatant was collected at each time and separated on the SDS gels and the proteins are visualized by silver staining (A) and identified by probing immunoblots with anti-peptide specific antibodies of

starch biosynthetic proteins as indicated (B) The numbers indicate the number of washings L=protein marker Target protein is indicated by the arrow in each

immunoblot

(A)

(B)

69

Figure 24 Analysis of the localization of proteins imbedded in the granule surface and loosely bound to the granules Approximately 1g of fresh weight of

starch from the amyloplast lysates was subjected to a series of washings with the 03 mL of 100mM rupturing buffer for 9 times The supernatant (soluble fraction) and 005g of the pellet was denatured in 2XSDS (200 μL) at each

washing (granule association) was collected at each time and separated on the SDS gels and the proteins are visualized by silver staining and identified by

probing the immunoblots with anti-SP and anti-SSIIa peptide specific antibodies of starch biosynthetic proteins as indicated in the blots The numbers indicate the number of washings L=protein marker

232 The synthetic activity of SP in developing maize endosperm

The synthetic activity of plastidial SP in developing maize endosperm was

determined by native affinity zymogram containing 01 glycogen in the gel

The amyloplast lysates were extracted from the endosperm at 12 15 19 22

days after anthesis (DAA) Activity bands were observed for all the

developmental stages tested (Fig 24A) The immunoblot was probed with

peptide specific anti-SP antibodies confirmed the activity bands are due to

plastidial SP (Fig 24B) The equal volumes of amyloplast lysates (30 μLwell)

70

were loaded on the gel The activities of SP shown on the gel did not vary over

the various developmental stages tested Synthetic activity of SP (22 DAA) was

slightly reduced when SSIIa was removed from amyloplast lysates but not the

SSIV (Appendix 01)

The SP in amyloplast lysates at 22 DAA age showed both synthetic and

phosphorolytic activities when both activities were tested in a glycogen affinity

native zymogram containing 01 glycogen in the gel (Fig 26) In synthetic

and degradative directions the gels were incubated at 1 2 5 10 and 20 mM

G-1-P and sodium phosphate dibasic (Na2HPO4) respectively When the activity

bands were visualized by Lugolrsquos solution the dark synthetic activity bands were

shown in all concentrations of G-1-P tested and the activity band was clear at all

concentrations of Na2HPO4 Both synthetic and degradative activities were

increased with increasing substrate concentrations (Fig 26)

Figure 25 The activity of Pho1 was observed in developing wild-type maize amyloplast lysates isolated 12-22 days after anthesis (DAA) using non-denaturing affinity native zymogram containing 01 glycogen in the gel (A)

Immunoblot of the zymogram gel was probed by peptide specific anti-SP antibodies to detect the SP protein in maize amyloplast (B) Pho1 is localized in

the amyloplast stroma and showed consistent activity in all the developmental stages of amyloplast measured

Days After Anthesis

12 15 19 22 12 15 19 22

A B

Days After AnthesisDays After Anthesis

12 15 19 22 12 15 19 22

A B

Days After Anthesis(A) (B)

71

Figure 26 The activity of SP in amyloplast lysates at 22 DAA age in the synthetic and phosphorolytic direction was tested on glycogen affinity native

zymogram contained 01 glycogen in the gel Following electrophoresis the gel was incubated overnight at 280C with the incubation buffer containing 01 glycogen and 1 2 5 10 and 20 mM glucose-1-phosphate (G-1-P) or sodium

phosphate (Na2HPO4) in synthetic and phosphorolytic directions respectively The activity bands were visualized by Lugolrsquos solution Arrows indicate the bands

corresponding plastidial SP

323 Investigating the regulation of SP by protein phosphorylation

The activities of the phosphorylated and dephosphorylated isoforms of SP

were analyzed on 01 glycogen affinity SP-native zymogram using amyloplast

lysates endosperm crude extracts and leaf crude extracts collected at 22 DAA

The soluble form of plastidial (Pho1) isoforms from maize endosperm

amyloplasts (Fig 27A1) both plastidial (Pho1) and cytosolic (Pho2) isoforms of

SP in the whole cell extract of endosperm (Fig 27B1) and the isoforms in

transient starch biosynthetic maize leaves (Fig 27C1) did not show any

detectable qualitative differences in the activities in both phosphorylated

20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4

SP Synthetic Activity SP Phosphorylitic Activity

20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4

SP Synthetic Activity SP Phosphorylitic Activity SP Synthetic Activity SP Phosphorolytic Activity

72

(treated with 1 mM ATP) and dephosphorylated (treated with 25 units of APase)

extracts when compared with the untreated controls (Fig 27) Immunoblot

analyses of the zymograms are respectively shown in A2 B2 and C2 which are

probed with peptide specific anti-Pho1

The mobility shift detection of proteins on phosphate affinity SDS-PAGE

using ligand bound Acrylamide-pendant Phos-TagTM showed no retardation in the

mobility of ATP-treated and untreated SP from amyloplast lysates (Fig 28)

73

Figure 27 Determination of the different activity levels of plastidial (Pho1) and cytosolic (Pho2) isoforms of SP following treatment with ATP and APase

The amyloplast lysates seed crude extract and leaf crude extracts collected at 22 DAA were treated with either 1mM ATP or with alkaline phosphatase (APase) (25unitml) and incubated for 1hr at room temperature The activity was

compared with the untreated controls on native affinity zymograms (01 glycogen) in the synthetic reaction The activities of amyloplast lysates soluble

protein fractions of kernel crude extract and leaf crude extract (90 μg of proteins were loaded in a well) on zymograms are shown in A1 B1 and C1 respectively with their respective immunoblots A2 B2 and C2 which are probed

with peptide specific anti-Pho1 antibodies APase was used as a negative control

Control ATP APaseAPase

only Control ATP APaseAPase

only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2



only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2

74

Figure 28 Mobility shift detection of phosphorylated proteins by Phosphate affinity SDS-PAGE using Phos-TagTM

Amyloplast lysates (22 DAA) treated with either 1 mM ATP APase (25unitml) or ATP+ PI (phosphatase inhibitor) 30 μg of proteins were loaded in each well

The gel was immunoblot following electrophoresis and probed with peptide-specific anti-SP antibodies and the mobility of the bands was compared with the untreated amyloplast lysates

234 Gel filtration chromatography (GPC) analysis of SP

Maize amyloplasts lysates (at 22 DAA) treated with ATP or APase (500

μgmL of proteins in each) were eluted through a Superdex 200 10300GL gel

permeation column to determine whether ATP or APase treatment influenced the

multimeric state of SP Fractions collected were run on the SDS-PAGE and the

elution pattern of the major starch biosynthetic enzymes were analyzed on the

immunoblots using peptide specific anti-SP SSI SSII SSIV SBEI and SBEIIB

antibodies (Fig 210BCDE) The elution patterns of SP at early (15 DAA) and

75

late developmental stages (35 DAA) in whole cell crude extracts of the maize

endosperm are shown in Fig 210A The gel permeation column was connected

to an AKTA Explorer FPLC was calibrated using commercial protein standards

from 137 kDa to 440 kDa and the calibration curve developed to estimate the

molecular weights of the proteins eluted by GPC is shown in Fig 29

Both in early and later stages of endosperm development SP eluted in

fractions (fraction 21-23) where the molecular weight corresponds to the

tetrameric form of SP (448 kDa) Dimeric forms were not visualized Amyloplast

lysates at 22 DAA the elution profile of SP was equal in untreated control

(fractions from 7-12) where as the ATP treated and APase treated fractions were

respectively from 8-13 and 6-12 (Fig 210B) The estimated molecular weights

of the eluted SP fractions showed the existence of monomeric (112 kDa)

dimeric (112 kDa X 2) and tetrameric forms (112 kDa X 4) of SP The elution

profile of SSI SSIV SBEI and SBEIIb were identical regardless of ATP or APase

treatments In contrast ATP-treated SSII eluted comparatively in low molecular

fractions (6-10) compared to APase treated fraction profile (fraction 4-8) (Fig

210C) Reprecentative graph of the elution from GPC is shown in Appendix 10

GPC-fractionated amyloplast lysates (22 DAA) were run on native affinity

zymograms The results indicated that ATP-treated SP eluted in fraction number

25-26 showed SP activity where as untreated or APase treated fractions

showed SP synthetic activity in fraction number 23-24 Approximate molecular

weights of these fractions were investigated as fraction 23-24 are tetrameric

and 25-26 fractions were dimeric forms of SP (Fig 211)

76


the proteins eluted by GPC Superdex 200 10300GL gel permeation column was calibrated using commercial protein standards from 137 kDa to 440 kDa The

graph shows the relationship between natural log values of the molecular weight of the commercial proteins versus fraction numbers

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number

Lo

gM

W

77

Figure 210A Gell filtration chromatography (GPC) analysis of maize whole cell

crude extracts at 15 DAA and 35 DAA 045 mg of proteins were separated by GPC through a Superdex 200 10300GL gel permeation column Elution of SP was detected by immunoblot are shown The size of the proteins in each fraction

was determined by calibrating the column using commercial protein standards from 137 kDa to 669 kDa and the sizes of the standards are also indicated L=

protein marker AP=amyloplast lysates C=crude extracts before loading onto the column

(A)

78

Figure 210B Gel filtration chromatography (GPC) analysis of amyloplast

lysates Maize amyloplast lysates at 22 DAA were treated with 1mM ATP or alkaline phosphatase (APase) (25unitmL) to the extracts and incubated for 1hr in room temperature 049 mg of protein was separated through a Superdex 200

10300GL gel permeation column In total 45 (500μL each) fractions were collected from each running for the analysis in total only the fractions where

the protein was detected by immunoblot analysis are shown The SP bands were shown by the arrows The size of the proteins in each fraction was determined by calibrating the column using commercial protein standards from 137 kDa to

669 kDa and the sizes of the standards are also indicated L= protein marker AP=amyloplast lysates C=crude extracts before loading onto the column

Arrows indicate the locations of the corresponding proteins

(B)

79

Figure 210C Gel filtration chromatography (GPC) separation of amyloplast stromal proteins Immunoblots probed with anti-SSI (74 kDa) and anti-SSIIa (85 kDa) antibodies of untreated ATP- or APase-treated maize amyloplast

lysates separated by GPC through a Superdex 200 10300GL gel permeation column The protein bands were shown by the arrows The size of the proteins in

each fraction was determined by calibrating the column by commercial protein standards from 137 kDa to 669 kDa and the sizes of the standards are also indicated L= protein marker AP=amyloplast lysates before loading onto the

column

(C)

80

Figure 210D Gel filtration chromatography (GPC) separation of amyloplast

stromal proteins Immunoblots probed with anti-SSIV (104 kDa) and anti-SBEI (80 kDa) antibodies of untreated ATP- or APase-treated maize amyloplast

lysates separated by GPC through a Superdex 200 10300GL gel permeation column The protein bands were shown by the arrows The size of the proteins in each fraction was determined by calibrating the column by commercial protein

standards from 137 kDa to 669 kDa and the sizes of the standards are also indicated L= protein marker AP=amyloplast lysates before loading onto the

column Arrows indicate the location of the corresponding proteins

(D)

81

Figure 210E Gel filtration chromatography (GPC) analysis of amyloplast lysates Immunoblots probed with anti-SBEIIb (85 kDa) antibodies of untreated

ATP- or APase-treated maize amyloplast lysates separated by GPC through a Superdex 200 10300GL gel permeation column The protein bands were shown by the arrows The size of the proteins in each fraction was determined by

calibrating the column by commercial protein standards from 137 kDa to 440 kDa and the sizes of the standards are also indicated L= protein marker

AP=amyloplast lysates before loading onto the column Arrows indicate the locations of the protein

(E)

82

Figure 211 Native affinity synthetic activity SP zymogram of the amyloplast lysates separated by GPC Untreated ATP- or APase-treated GPC fractions (10

μg of proteins) were run on native gels containing 01 glycogen Arrows indicate the synthetic activity bands

235 The synthetic and phosphorolytic activities of SP with different

glucan substrates

The glucan synthetic activity of ATP- or APase-treated SP was

quantitatively measured using maltoheptaose glycogen and amylopectin as

glucan primers at 25 mgmL concentration Amyloplast lysates were used as the

SP source and [U14_C]-G-1-P as the glucan donor (Table 25) The means of

enzyme activities were statistically compared using the One-Way ANOVA (at

Plt005 level F=8274 P=000028) (See Appendix 08 for the statistical

analysis of ANOVA by Statistix 9 statistical analysis program) The results

indicated that synthetic activities of SP were not significantly different in three

different glucans in SP present in untreated amyloplast lysates at 25 mgmL of

substrate concentration The synthetic activity was significantly higher with

amylopectin (1433152 nmolmgmin) compared to maltoheptaose

20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29

Untreated Control ATP Treated APase Treated

20 21 22 23 24 25 26 27 28 29

Fraction NumbersFraction NumbersFraction Numbers20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29


20 21 22 23 24 25 26 27 28 29

Fraction NumbersFraction NumbersFraction Numbers

83

(6000456 nmolmgmin) when amyloplast lysates were treated with ATP

There was no significant difference in the synthetic activity between

maltoheptaose and glycogen within untreated or ATP-treated amyloplasts SP

activities were decreased in all substrates in APase-treated amyloplast lysates

compared to both untreated and ATP-treated samples In addition synthetic

activity was significantly decreased when treated with APase with amylopectin

and glycogen compared to ATP-treated SP The synthetic activity was not

significantly decreased in ATP or APase-treated SP when maltoheptaose was

used as the glucan primer (Table 25)

Phosphatase inhibitor cocktail (PI) was added to inhibit the activity of

endogenous phosphatase present in the amyloplast (see Appendix 09 for the

details about PI) However ATP+PI treated SP recorded lower activity compared

to ATP treated SP in all three substrates The APase used was bound to agarose

beads (insoluble APase) and it was removed after amyloplast lysates were

treated for 1 hour to prevent under estimation of the enzyme activity due to

continuous dephosphorylation of substrates in the assays In general plastidial

SP had greater activities in synthetic direction over phosphorolytic direction

despite ATP or APase treatments or in high or low molecular glucan polymers at

25 mgmL concentration SP phosphorolytic activity was not significantly altered

within untreated ATP-treated or APase-treated samples when maltoheptaose

was used as the glucan primer (Table 25) Phosphorolytic activities of untreated

and ATP-treated SP were significantly different from APase-treated SP with

amylopectin (Table 25) (see Appendix 08 for the statistical analysis on One-way

ANOVA F= 3557 P= 00004)

84

The enzyme followed typical saturation kinetics toward amylopectin and

maltoheptaose when activity was measured in the phosphorolytic direction The

kinetic data were analyzed using the MichaelisndashMenten equation The Km and

Vmax values of SP in the phosphorolytic direction were analyzed at a range of

(5-25 mgmL) maltoheptaose and amylopectin concentrations using

LineweaverndashBurk plots (Table 26) SP had a higher Km value with

maltoheptaose compared to amylopectin in untreated or ATP-treated or APase

treated samples Km values increased with both maltoheptaose and amylopectin

with ATP treatment and decreased with APase treatment compared with the

untreated sample values The Vmax was increased in both substrates following

ATP treatment compared to the untreated samples by 13 and 12 times in

amylopectin and maltoheptaose respectively (Table 26)

Table 25 Synthetic and phosphorolytic activities of SP in different glucan substrates Both activities were calculated as nmolmgmin Significantly different means (at Plt005) from the One-way ANOVA followed by LSD are

indicated by identical symbols for synthetic and phosphorolytic activities

Substrate

Pre-treatment

Untreated ATP ATP+PI PI APase

Synthetic

Activity

Maltoheptaose

Amylopectin

Glycogen

69060

99453

100526

60056

143352Dagger

114410dagger

60250

90450

73312

59156

75874

74208

27238

32845Dagger

37420dagger

Phosphorolytic

Activity

Maltoheptaose

Amylopectin

24615

46913

27014

58840

- -

- -

16634

33418

85

Table 26 Km and Vmax values of SP in amyloplast lysates in the phosphorolytic direction The phosphorolytic activity of SP was assayed by spectrophotometry

and amount of NADH released was analyzed at 340nm as the amount of G-1-P produced in the reaction Amylopectin and maltoheptaose concentrations at 5-

25 mgmL were considered in the calculations Km and Vmax values were calculated using LineweaverndashBurk plots

Glucan Substrate

Treatment

Km (mgmL)

Vmax

(nmolmgmin)

Amylopectin

Untreated

ATP

APase

18plusmn002

31plusmn001

13plusmn005

483plusmn02

654plusmn006

337plusmn02

Maltoheptaose

Untreated

ATP

APase

33plusmn002

67plusmn0001

23plusmn0001

279plusmn001

339plusmn0003

176plusmn002

236 Immunoprecipitation of SP

The immunoprecipitation of SP from the amyloplast lysates was

attempted using peptide specific anti-SP antibodies Native SP protein was not

immunoprecipitated by protein A-Sepharose beads (Fig 212) therefore co-

immunoprecipitation was not possible SP was not immunoprecipitated by anti-

SP antibodies bound to Protein A-sepharose beads after removing the SSIIa

present in amyloplast lysates indicated that SSIIa is not cover up antibodies

binding epitopes of SP (Appendix 02) Recombinant maize SP with a S-tag was

produced by over expressing the protein in Ecoli The biochemical and

proteomic characterization and protein-protein interaction studies using the

recombinant SP is discussed in Chapter 3

86

Figure 212 Immunoprecipitation of SP by peptide specific anti-SP antibodies

(30 mgmL) with 1 mL amyloplast lysates 40 μL of 50 (wv) Protein A-Sepharose beads slurry made in phosphate buffer saline (PBS) was used to pull down the Protein A-Sepharose-antibody-proteins complex The

immunoprecipitated pellet was boiled in 2X SDS loading buffer and separated on SDS-PAGE followed by immunoblot analysis Pre-immune serum was used as a

control to show the specificity of the purified antibodies Figure illustrates the immunoblot probed with SP-specific antibodies The arrows denote the SP band L= protein marker

87

24 Discussion


The overall objective of the study was to elucidate the role and regulation

of plastidial starch phosphorylase (SP) and to investigate the possible post

translational modifications of SP in wild-type maize endosperm The subcellular

localization of SP was tested at 22 DAA which corresponds with the maximal

period of starch synthesis in maize endosperm (9-24 DAA) (Yu et al 2001 Tsai

and Nelson 1968) and the time when all the major starch biosynthetic enzymes

are expressed and active in amyloplasts (Liu et al 2009 Hennen-Bierwagen et

al 2008) The peptide specific anti-SP antibodies recognized the plastidial SP

only in the storage starch synthesizing amyloplasts and not in the transient

starch synthesizing chloroplasts (Fig 22B) The plastidial form of SP in

chloroplasts may be structurally different from the SP in the amyloplasts within

the same species Degradation of the proteins in crude extracts may be a

possible reason for SP not being detected effectively by antibodies Mutant

analysis suggested that the plastidial SP present in Arabidopsis thaliana leaves is

not involved in transient starch biosynthesis or degradation (Zeeman et al

2004) The SP mutants of Arabidopsis showed no change in the activity of other

enzymes of starch metabolism or show any significant change in the total

accumulation of starch or the starch structure during the day or its

remobilization at night (Zeeman et al 2004) Also leaves contain the cytosolic

form of SP (Pho2) abundantly compared to the plastidial SP (Satoh et al 2008)

which was not detected in leaf crude extracts with the antibodies

88

The results presented here confirmed the previous findings that the Pho1

is exclusively found in the amyloplast stroma (Satoh et al 2008 Grimaud et al

2008 Yu et al 2001) in the maize amyloplast (Fig 22) The existence of the SP

and other SSs and SBE in the interface of the soluble fraction and the granule

as the granule surface imbedded protein suggests their involvement in granule

synthesis The soluble protein fractions collected after the repeated washings of

the granules with the amyloplast rupturing buffer and tested on immunoblots

indicated that some of the major starch biosynthetic enzymes are present at the

granule surface (Fig 23) SP was present up to the fifth wash indicating the

tight association with the surface of the starch granule Similarly SSI and

SBEIIb (wash 1 to 3) and SBEI (wash 1 to 4) were also associated with the

granule periphery In contrast SSII was found clearly from wash 1 to 7 (Fig

22B) which is comparatively abundant in the granule surface By contrast SSIV

and the SSIII were found only in the first extract of the amyloplast and may be

regulated as purely soluble SSIV and SSIII are either not present in the granule

surface or present at the extremely low levels in the granule surface In the

wild-type maize amyloplast stroma it has been demonstrated that the protein

present in the assembly of heteromeric protein complexes (SSI SSII and

SBEIIb) are also entrapped in the starch granule (Liu et al 2009) However the

SP is regulated by protein complex formation with SBEI and SBEIIb in wheat

amyloplasts (Tetlow et al 2004) but the components of this protein complex do

not appear to become entrapped in the starch granule SP was only found as a

granule-associated protein in the ae- background when it was found to be

associated with SSI and SSIIa (Liu et al 2009 Grimaud et al 2008)

89

We were unable to conduct standard immuno-precipitation experiments

using the anti-maize SP antibodies since they did not appear to recognize the

native protein and could only detect the protein after SDS-PAGE (Fig 212) The

reason for this is unclear but the epitope (SVASDRDVQGPVS located at 73-85

amino acids in N-terminal) present on the SP monomer may well be hidden

when the native SP adopts is natural multimeric (dimeric and tetrameric)

configuration

As Tickle et al 2009 proposed SP may contribute to starch synthesis by

operating in two ways in the cereal endosperm First it has been suggested that

SP may degrade soluble malto-oligosaccharides in the stroma produced via the

action of DBE to G-1-P and then to ADP-glucose by ADP-glucose

pyrophosphorylase to produce starch Second SP may directly act at the surface

of the starch granule where it functions to phosphorolytically modify the

structure of starch to provide suitable substrates for other starch biosynthetic

enzymes ultimately producing G-1-P which can be recycled back to produce

starch Both of the models suggested the effect of SP on starch synthesis by

providing G-1-P via the degradative process to produce ADP-glucose by AGPase

enzyme Data in this thesis support a role of SP operating at the granule surface

as SP localization experiments clearly show SP associated with starch granule

(Fig 23 24) Analyses of metabolites in the amyloplast also indicate high PiG-

1-P levels which could suggest that the phosphorolytic SP reaction is favored in

vitro (Fettke et al 2010 Schupp and Ziegler 2004) In contrast previous

studies suggested that SP exists in the storage starch biosynthetic tissues and

operates in the synthetic reaction in monocots where it is available throughout

90

the endosperm development (Schupp and Ziegler 2004 Satoh et al 2008 Yu

et al 2001) Recently Hwang et al (2010) showed that the SP reaction in rice

endosperm amyloplasts is predominantly synthetic even in the presence of high

Pi levels

242 The synthetic activity of plastidial SP in developing maize

endosperm

The synthetic activity of SP in the amyloplast lysates was tested by SP-

native zymogram analysis using glycogen as a substrate SP was active

throughout the endosperm development at stages measured (12 15 19 and 22

DAA) (Fig 25) The activity of SP is being found as early as 9 DAA in the maize

endosperm and remains active throughout the endosperm development (Yu et

al 2001) In the 22 DAA amyloplast lysates SP showed activity in both

synthetic and degradative directions when tested on native affinity zymograms

which were respectively incubated with G-1-P and Pi (Fig 26) Accumulation of

Pho1 was detected throughout the endosperm development in maize was

similarly observed in wheat endosperm during 8-31 DAA and Pho1 was

undetectable until 8 DAA and reached to the maximum level at 18 DAA and

remained constant (Tickle et al 2009) The presence of Pho1 in cereal

endosperm correlates with the presence of other starch biosynthetic enzymes

SBEI SBEII AGPase and SSs (Liu et al 2009 Tetlow et al 2003 Morell et al

1997 Ainsworth et al 1995) suggesting that Pho1 may be involved in starch

biosynthesis or be involved in functional interaction with other starch

biosynthetic enzymes

91


SP in cereal endosperms has been found to be regulated by protein

phosphorylation (Liu et al 2009 Pollack 2009 Tetlow et al 2004) However

the activities of the ATP-treated and APase-treated isoforms of SP on 01

glycogen zymograms showed no detectable differences in the activities between

treatments (Fig 27) The glycogen affinity SP-native zymogram may not be

sensitive enough to detect subtle alterations in catalytic activity associated with

phosphorylation


using Acrylamide-pendant Phos-TagTM (10 μΜmL) (Fig 28) showed no

difference in mobility in ATP-treated or untreated SP suggesting SP is not

phosphorylated However the Pi present in the amyloplast lysates may have

affected the activity of SP

244 Gel filtration chromatography analysis of SP

Phosphorylases exist as homodimers or homotetramers and have similar

kinetic and structural properties but their regulatory mechanisms may vary

depending on the source of the enzyme in higher plants (Brisson et al 1989) in

bacterial forms (Dauvilleacutee et al 2006) and yeast (Tanabe et al 1987) Gel

filtration chromatography studies revealed that the native enzyme consisted of

two identical subunits in maize (Mu et al 2001) In the present study the SP

was found in multimeric tetrameric and dimeric forms in both early (15 DAA)

and late (35 DAA) developmental stages (Fig 210 A) in endosperm crude

extracts and at 22 DAA in maize wild-type amyloplast lysates (Fig 210B)

92

which has been observed previously (Liu et al 2009) Seed crude extracts of 15

DAA and 35 DAA had showed similar elution profiles for SP from 21-23 fractions

and amyloplast lysates at 22 DAA had wider elution profiles (from fraction 21 to

26) that may be due to less dimeric form of SP in crude extracts In addition

monomeric dimeric and tetrameric forms of SP separated by GPC were tested

for the synthetic activity by native SP zymogram (Fig 211) Synthetic activity

of ATP-treated SP showed an apparent molecular weight approximately similar

to the dimeric form (fraction 25-26) untreated and APase-treated SP showed

activity in (fraction 22-23) the fractions corresponding to a molecular weight

equal to a tetrameric form suggested that the dimeric forms were more active

compared to the tetrameric forms when the amyloplast lysates were treated

with ATP (Fig 211)

Phosphorylation may effect the multimeric status of SP However no

detectable difference in the elution profiles of phosphorylated and

dephosphorylated amyloplast lysates was observed (Fig 210B) The SP involved

in heteromeric protein complex formation with SSI and SSIIa recorded in the

ae1 mutant showed the same elution profile as in wild-type (Liu et al 2009)

suggesting that the observed elution profile of SP may be made up of a variety

of different SP-containing protein complexes as well as SP monomers

Immunoblot analysis of the GPC fractions illustrated that SSIV (104 kDa)

and SBEI (80 kDa) SBEIIb (85 kDa) showed no difference in their elution

profiles following ATP or APase treatment However SBEI and ATP-treated

SBEIIb eluted in two different molecular groups high apparent mass (greater

than the expected size of monomer) low apparent mass consistent with the

93

expected monomeric mass The results of SBEIIb elution can be explained by

the phosphorylation dependent SBEIIb complex formation previously observed

in maize and wheat amyloplasts (Liu et al 2009 Tetlow et al 2008) In

contrast there is no evidence for the existence of homo dimeric or multimeric

forms of SBEI in wild-type maize amyloplasts The higher molecular mass

fractions of SBEI are therefore probably due to the formation of heteromeric

protein complexes containing SBEI We observed no alteration of SSIV elution

following ATP or APase treatment (Fig210D) In ATP treated lysates SSI eluted

comparatively higher apparent molecular mass fractions (6-13) than in the

untreated and dephosphorylated treatments (Fig 210B) as previously observed

in Liu et al (2009) In wild-type maize amyloplast stroma SSI SSIIa and

SBEIIb form a phosphorylation-dependent heteromeric protein complex (Liu et

al 2009) By contrast SSIIa eluted in higher molecular fractions when the

enzyme was dephosphorylated (Fig 28B Table 21) suggesting that the

dephosphorylated SSIIa may form proteinndashprotein interactions or complex

formation in wild-type maize amyloplasts This suggestion is further supported

by Liu et al (2009) that the [γ-32P]ATP treated ae1 mutant and wild-type

amyloplast lysates immunoprecipitated with anti-maize SSIIa antibodies showed

that SBEIIb in wild-type and SBEI and SP in ae1 mutant were phosphorylated

but no evidence for phosphorylation of SSII in the complex

The effect of phosphorylation on the monomeric dimeric and tetrameric

forms of SP and their involvement of protein-protein interactions are discussed

in Chapter 3 using a catalytically active recombinant maize SP containing an S-

protein affinity tag

94

245 The synthetic and phosphorylitic activity of SP in different glucan

substrates

Glucan synthetic activity was significantly less with maltoheptaose

cpmpared with amylopectin and glycogen in untreated ATP or APase-treated SP

(Table 23) and the synthetic activity was significantly higher following ATP

treatment with amylopectin and glycogen compared to maltoheptaose indicating

that the activity of plastidial SP was greater with high molecular mass branched

glucans This was similarly observed in recombinant plastidial SP in rice the

ratio between the activities of synthetic and dedradative reaction rate

(equilibrium constant) was higher in amylopectin (45) compared to

maltopentaose (G5) maltohexaose (G6) maltoheptaose (G7) and amylose

respectively as 22 19 15 and 17 (Hwang et al 2010) Synthetic activity of SP

was inhibited by Pi produced in the reaction [inhibition constant (Ki) = 069 mM]

when amylopectin was used as the primer substrate but this inhibition is less

(Ki = 142 mM) when short α-glucan chains are used as primers and also

extends them to synthesize longer MOSs (DP= 4ndash19) (Hwang et al 2010) This

observation suggested that under physiological conditions of high PiG-1-P Pho1

extends the chain length of short MOSs which can then be used as subsequent

primer by starch synthase activities (Hwang et al 2010)


endogenous protein phosphatases But its addition did not increase the synthetic

activity compared with ATP-treated samples with glycogen and amylopectin

suggesting that some compound in PI cocktail mixture may have inhibited the

activity of SP

95

The activity of SP in ATP and APase-treated amyloplast lysates in

phosphorolytic direction was greater in amylopectin in untreated ATP or APase-

treated SP compared to maltoheptaose (Table 25) In contrast Km was greater

in maltoheptaose over amylopectin in ATP-treated SP (Table 26) Similarly the

kinetics analysis of purified SP from maize endosperm recorded that the

phosphorylitic reaction was favored over the synthetic reaction when malto-

oligosaccharides were used as the substrate (Mu et al 2001) The Vmax and Km

values recorded in this study were approximately 7 and 58 times lower than

with purified SP respectively (Mu et al 2001) Unlike in the purified form of SP

the activity of SP present in amyloplast lysates may be regulated by other

enzymes [eg SBEs (Nakamura et al 2012)] and other metabolites For

instance G-1-P and Pi present in amyloplast lysates and high PiG-1-P ratios are

considered as the controlling mechanism of SP activity (Tiessen et al 2011 Mu

et al 2001 Fettke et al 2009 Schupp and Ziegler 2004) However according

to the findings of Hwang et al (2010) incorporation of [U14_C]-G-1-P into starch

was only partially affected by the concentration of Pi in rice Even under

physiological G-1-P substrate levels (02 mM) and 50-fold excess of Pi in vitro

than the normal physiological level the Pho1 from of rice was able to carry out

the biosynthetic reaction (Hwang et al 2010) ADP-glucose the major precursor

for starch biosynthesis inhibits the activity of SP in the synthetic direction

(Dauvilleacutee et al 2006 Matheson and Richardson 1978) and may reduce the

activity of plastidial SP in amyloplast lysates The effect of ADP-glucose on

plastidial SP in maize was not tested in this study

96

The preference of SP for different α-glucans has been studied in many

plant species (Young et al 2006 Dauvilleacutee et al 2006 Yu et al 2001 Mori et

al 1993 Shimomura et al 1982 Liddle et al 1961) In contrast to maize SP in

sweet potato tubers plastidial SP showed a low binding affinity toward starch

and a high affinity toward low molecular weight linear malto-oligosaccharides

(MOS) (Young et al 2006) In contrast the cytosolic isoform has a high affinity

towards highly branched polyglucan amylopectin (Young et al 2006) The

synthetic activity of SP with amylopectin (Km =013 mgmL) is higher when

compared to the highly branched glycogen (Km=045 mgmL) in maize (Yu et

al 2001) in potato tubers (Liddle et al 1961) and in spinach leaves

(Shimomura et al 1982) In sweet potato tubers the L-78 amino acid peptide

insertion located in the middle of the plastidial form of SP appears to block the

binding site of SP to high molecular weight α-glucans (Young et al 2006) We

found no evidence for the proteolytic cleavage of the L-78 peptide in maize

endosperm amyloplasts

In this chapter experiments were carried out to investigate the regulatory

properties of SP in maize amyloplasts Plastidial SP is present only in the

amyloplast stroma and is not found as a granule associated protein which is in

agreement with previous studies (Grimaud et al 2008) SP remains active

throughout the endosperm development and it is present in homodimeric or

tetrameric configurations throughout the developmental stages analyzed in this

study This study suggested that the tetrameric and dimeric forms have different

catalytic activities and may be involved in starch biosynthesis by differential

regulation The SP elution profile by GPC was not altered by ATP or APase

97

treatments suggesting phosphorylation may not alter the multimeric status of

SP The synthetic and phosphorylitic activity assays showed that SP was active

in both directions However SP showed greater activities with amylopectin

compared to glycogen and maltoheptaose in both synthetic and phosphorylitic

directions ATP treated SP showed higher activities in both directions with

amylopectin indicating that ATP may be involved in regulating SP by

phosphorylation Protein-protein interactions with the plastidial enzyme could

not be detected by co-immunoprecipitation since the native SP was unable to

be immunoprecipitated by Protein-A sepharose beads The development of a S-

tagged recombinant SP was used in future experiments to analyze protein-

protein interactions involving SP these experiments are described in Chapter 3

98

CHAPTER 3

99

Using recombinant plastidial SP to understand the regulation of starch

biosynthesis

31 Introduction

Glucan-phosphorylases are widely distributed enzymes in bacteria plant

and animal tissues (Dauvilleacutee et al 2006 Weinhaumlusel et al 1996 Newgard et

al 1989 Tanabe et al 1987 Preiss 1984) SP catalyzes both synthesis and

degradation of the α-glucan polymers The structure and the function of these

enzymes are best understood for glycogen phosphorylases the SP counterpart

of animals and bacteria (Dauvilleacutee et al 2006 Weinhaumlusel et al 1996 Newgard

et al 1989) Glycogen phosphorylase (GP) plays an important role by initiating

the degradation of glycogen in glycogen metabolism (Dauvilleacutee et al 2006

Roach 2002 Fischer et al 1971) Predominantly the physiological function of

SP was considered phosphorolytic rather than to synthesize glucan polymers is

based on the observations in glycogen phosphorylase in animal system and that

SP has a low affinity for G-1-P (Schupp and Ziegler 2004) Preiss and Sivak

1996)

SP has been shown to be regulated by protein phosphorylation in plants

(Pollack 2009 Grimaud et al 2008 Tetlow et al 2004) GP in animal systems

is found to be coordinated with the activity of glycogen synthase GP is required

to be phosphorylated in order to activate the glycogen synthases (Carabaza et

al 1992 Johnson 1992 Madsen 1991) to regulate glycogen synthesis and

breakdown Structural changes of GP occur at the interface of the subunits as a

result of conformational transition at the amino terminus by protein

100

phosphorylation residues surrounding the phosphorylation site (serine-14) that

participate in intrasubunit interactions in the dephopsphorylated state are

observed to adapt alternate side-chain conformations in the phosphorylated

state enabaling them to form intersubunit interactions to form homodimeric

structure of GP (Sprang et al 1988)

SP present in storage starch synthesizing tissues in plants is suggested to

be involved in starch synthesis since SP is active throughout endosperm

development in cereals (Tickle et al 2009 Satoh et al 2008 Schupp and

Ziegler 2004 Mu et al 2001) Also the shrunken 4 mutants which lack SP

activity in maize endosperm resulted in reduced starch contents (Tsai and

Nelson 1969) and SP mutants in rice produced shrunken endosperm

phenotypes with low starch contents (Satoh et al 2008) Further SP does not

appear to influence the starch degradation in Arabidopsis thaliana (Zeeman et

al 2004) suggesting SP plays a more dominant role in the storage starch

biosynthesis In addition the SP-L isoform (plastidial form of SP which has lower

affinity towards the high molecular starch) in potato tubers and the chimeric

form of SP-L enzyme which was developed by replacing the 18 residue

sequence of the SP-L isoform including a part of 78-residue insertion were over

expressed in Ecoli and the affinities of purified forms of recombinant proteins

were compared by Mori et al (1993) The purified chimeric phosphorylase was

five times less active in synthetic direction than the parental type SP-L isoform

However the affinity of the chimeric phosphorylase for glycogen (Km= 238

mgmL) and amylopectin (Km=53 mgmL) was much higher than that of the

type SP-L isoform (Km=10400 Km=82 mgmL mgmL respectively in glycogen

101

and amylopectin) and only slightly lower than that of the cytosolic SP-H the

high affinity isoform These results provide evidence for the role of the unique

78-residue insertion present in plant plastidial SP sequences which lowers the

affinity of the enzyme for large branched substrates (Mori et al 1993)

A possible function of SP in starch biosynthesis is that SP acts on malto

oligosaccharide (MOS) which are liberated by the activity of debranching

enzymes (DBE) to produce linear maltodextrin (MD) of a length sufficient for a

subsequent branching reaction by starch branching enzymes (SBE) (Nakamura

et al 2012 Ball and Morell 2003) In addition functional interactions between

SP and SBE isoforms were observed in rice endosperm strongly suggesting that

SP and SBE have mutual capacities for chain elongation and chain branching

(Nakamura et al 2012) Purified SP from rice endosperm synthesized glucans

from G-1-P in the presence of SBE without any exogenous glucan primer and

glucan production was higher with SBEI compared to SBEIIa or SBEIIb

(Nakamura et al 2012) Physical interaction between SP SBEI and SBEIIb was

also recorded in wheat amyloplasts and this protein complex was assembled in a

phosphorylation dependent manner (Tetlow et al 2004) Based on the

observations in ss4 and ss4sp mutants in Arabidopsis leaves which produce

reduced numbers of starch granules with increased granule surface (Roland et

al 2008 Planchot et al 2008) it has been suggested that SP may be involved

in granule initiation in starch biosynthesis process via functional or physical

interactions between SP and SSIV (Roland et al 2008 Planchot et al 2008)

Investigating possible interactions of SP with other starch biosynthetic enzyme

102

isoforms is therefore important to elucidate the role and regulation of SP in

storage starch biosynthesis in maize amyloplasts

All phosphorylases exist as dimers or tetramers of identical subunits

(Dauvilleacutee et al 2006 Mu et al 2001 Brisson et al 1989 Sprang et al 1988

Tanabe et al 1987) In Chlamydomonas reinhardtii identical subunits of dimeric

form have similar kinetic and structural properties but their regulatory

mechanisms may vary (Dauvilleacutee et al 2006) In maize amyloplasts SP is

present as dimeric and tetrameric assembles (Mu et al 2001 Liu et al 2009)

However catalytic and regulatory mechanisms of these individual configurations

are not well characterized in higher plants

Previous work showed that available SP antibodies are not capable of

effectively immunoprecipitating native SP in protein-protein interaction

experiments We therefore decided to provide a recombinant maize SP for such

studies In this chapter we discuss the production of catalytically active S-

tagged SP recombinant proteins from wild-type maize endosperm and the

biochemical characterization of the recombinant SP and the investigations of the

possible interactions of SP with other starch biosynthetic enzymes GPC analysis

showed that the S-tagged recombinant SP is present in tetrameric and dimeric

forms which were also observed in the amyloplast lysates and these fractions

were found as valuable tools in understanding their diverse regulatory

mechanisms The synthetic and degradative activities of these different

recombinant SP configurations in different glucan polymers and their regulation

by protein-protein interactions are discussed

103


321 RNA extraction from maize endosperm and synthesis of cDNA

The RNA was extracted from maize endosperm at 22 DAA by using the QIAGEN

RNeasy Plant Mini Kit (Catalog No 74104) Approximately 100 mg of the frozen

maize endosperm was used in a sample First strand cDNA was synthesized from

RNA by using Fermentas RevertAidTM H Minus Strand cDNA Synthesis kit

(Catalog No K1631) following manufacturerrsquos recommendations with some

modifications The mixture of 5 μL RNA (100 μgmL) 1 μL Oligo DT primer (05

μg μL) 6 μL RNase free H2O was mixed and incubated at 700C for 5 min and

chilled on ice Then 4 μL 5X reaction buffer 1 μL RiboLock ribonuclease

inhibitor 2 μL 10 mM dNTPs were added to the mix and incubated 370C for 5

min 1 μL RevertAidTM H Minus M-MuLV-RT reverse transcriptase was added and

incubated further at 420C for 1hr After stopping the reaction by heating at 700C

for 10 min the complementary RNA was removed by RNase H (05 Μl 29 μL

reaction) and further incubated 370C for 20 min The cDNA was stored in -200C

322 Quantification of nucleic acid

The amount of RNA and DNA were measured in a NanoDrop 2000 (Thermo

Fisher Scientific) spectrophotometer at the wavelength of 260 nm the optical

density (OD) of 1 corresponds to a concentration of 50 μgmL for double-

stranded DNA and 38 μgmL for the RNA

104

323 Agarose gel electrophoresis

Agarose gel electrophoresis was carried out using standard procedures

commonly use Agarose was added to TAE buffer (004M TRIS-acetate 1 mM

EDTA pH 80) to make the final concentration of 08-1 (wv) and heated in a

microwave until completely dissolved The resulting solution was allowed to cool

for approximately 5 minutes before the addition of ethidium bromide to a final

concentration of 02 μgmL

and pouring into an appropriately sized horizontal

electrophoresis unit Upon setting the gel was overlaid with TAE buffer Samples

were subsequently mixed with 016 volumes loading buffer (30 glycerol [vv]

025 bromophenol blue [wv]) and loaded onto the gel Electrophoresis was

carried out at 80V for 1-15 hours Nucleic acids immobilized in agarose gels

were visualized on a gel documentation system

324 Designing oligo-nucleotide primers and RT-PCR

The complete mRNA sequence (3053 bp) of plastidial maize SP (GenBank

EU8576402) was taken from the National Center for Biotechnology Information

data base (NCBI) The transit peptide (TP) sequence was detected as 70 amino

acids by using ChloroP 11 sequence analytical server after analyzing the correct

protein frame in the GeneRunner program The coding sequence including a part

of TP sequence was isolated using the forward primer (SP-F1) 5rsquo

GCGGAGGTGGGGTTCTCCT 3rsquo and the reverse primer (SP-R1) 5rsquo

GCGAAAGAACCTGATATCCAC 3rsquo The PCR product was purified from the agarose

gel by using QIAquick Gel Extraction Kit (QIAGEN Cat No 28704) 50-100

ngmL-1

was used as the template in next PCR to obtain the complete mRNA

105

sequence of the plastidial SP The next PCR primers were specifically designed

for the CloneEZreg PCR Cloning Kit (GenScript Cat No L00339) with a 15 bp

overhang sequence from the vector system pET29a on both forward (SP-F2) and

reverse (SP-R2) primers as the forward (SP-F2)

5rsquoGGTTCCATGGCTGATTCAGCGCGCAGCG 3rsquo and the reverse (SP-R2)

5rsquoGAATTCGGATCCGATCTAGGGAAGGATGGC 3rsquo (15 bp overhangs are

underlined) All forward and reverse primers were used as 30 pmol μL final

concentration in a 50 μL of the PCR reaction contained final concentration of 50-

100 ngmL-1

of the DNA template with 10 μL DMSO 4 μL of 25 mM MgSO4 10 μL

of 2 mM dNTPs and 2 μL of KOD Hot Start DNA Polymerase (Novagen 200 U

Cat No 71086-3) The same PCR program was run with both sets of primers as

3 cycles of Loop 1 980C for 15 seconds 420C for 30 seconds and 680C for

35min followed by 35 cycles of Loop 2 980C for 15 seconds 600C for 30

seconds and 680C for 35min and the reaction was further extended at 680C for

10 min The PCR product was purified from the gel as described before to use in

the ligation The consensus and complementary cDNA sequences and the

primers designed are shown in Fig 31

106

Figure 31 Schematic diagram of the consensus and complementary strands

showing the forward and reverse primers use to isolate the complete cDNA sequence of the plastidial SP from maize The coding sequence including a part of TP sequence was isolated using the forward primer (SP-F1) 5rsquo

GCGGAGGTGGGGTTCTCCT 3rsquo and the reverse primer (SP-R1) 3rsquoCACCTATAGTCCAAGAAAGCG 5rsquo The PCR product was purified from the

agarose gel and used in next PCR with forward (SP-F2) 5rsquoGGTTCCATGGCTGATTCAGCGCGCAGCG 3rsquo and reverse (SP-R2) 3rsquoGAATTCGGATCCGATCTAGGGAAGGATGGC 5rsquo primers with a 15 bp overhang

sequence from the vector system pET29a on both primers

Forward Primers

2291bp 5rsquo GCCATCCTTCCCTAG ACCAGGTGGATATCAGGTTCTTT CGCC TATATTT C 3rsquo- 3039bp3rsquo CGGTAGGAAGGGATCTGGTCCACCTATAGTCCAAGAAAGCGGATATAAAG ndash 5rsquo

Consensus DNA sequence

Complementary DNA sequence

Reverse Primers

CACCTATAGTCCAAGAAAGCGSP-R1

GAATTCGGATCCGATCTAGGGAAGGATGGC

SP-R2-3005bp

-2291bp15bp overhang from

pET29a vector

3030bp--3010bp

121bp- 5rsquoGTGGCGGCGGAGGTGGGGTTCTCCTCGCTGGCGGTTCCGGAGGTGGGGTCGCGCCAGGTTGGGGTCGAGGGCGATTGCAGCGGCGGGTGTCAGCGCGCAGCGTGGCGA 5rsquo -230bp3rsquoCACCGCCGCC TCCACCCCAAGAGGAGCGACCGCCAAGGCCTCCACCCCAGCGCGGTCCAACCCCAGCTCCCGCTAACGTCGCCGCCCACAGTCGCGCGTCGCACCGCT 3rsquo

SP-F2SP-F1

GCGGAGGTGGGGTTCTCCT127bp- -145bp

GGTTCCATGGCTGATTCAGCGCGCAGCG

15bp overhang from

pET29a vector-221bp-209bp



Forward Primers




Reverse Primers




SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1


SP-F1

GCGGAGGTGGGGTTCTCCTGCGGAGGTGGGGTTCTCCT127bp- -145bp


15bp overhang from




107


vector and transformation to DH5α competent cells

The complete coding sequence of SP in the PCR product was confirmed by

gene sequence analysis (Appendix 01) The pET29a expression vector encoded a

15 amino acid S-tag (LysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSer) at the

N-terminus with a thrombin digestion site (LeuValProArgGlySer) and a T7

promoter (TAATACGACTCACTAT) (Fig 32) 20 μL of ligation mixture was

prepared by adding 8 μL of purified PCR (300 ng μL) 8 μL of linearized vector

(100-200 ngμl) 2 μL 10X CloneEZreg buffer 2 μL CloneEZreg ligation enzyme in

the CloneEZreg PCR Cloning Kit (GenScript Cat No L00339) and incubated in

room temperature for 40 min and transferred to ice for 5 min Then 8 μL of

ligated mix was added to 50 μL of DH5α competent cells and the mix was kept

on ice for 30 min The transformation was done by a heat shock at 420C for 90

seconds with a quick transfer to ice for 5 min and 600 μL of SOC bacterial

growth media (super optimal broth with catabolic repressor 20 mM glucose)

was added to the transformed mix and incubated at 370C on a rotor for 1hr

Then the cells were plated on 10 mL solid LB media contained 10 μL of 50 mM

kanamycin after remove the excess media by centrifugation and incubated

overnight at 370C A single colony was grown in 6 mL of LB media contained 6

μL of 50 mM kanamycin overnight at 370C and the plasmid DNA was extracted

by using QIAprep Spin Miniprep Kit Successful insertion of the SP sequence was

identified after restricted enzyme digested plasmid DNA was run on an agarose

gel Then 2 μL of 100-150 ngmL of the plasmid DNA with the correct size of

the insert was used for transformation into the Arctic Express expression cells

108

described above The transformed cells were grown on a plate contained 10 mL

of solid LB media 10 μL of 50 mM of kanamycin and 10 μL of 100 mM of

gentamycin and incubated overnight at 370C

326 Expression of plastidial maize SP in Escherichia coli

An individual colony of the Arctic express Ecoli with the insert was grown

in 6 μL of liquid LB broth with 6 μL of 50 mM kanamycin and 6 μL of 100 mM of

gentamycin and incubated overnight at 370C on a shaker Then the cultures

were further grown in LB liquid media without the selection antibiotics and the

expression of the recombinant protein was induced by adding the final

concentration of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) when the

density of the culture was at 05-06 at OD600 The cultures were further kept

in 100C and shaken at 250 rpm for 16 hrs The E coli cells were collected by

centrifugation (at 13000xg at for 20 min) lysed using lsquoBugBusterrsquo Protein

Extraction Reagentrsquo (Novagen catalogue no 70584) and the soluble fraction

containing recombinant SP was collected The expression level of the protein

was tested by running on SDS-PAGE gel followed by Coomassie staining

(Appendix 05 shows the alignment comparison of the predicted amino acid

sequence of SP with the amino acid sequence of the recombinant SP produced in

the study)

109


using glycogen affinity native zymogram

The glycogen affinity zymogarm analyses were carried out by using

soluble recombinant protein of SP The zymogram gel preparations

electrophoresis and incubation were carried out as described in chapter 2

(22253) to test the synthetic and degradative activity

328 Gel filtration chromatography analysis of the recombinant SP

The soluble extract of recombinant SP (15 mg of total protein) was eluted

through a Superdex 200 10300GL gel permeation column pre-equilibrated with

two column volumes of rupturing buffer using an AKTA- FPLC system

(Amershamp Pharmacia Biotech model No 01068808) In total 30 (500 μL

each) fractions were collected The column was calibrated using commercial

protein standards from 137 kDa to 669 kDa (GE Healthcare Gel Filtration

Calibration Kits low molecular and high molecular weight) and the fractions

contained different multimeric forms of SP were identified using immunoblotting

329 Immobilization of recombinant SP on S-Protein Agarose beads

and pulldown assay

The S-tagged GPC fractions of tetrameric dimeric and monomeric forms

of SP were each immobilized to S-protein agarose beads (Novagen catalogue

no 69704) as described by Liu et al (2009) with some modifications 675 μg of

different recombinant SP GPC fractions were incubated in room temperature on

a rotator with 05 mgmL of amyloplast lysates pretreated earlier with 1mM ATP

110

or alkaline phosphatase (APase the insoluble form of suspension in (NH4)SO4 in

agarose beads final conc 25 units1ml) or untreated amyloplast lysates The

APase in beads were removed after incubation by centrifugation 250 μL of 50

(vv) S-protein agarose beads slurry prepared in buffer (20 mM Tris-HCl pH

75 150 mM NaCl 01 (vv) Triton X-100 01 (wv) Na azide) was added

and further incubated for 1 hour The controls were prepared by incubating the

ATP APase and untreated amyloplast lysates with S-agarose beads without the

recombinant GPC fractions The mixture was transferred onto a 10 mL Bio-Rad

Polyprep chromatography column (Bio-Rad catalogue no 731-1550) and

washed with 300 mL washing buffer [20 mM TRIS-HCl pH 75 150 mM NaCl

01 (vv) Triton X-100)] to remove non-specifically bound proteins from the

beads The controls were prepared by incubating the amyloplast lysates with the

same amount of S-agarose beads without the recombinant GPC fractions The

washed pellets of S-agarose protein bead complex was then transferred back

into a micro-centrifuge tube and centrifuged at 40C for 5 min at 500xg micro

centrifuge Following the removal of the supernatant the pellet was boiled in

100 μL of 20mM Tris-HCl pH 75 and 5X SDS-loading buffer (031M Tri-HCl pH

675 25 (vv) 2-mercaptoethanol 10 (wv) SDS 50 (vv) glycerol

0005 (wv) Bromophenol Blue) for 6 min at 950C The proteins in the samples

were analyzed by SDS-PAGE and immunoblotting with primary antibodies of anti

SSI -SSII SSIII SSIV SBEI SBEIIa SBEIIb SP and S-tag specific antibodies

111

Figure 32 Novagen pET29a vector used to overexpress plastidial SP The

expression vector contained a 15 amino acid S-tag on the N-terminus with a thrombin digestion site and a T7 promoter


The synthetic activity of the SP recombinant protein in amylopectin

glycogen and maltoheptaose substrates was analyzed in vitro by using the

tetrameric and dimeric forms of the enzyme obtained from the GPC analysis by

using the procedure described earlier in Chapter 2 section 222421 Total

protein content in a reaction was 1515 μg


SP phospholytic activity was determined as previously described in Chapter 2

section 222422 by using dimeric and tetrameric forms of recombinant SP

112

3 3 Results

331 Comparison of the protein sequence of plastidial SP of maize

endosperm from the cytosolic form and other species

The protein sequences of the plastidial SP of maize endosperm (SP1

Gene Bank ACF946921) Ipomoea batatas tubers (GenBank AAA632711)

Oryza sativa endosperm (Japonica type GenBank BAG493281) Triticum

aestivum endosperm (GenBank ACC592011) Solanum tuberosum tuber

(GenBank CAA520361) and the cytosolic form of maize (SP2 Gene Bank

ACF946911) were aligned by using CLUSTAL 21 multiple sequence alignment

program (Fig 33) The transit peptide sequence (TP) of maize SP was analyzed

and predicted to be 70 amino acids in size using the ChloroP11 sequence

analytical server and is indicated in green (Fig 33) The L-80 insertion of

plastidial form of maize is located at 510-590 amino acids (highlighted in red)

The epitope sequence of the synthetic peptide used to develop anti SP specific

antibodies (YSYDELMGSLEGNEGYGRADYFLV) is located at 916-940 amino acids

in the C-terminus In addition the serine threonine and tyrosine residues of

predicted phosphorylation sites of plastidial SP were analyzed using NetPhos 20

Server The results indicated that 28 serine residues are present in the protein

sequence except the TP and 25 of the total serine residues are located in the

L-80 insertion Also 285 of the total threonine residues are present in the L-

80 insertion but none of the tyrosine residues are located in the insert (Fig 34)

113

CLUSTAL 21 multiple sequence alignment

sweetpotato -----------------MSRLSG---ITPRARDDRSQFQNPR--LEIAVPDRTAGLQRTK 38

potato -----------------MATANGAHLFNHYSSNSRFIHFTSRNTSSKLFLTKTSHFRRPK 43

SP1 LISPHASHRHSTARAAMATTTSPPLQLASASRPHAS--ASGGGGGGGVLLAGGSGGGVAP 58

rice -----------------MATASAPLQLATASRPLPVGVGCGGGGGGGLHVGGARGGGAAP 43

wheat -----------------MATASPP--LATAFRPLAA---AGGAGGGGAHAVGAAG-RVAP 37

SP2 ------------------------------------------------------------

sweetpotato --------RTLLVKCVLDETKQTIQHVVTEKN-----EGTLLDAASIASSIKYHAEFSPA 85

potato --------RCFHVNNTLSEK---IHHPITEQGGESDLSSFAPDAASITSSIKYHAEFTPV 92

SP1 GWGRGRLQRRVSARSVASDRD--VQGPVSPAE-GLPSVLNSIGSSAIASNIKHHAEFAPL 115

rice ------ARRRLAVRSVASDRG--VQGSVSPEE-EISSVLNSIDSSTIASNIKHHAEFTPV 94

wheat R----RGRRGFVVRSVASDRE--VRGPASTEE-ELSAVLTSIDSSAIASNIQHHADFTPL 90

SP2 ---------MPEIKCGAAEK---VKPAASPEA---------EKPADIAGNISYHAQYSPH 39

sweetpotato FSPERFELPKAYFATAQSVRDALIVNWNATYDYYEKLNMKQAYYLSMEFLQGRALLNAIG 145

potato FSPERFELPKAFFATAQSVRDSLLINWNATYDIYEKLNMKQAYYLSMEFLQGRALLNAIG 152

SP1 FSPDHFSPLKAYHATAKSVLDALLINWNATYDYYNKMNVKQAYYLSMEFLQGRALTNAIG 175

rice FSPEHFSPLKAYHATAKSVLDTLIMNWNATYDYYDRTNVKQAYYLSMEFLQGRALTNAVG 154

wheat FSPEHSSPLKAYHATAKSVFDSLIINWNATYDYYNKVNAKQAYYLSMEFLQGRALTNAIG 150

SP2 FSPFAFGPEQAFYATAESVRDHLIQRWNETYLHFHKTDPKQTYYLSMEYLQGRALTNAVG 99

sweetpotato NLELTGEYAEALNKLGHNLENVASKEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 205

potato NLELTGDFAEALKNLGHNLENVASQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 212

SP1 NLEITGEYAEALKQLGQNLEDVASQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 235

rice NLELTGQYAEALQQLGHSLEDVATQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 214

wheat NLELTGQYAEALKQLGQNLEDVASQEPDPALGNGGLGRLASCFLDSMATLNYPAWGYGLR 210

SP2 NLGITGAYAEAVKKFGYELEALAGQEKDAALGNGGLGRLASCFLDSMATLNLPAWGYGLR 159

sweetpotato YKYGLFKQRITKDGQEEVAEDWLELGNPWEIIRMDVSYPVKFFGKVITGSDGKKHWIGGE 265

potato YKYGLFKQRITKDGQEEVAEDWLEIGSPWEVVRNDVSYPIKFYGKVSTGSDGKRYWIGGE 272

SP1 YEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVEGTDGRKHWIGGE 295

rice YKHGLFKQIITKDGQEEVAENWLEMGNPWEIVRTDVSYPVKFYGKVVEGTDGRMHWIGGE 274

wheat YRYGLFKQIIAKDGQEEVAENWLEMGNPWEIVRNDVSYPVKFYGKVVEGTDGRKHWIGGE 270

SP2 YRYGLFKQHIAKEGQEEVAEDWLDKFSPWEIPRHDVVFPVRFFGHVEILPDGSRKLVGGE 219

sweetpotato DILAVAYDVPIPGYKTRTTISLRLWSTKVPSEDFDLYSFNAGEHTKACEAQANAEKICYI 325

potato DIKAVAYDVPIPGYKTRTTISLRLWSTQVPSADFDLSAFNAGEHTKACEAQANAEKICYI 332

SP1 NIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAYEAHLNAKKICHI 355

rice NIKVVAHDIPIPGYKTKTTNNLRLWSTTVPSQDFDLEAFNAGDHASAYEAHLNAEKICHV 334

wheat NIKAVAHDVPIPGYKTKTTNNLRLWSTTVPSQNFDLGAFNAGDHAKANEAHLNAEKICHV 330

SP2 VLKALAYDVPIPGYKTKNAISLRLWEAKATAEDFNLFQFNDGQYESAAQLHARAQQICAV 279

sweetpotato LYPGDESIEGKILRLKQQYTLCSASLQDIIARFERRSGEYVK--WEEFPEKVAVQMNDTH 383

potato LYPGDESEEGKILRLKQQYTLCSASLQDIISRFERRSGDRIK--WEEFPEKVAVQMNDTH 390

SP1 LYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLN--WEDFPSKVAVQMNDTH 413

rice LYPGDESPEGKVLRLKQQYTLCSASLQDIIARFERRAGDSLS--WEDFPSKVAVQMNDTH 392

wheat LYPGDESSEGKILRLKQQYTLCSASLQDIISRFESRAGDSLN--WEDFPSKVAVQMNDTH 388

SP2 LYPGDATEEGKLLRLKQQFFLCSASLQDMIARFKERKSDRVSGKWSEFPTKVAVQLNDTH 339

sweetpotato PTLCIPELIRILIDLKGLSWKEAWNITQRTVAYTNHTVLPEALEKWSYELMEKLLPRHIE 443

potato PTLCIPELMRILIDLKGLNWNEAWNITQRTVAYTNHTVLPEALEKWSYELMQKLLPRHVE 450

SP1 PTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 473

rice PTLCIPELMRILIDVKGLSWNEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 452

wheat PTLCIPELMRILMDIKGLSWNEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 448

SP2 PTLAIPELMRLLMDEEGLGWDEAWDITYRTISYTNHTVLPEALEKWSQIVMRKLLPRHME 399

114

sweetpotato IIEMIDEQLINEIVSEYGTSDLDMLEKKLNDMRILENFDIPSSIANLFTKPKETSIVDPS 503

potato IIEAIDEELVHEIVLKYGSMDLNKLEEKLTTMRILENFDLPSSVAELFIKP-EISVDDDT 509

SP1 IIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVKPKDKKESPAK 533

rice IIEKIDGELMNIIISKYGTEDTSLLKKKIKEMRILDNIDLPDSIAKLFVKPKEKKESPAK 512

wheat IIETIDEKLMNNIVSKYGTADISLLKQKLKDMRILDNVDLPASVAKLFIKPKEKTG---- 504

SP2 IIEEIDKRFKELVISKH-----KEMEGKIDSMKVLD------------------------ 430

sweetpotato EEVEVSGKVVTESVEVSDKVVTESEKDE----------LEEKDTELEKDED--------P 545

potato ETVEVH-----DKVEASDKVVTNDEDDTGKKTSVKIEAAAEKDIDKKTPVS--------P 556

SP1 SKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSED---ELDPFV 590

rice LKEKLLVKSLEPSVVVEEKTVSKVEINEDSEEVEVDSE-EVVEAENEDSED---ELDPFV 568

wheat ---KLLVQSLESIAEGDEKTESQEEENILSETAEKKGGSDSEEAPDAEKEDPVYELDPFA 561

SP2 ------------------------------------------------------------

sweetpotato VPAPIPPKMVRMANLCVVGGHAVNGVAEIHSDIVKEDVFNDFYQLWPEKFQNKTNGVTPR 605

potato EPAVIPPKKVRMANLCVVGGHAVNGVAEIHSEIVKEEVFNDFYELWPEKFQNKTNGVTPR 616

SP1 KSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNKTNGVTPR 650

rice KSDPKLPRVVRMANLCVVGGHSVNGVAAIHSEIVKEDVFNSFYEMWPAKFQNKTNGVTPR 628

wheat KYDPQLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNKTNGVTPR 621

SP2 NSNPQKP-VVRMANLCVVSSHTVNGVAELHSNILKQELFADYVSIWPTKFQNKTNGITPR 489

sweetpotato RWIRFCNPALSNIITKWIGTEDWVLNTEKLAELRKFADNEDLQIEWRAAKRSNKVKVASF 665

potato RWIRFCNPPLSAIITKWTGTEDWVLKTEKLAELQKFADNEDLQNEWREAKRSNKIKVVSF 676

SP1 RWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKANKMKVVSL 710

rice RWIRFCNPELSAIISKWIGSDDWVLNTDKLAELKKFADDEDLQSEWRAAKKANKVKVVSL 688

wheat RWIRFCNPELSAIISKWIGSDDWILNTDKLAGLKKFADDEDLQSEWRTAKRNNKMKVVSL 681

SP2 RWLRFCNPELSEIVTKWLKSDQWTSNLDLLTGLRKFADDEKLHAEWAAAKLSCKKRLAKH 549

sweetpotato LKERTGYSVSPNAMFDIQVKRIHEYKRQLLNILGIVYRYKQMKEMSAREREAKFVPRVCI 725

potato LKEKTGYSVVPDAMFDIQVKRIHEYKRQLLNIFGIVYRYKKMKEMTAAERKTNFVPRVCI 736

SP1 IREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKSFVPRVCI 770

rice IREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSAKDRINSFVPRVCI 748

wheat IRDKTGYVVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSAKDRIKSFVPRVCI 741

SP2 VLDVTGVTIDPTSLFDIQIKRIHEYKRQLLNILGAVYRYKKLKGMSAEEK-QKVTPRTVM 608

sweetpotato FGGKAFATYVQAKRIAKFITDVGATINHDPEIGDLLKVIFVPDYNVSAAELLIPASGLSQ 785

potato FGGKAFATYVQAKRIVKFITDVGATINHDPEIGDLLKVVFVPDYNVSVAELLIPASDLSE 796

SP1 FGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALIPASELSQ 830

rice FGGKAFATYVQAKRIVKFITDVAATVNHDPEIGDLLKVVFIPDYNVSVAEALIPASELSQ 808

wheat FGGKAFATYVQAKRIVKFITDVAATVNYDPDVGDLLKVVFVPDYNVSVAEKLIPASELSQ 801

SP2 IGGKAFATYTNAKRIVKLVNDVGAVVNNDPEVNKYLKVVFIPNYNVSVAEVLIPGSELSQ 668

sweetpotato HISTAGMEASGQSNMKFAMNGCILIGTLDGANVEIRQEVGEENFFLFGAEAHEIAGLRKE 845

potato HISTAGMEASGTSNMKFAMNGCIQIGTLDGANVEIREEVGEENFFLFGAQAHEIAGLRKE 856

SP1 HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHEIAGLRKE 890

rice HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHEIAGLRKE 868

wheat HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAHAPEIAGLRQE 861

SP2 HISTAGMEASGTSNMKFSLNGCVIIGTLDGANVEIREEVGEDNFFLFGAKADEVAGLRKD 728

sweetpotato RAEGKFVPDERFEEVKEFIKRGVFGSNTYDELLGSLEGNEGFGRGDYFLVGKDFPSYIEC 905

potato RADGKFVPDERFEEVKEFVRSGAFGSYNYDDLIGSLEGNEGFGRADYFLVGKDFPSYIEC 916

SP1 RAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELMGSLEGNEGYGRADYFLVGKDFPSYIEC 950

rice RAQGKFVPDPRFEEVKRFVRSGVFGTYNYDDLMGSLEGNEGYGRADYFLVGKDFPSYIEC 928

wheat RAEGKFVPDPRFEEVKEYVRSGVFGTSNYDELMGSLEGNEGYGRADYFLVGKDFPSYIEC 921

SP2 RENGLFKPDPRFEEAKQFIRSGAFGSYDYEPLLDSLEGNSGFGRGDYFLVGYDFPSYIDA 788

sweetpotato QEKVDEAYRDQKIWTRMSILNTAGSYKFSSDRTIHEYAKDIWNIQPVVFP 955

potato QEKVDEAYRDQKRWTTMSILNTAGSYKFSSDRTIHEYAKDIWNIEAVEIA 966

SP1 QEKVDEAYRDQKLWTRMSILNTAGSSKFSSDRTIHEYAKDIWDISPAILP 1000

rice QEKVDKAYRDQKLWTRMSILNTASSSKFNSDRTIHEYAKDIWDIKPVILP 978

wheat QQKVDEAYRDQKLWTRMSILNTAGSPKFSSDRTIHEYAKDIWDISPVIMP 971

SP2 QDRVDAAYKDKKKWTKMSILNTAGSGKFSSDRTIAQYAKEIWDIKASPVV 838

115

Figure 33 The protein sequences of the plastidial SP of maize endosperm (SP1 Gene Bank ACF946921) the cytosolic form of maize (SP2 Gene Bank ACF946911) Ipomoea batatas tubers (GenBank AAA632711) Oryza sativa

endosperm (Japonica type GenBank BAG493281) Triticum aestivum endosperm (GenBank ACC592011) and Solanum tuberosum tuber (GenBank

CAA520361) were aligned by using CLUSTAL 21 multiple sequence alignment program The Transit peptide sequence (TP) of maize SP (70 amino acids) is indicated in green The L-80 insertion of plastidial form of maize is located at

510-590 amino acids (highlighted in red) The epitope sequence for the synthetic peptide used to develop anti SP specific antibodies

(YSYDELMGSLEGNEGYGRADYFLV) is located at 916-940 amino acids

116

Serine predictions

Name Pos Context Score Pred

_________________________v_________________

Sequence 3 --LISPHAS 0014

Sequence 7 SPHASHRHS 0927 S

Sequence 11 SHRHSTARA 0996 S

Sequence 22 ATTTSPPLQ 0159

Sequence 29 LQLASASRP 0126

Sequence 31 LASASRPHA 0020

Sequence 36 RPHASASGG 0363

Sequence 38 HASASGGGG 0637 S

Sequence 52 LAGGSGGGV 0006

Sequence 70 QRRVSARSV 0995 S

Sequence 73 VSARSVASD 0987 S

Sequence 76 RSVASDRDV 0989 S

Sequence 85 QGPVSPAEG 0993 S

Sequence 92 EGLPSVLNS 0160

Sequence 96 SVLNSIGSS 0495

Sequence 99 NSIGSSAIA 0006

Sequence 100 SIGSSAIAS 0023

Sequence 104 SAIASNIKH 0058

Sequence 117 APLFSPDHF 0144

Sequence 122 PDHFSPLKA 0242

Sequence 133 ATAKSVLDA 0898 S

Sequence 161 AYYLSMEFL 0018

Sequence 199 EDVASQEPD 0852 S

Sequence 216 GRLASCFLD 0007

Sequence 221 CFLDSLATL 0003

Sequence 272 RNDVSYPVK 0018

Sequence 321 LRLWSTTVP 0075

Sequence 336 AAFNSGDHT 0018

Sequence 362 PGDESLEGK 0826 S

Sequence 378 YTLCSASLQ 0009

Sequence 380 LCSASLQDI 0882 S

Sequence 390 ARFESRAGE 0713 S

Sequence 395 RAGESLNWE 0546 S

Sequence 403 EDFPSKVAV 0004

Sequence 432 VKGLSWSEA 0992 S

Sequence 434 GLSWSEAWS 0040

Sequence 438 SEAWSITER 0375

Sequence 460 LEKWSLDIM 0004

Sequence 488 NNIVSKYGT 0777 S

Sequence 516 DLPASISQL 0296

Sequence 518 PASISQLFV 0004

Sequence 530 DKKESPAKS 0994 S

Sequence 534 SPAKSKQKL 0584 S

Sequence 542 LLVKSLETI 0725 S

Sequence 563 AEVLSEIEE 0985 S

Sequence 572 EKLESEEVE 0973 S

Sequence 581 AEEESSEDE 0996 S

Sequence 582 EEESSEDEL 0993 S

Sequence 592 PFVKSDPKL 0138

Sequence 612 VGGHSVNGV 0038

Sequence 621 AEIHSEIVK 0012

Sequence 631 DVFNSFYEM 0041

Sequence 661 NPALSALIS 0019

Sequence 665 SALISKWIG 0004

Sequence 670 KWIGSDDWV 0009

Sequence 694 EDLHSEWRA 0465

Sequence 709 MKVVSLIRE 0953 S

Sequence 720 GYIVSPDAM 0052

Sequence 756 MKEMSTEER 0996 S

Sequence 763 ERAKSFVPR 0944 S

Sequence 800 VNHDSDIGD 0526 S

Sequence 817 DYNVSVAEA 0179

Sequence 826 LIPASELSQ 0075

Sequence 829 ASELSQHIS 0164

Sequence 833 SQHISTAGM 0013

Sequence 840 GMEASGTSN 0020

Sequence 843 ASGTSNMKF 0053

Sequence 911 EFVRSGVFG 0433

Sequence 918 FGTYSYDEL 0124

Sequence 925 ELMGSLEGN 0913 S

Sequence 946 KDFPSYIEC 0610 S

Sequence 968 WTRMSILNT 0561 S

Sequence 975 NTAGSSKFS 0933 S

Sequence 976 TAGSSKFSS 0468

Sequence 979 SSKFSSDRT 0988 S

Sequence 980 SKFSSDRTI 0808 S

Sequence 995 IWDISPAIL 0037

NetPhos 20 Server - prediction results

117

_________________________^________________

Threonine predictions


_________________________v_________________

Sequence 12 HRHSTARAA 0985 T

Sequence 19 AAMATTTSP 0074

Sequence 20 AMATTTSPP 0697 T

Sequence 21 MATTTSPPL 0660 T

Sequence 130 AYHATAKSV 0546 T

Sequence 145 NWNATYDYY 0020

Sequence 171 GRALTNAIG 0117

Sequence 180 NLEITGEYA 0032

Sequence 224 DSLATLNYP 0021

Sequence 246 KQIITKDGQ 0099

Sequence 285 VVEGTDGRK 0186

Sequence 311 PGYKTRTTN 0063

Sequence 313 YKTRTTNNL 0107

Sequence 314 KTRTTNNLR 0341

Sequence 322 RLWSTTVPA 0615 T

Sequence 323 LWSTTVPAQ 0024

Sequence 340 SGDHTKAYE 0029

Sequence 375 KQQYTLCSA 0238

Sequence 412 QMNDTHPTL 0028

Sequence 415 DTHPTLCIP 0513 T

Sequence 440 AWSITERTV 0309

Sequence 443 ITERTVAYT 0150

Sequence 447 TVAYTNHTV 0013

Sequence 450 YTNHTVLPE 0063

Sequence 477 EIIETIDEE 0921 T

Sequence 492 SKYGTTDTE 0274

Sequence 493 KYGTTDTEL 0367

Sequence 495 GTTDTELLK 0233

Sequence 545 KSLETIVDV 0637 T

Sequence 553 VEEKTELEE 0855 T

Sequence 638 EMWPTKFQN 0195

Sequence 644 FQNKTNGVT 0031

Sequence 648 TNGVTPRRW 0569 T

Sequence 677 WVLNTDKLA 0471

Sequence 715 IREKTGYIV 0920 T

Sequence 757 KEMSTEERA 0420

Sequence 778 KAFATYIQA 0089

Sequence 790 VKFITDVAA 0051

Sequence 795 DVAATVNHD 0134

Sequence 834 QHISTAGME 0075

Sequence 842 EASGTSNMK 0158

Sequence 857 ILIGTLDGA 0499

Sequence 916 GVFGTYSYD 0027

Sequence 965 QKLWTRMSI 0007

Sequence 972 SILNTAGSS 0033

Sequence 983 SSDRTIHEY 0468

_________________________^_________________

Tyrosine predictions


_________________________v_________________

Sequence 127 PLKAYHATA 0057

Sequence 146 WNATYDYYN 0411

Sequence 148 ATYDYYNKM 0262

Sequence 149 TYDYYNKMN 0559 Y

Sequence 158 VKQAYYLSM 0035

Sequence 159 KQAYYLSME 0385

Sequence 183 ITGEYAEAL 0332

Sequence 227 ATLNYPAWG 0043

Sequence 232 PAWGYGLRY 0042

Sequence 236 YGLRYEYGL 0176

Sequence 238 LRYEYGLFK 0010

Sequence 262 LEMGYPWEV 0023

Sequence 273 NDVSYPVKF 0124

Sequence 278 PVKFYGKVV 0016

Sequence 309 PIPGYKTRT 0269

Sequence 343 HTKAYEAHL 0125

Sequence 357 CHILYPGDE 0013

Sequence 374 LKQQYTLCS 0035

Sequence 446 RTVAYTNHT 0780 Y

Sequence 490 IVSKYGTTD 0134

Sequence 633 FNSFYEMWP 0768 Y

Sequence 717 EKTGYIVSP 0980 Y

Sequence 735 RIHEYKRQL 0049

Sequence 747 LGIVYRYKK 0009

Sequence 749 IVYRYKKMK 0033

Sequence 779 AFATYIQAK 0207

Sequence 814 FVPDYNVSV 0357

Sequence 917 VFGTYSYDE 0025

Sequence 919 GTYSYDELM 0045

Sequence 932 GNEGYGRAD 0911 Y

Sequence 937 GRADYFLVG 0162

Sequence 947 DFPSYIECQ 0744 Y

Sequence 958 VDEAYRDQK 0770 Y

Sequence 987 TIHEYAKDI 0017

_________________________^_________________

Figure 34 The predicted phosphorylation sites of the plastidial maize SP protein sequence were analyzed using NetPhos 20 Server

118

332 Development of recombinant SP

3321 PCR

The complete mRNA sequence (3053 bp) of plastidial SP of maize

(GenBank EU8576402) was obtained from the National Center for

Biotechnology Information data base (NCBI) Initially the coding sequence

including a part of TP sequence was isolated using the forward primer (SP-F1) 5rsquo


GCGAAAGAACCTGATATCCAC 3rsquo and the purified PCR product was used as the

template in next PCR to obtain the 2805 bp of complete mRNA coding sequence

which produces plastidial SP with 935 amino acids Fig 35 shows the PCR

product of the full length sequence (2805 bp) of SP visualized on a agarose gel

For the next PCR the primers were specifically designed for the CloneEZreg PCR

Cloning Kit (GenScript Cat No L00339) with a 15 bp overhang sequence from

the vector system pET29a on both forward (SP-F2) and reverse (SP-R2) primers

to facilitate the homologous recombination (Appendix 03 and 04 shows the

sequences of all the primers used in the study in PCR and sequence analysis)

119



ethidium bromide

3322 Testing the expression level and the synthetic and degradative

activity of recombinant SP on glycogen affinity zymogram

The expression level of the cloned gene was qualitatively tested by SDS-

PAGE analysis of produced proteins (Fig 36) Soluble extract of recombinant SP

obtained after the culture was induced by 1mM IPTG was run on the gel (30 μg

of total protein per well) and compared with equal amounts of soluble proteins

obtained from the uninduced cultures (control) the induced Arctic Expression

Ecoli cells without the plasmid induced Arctic Expression Ecoli cells only with

2805bp

5000

3000

2000

bp

2805bp

5000

3000

2000

bp

120

the plasmid but without the insert (Fig 36A) Induced Ecoli cells with the insert

(Lane 1) showed higher level of expression and the immunoblot probed with

anti-SP specific antibodies confirmed the higher expression was due to

recombinant SP (Fig 36B)

The synthetic activity of the recombinant SP was analyzed on 01

glycogen affinity zymogram (Fig 37A) 90 μg of proteins were run on the

zymogram The soluble recombinant proteins obtained after the cultures were

induced by 1 mM IPTG (Lane 1) showed higher activity than the amyloplast

lysates (Lane 6) There was no activity observed in the soluble fractions of

induced Arctic Express cells without plasmid (Lane 2) uninduced Arctic Express

cells with both the plasmid and the insert (Lane 3) and induced Arctic Express

cells with the plasmid (Lane 4) or in uninduced Arctic Express cells with the

plasmid but without the insert (Lane 5) The immunoblot of the zymogram

probed with anti-SP specific antibody recognized the SP in the recombinant

soluble fraction (Fig 37B) However the faint band in lane 3 in uninduced

culture in the immunoblot is due to the leaky promoter since there was no band

observed in other samples (Fig 37B) Corresponding immunoblots of the native

zymogram of SP recombinant proteins showed four distinct bands and may

represent the monomeric dimeric tetrameric and multimeric (consisting of

more than four subunits) configurations of the recombinant SP (Fig 37B)

The synthetic activity and degradative activity of the recombinant protein

was qualitatively tested on the zymogram by incubating the zymogram gel in 20

mM of G-1-P and Na2HPO4 as the inorganic phosphate substrate respectively

(Fig 38) Multiple bands on the samples may correspondent to the different

121

multimeric forms (dimeric and tetrameric) of SP The observation that the

activity bands shown in synthetic activity zymogram disappeared in the

degradative activity zymogram (38D) indicates that the recombinant SP is

active in both synthetic and degradative directions in a manner that is similar to

the SP presence in the amyloplast lysates (Fig 38)

122


analyzed by running the soluble recombinant protein on a 10 SDS gel followed by Coomassie staining (A) and immunoblot analyses by probing with anti-SP specific antibodies (B) 30 μg of proteins were run in each lane The expression

of the recombinant protein was induced by adding the final concentration of 1 mM IPTG (Lane 1) Uninduced cultures (Lane 2) IPTG induced Arctic Express

cells without the plasmid (Lane 3) IPTG induced Arctic Express cells with the plasmid but without the insert (Lane 4 and 5) and the amyloplast lysates(Lane 6) are shown Arrow indicated the expressed SP in lane 1

(A) (B)

kDa

150

100

75

50

L 1 2 3 4 5 6 L 1 2 3 4 5 6

L ndash Protein marker

1 Recombinant SP obtained after the cultures were induced by 1mM IPTG

2 Uninduced control

3 Only the induced Arctic Expression E-coli cells without the plasmid

4 and 5 Induced Arctic Expression E-coli cells with the plasmid no insert

6 Amyloplast lysates

123

Figure 37 The synthetic activity of recombinant SP in a glycogen affinity

native zymogram that contained 01 glycogen in the gel (A) and corresponding immunoblot of the native zymogram probed with anti-SP specific

antibodies (B) are shown 90 μg of proteins were run in a well and following electrophoresis the native gel was incubated overnight at 280C with the incubation buffer containing 01 glycogen and 20 mM G-1-P in the synthetic

direction The activity bands were visualized by Lugolrsquos solution and are indicated with arrows (A) Multiple bands which were recognized by SP-specific

antibodies on immunoblot are shown by arrows (B)

(B)

(A)

124

Figure 38 Testing the synthetic and degradative activity of recombinant SP on

glycogen affinity native zymogram The synthetic activity of recombinant SP in glycogen affinity native zymogram (A) and the corresponding immunoblot of the zymogram probed with anti-SP specific antibodies (B) immunoblot probed with

anti-S-tag antibodies (C) and degradative activity on zymogram (D) are shown 30 μg of protein were run in a well and following electrophoresis the native gel

was incubated overnight at 280C with the incubation buffer contained 20 mM G-1-Pin the synthetic direction (A) and 20 mM sodium phosphate dibasic (Na2HPO4) in phosphorylitic direction (D) Bands were visualized by Lugolrsquos

solution Suggested dimeric and multimeric forms of SP and are indicated with arrows

(A) (B) (C) (D)

SP synthetic

activity zymogramImmunoblot of SP

synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

125

333 Gel filtration chromatography analysis of recombinant SP

The soluble fraction of the recombinant SP was separated through a

Superdex 200 10300GL gel permeation column and the fractions collected were

analyzed by SDS-PAGE and immunoblotting using peptide specific anti-SP

antibodies (Fig 39A) Recombinant SP was eluted in for different peaks and the

predicted molecular weights of the eluted SP fractions (based on the elution of

the standards) showed the existence of monomeric (112 kDa) dimeric (112 kDa

X 2) tetrameric (112 kDa X 4) and multimeric forms (more than four subunits)

The synthetic activity of the various multimers of recombinant SP was tested on

the native zymograms by loading the equal amounts of proteins on the gel (Fig

39B) Activity bands were observed in the dimeric tetrameric and multimeric

forms but no activity was detected in the monomeric form on the zymogram

(Fig 39B)

126

Figure 39 Gel filtration chromatography (GPC) analysis of recombinant SP Recombinant SP soluble fraction was separated by GPC through a Superdex 200 10300GL gel permeation column The fractions were run (30 μg of proteins in a

well) on SDS-PAGE followed by immunoblot analysis with anti-SP antibodies Monomeric (112 kDa) dimeric tetrameric and multimeric forms of SP were

detected (A) Fractions containing SP were tested for synthetic activity on a glycogen affinity zymogram (B) and corresponding immunoblot of the zymogram probed with the anti-SP specific antibodies (C) The SP bands correspond to the

various SP multimers and are shown by the arrows and the fraction numbers of the bands were shown The sizes of the known protein standards eluted in the

column were indicated in the boxes AP=amyloplast lysates

(A)

(B)

(C)

127



of SP were separately immobilized to S-protein agarose beads following

incubation with 05 mgmL of pretreated amyloplast lysates The success of

immobilization of the recombinant tetrameric and dimeric forms to the S-

agarose beads was tested by probing immunoblots of washed beads with anti-SP

specific and anti S-tag specific antibodies (Fig 310) Both the dimeric and

tetrameric SP incubated with both untreated and ATP-treated amyloplast lysates

showed very strong immuno-reactive bands The tetrameric form showed

nonspecific binding with the proteins in the amyloplast lysates however the

level of binding is negligible when compare with the immobilized samples (Fig

310)

To test the protein-protein interactions of the tetrameric and dimeric

forms of recombinant SP with major starch biosynthetic enzymes the beads

containing protein complexes were separated on SDS-PAGE gels and

immunoblots probed with various peptide-specific antibodies Interactions were

observed between recombinant SP forms only with SSIIa SBEI and SBEIIb (Fig

311) The tetrameric form of recombinant SP interacted with SSIIa and SBEI

when the amyloplast was treated with ATP but not in the untreated amyloplast

lysates or APase treated samples In contrast there was no interaction with

SBEIIb and the tetrameric form In ATP-treated amyloplasts SBEI and SBEIIb

interact with the dimeric form but not with the ATP treated SSIIa SSIIa

interacted with the dimeric form of SP in the untreated amyloplast lysates

Further the interaction between SBEI and dimeric forms was independent of

128

ATP treatment The dimeric form of SP showed much stronger interaction with

SBEIIb in ATP-treated sample than in the untreated samples The APase-treated

samples did not show any interaction with any of the enzymes tested Fig 313

is a schematic diagram summarizing the possible interactions of the recombinant

forms of SP with SSIIa SBEI and SBEIIb enzymes

129

Figure 310 Immunoblots probed with anti-SP and anti-S-tag peptide specific antibodies to confirm the immobilization of the recombinant GPC fractions by S-Agarose beads The S-tagged GPC fractions (675 μg of protein) were

immobilized to S-protein agarose beads after the fractions were incubated with 05 mgmL of untreated and pretreated amyloplast lysates with 1mM ATP

APase The ATP or APase and untreated amyloplast lysates were incubated with the S-agarose beads without the recombinant fractions as the controls (lane 4-6) The washed pellets of S-agarose protein bead complexes were subjected to

SDS-PAGE and immunoblot analysis L= protein marker and the size of SP is 112 kDa

Dimeric form of SPTetrameric form of SP

1 Untreated amyloplast lysates (AP) incubated with recombinant SP forms

2 ATP Treated AP incubated with recombinant SP forms

3 APase Treated incubated with recombinant SP forms

4 Beads+ Untreated AP (no recombinant SP forms)

5 Beads+ ATP treated AP (no recombinant SP forms)

6 Beads+ APase treated AP (no recombinant SP forms)

7 SP forms + beads only


L 1 2 3 4 5 6 7 8

kDa

150

100

75

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8

kDa

150

100

75

Anti-SP

Anti-S-Tag

130

Figure 311 Immunoblots of the immobilized GPC fractions of the recombinat

SP by S-Agarose beads probed with anti-SSIIa anti-SBEI and anti-SBEIIb peptide specific antibodies The S-tagged tetrameric and dimeric GPC fractions

(675 μg of protein) were immobilized to S-protein agarose beads after the fractions were incubated with 05 mgmL of untreated and pretreated amyloplast lysates with 1mM ATP or APase The ATP APase and untreated

amyloplast lysates were incubated with the S-agarose beads without the recombinant fractions as the controls The washed pellets of S-agarose protein

bead complexes were subjected to SDS-PAGE and immunoblot analysis to test the protein-protein interactions L= protein marker The arrows indicate the enzyme SSIIa at 76 kDa SBEI at 80 kDa and SBEIIb at 85 kDa

131


of SP by peptide specific anti-SP antibodies bound to Protein A- Sepharose beads

132

Figure 313 Schematic diagram summarizing the protein-protein interactions between tetrameric and dimeric forms of recombinant SP with starch biosynthetic enzymes present in the amyloplast lysates

335 The glucan synthetic and phospholytic activity of recombinant SP

The synthetic activity of the tetrameric dimeric and monomeric forms of

recombinant SP was analyzed in vitro by using [U14

C]-G-1-P as the substrate

The transfer of glucosyl units from radio labeled G-1-P to glycogen amylopectin

and maltoheptaose were assayed using 25 mgmL substrate concentration for

30 minutes and the synthetic activity was calculated as nmolmghr (Fig 314)

The tetrameric form of SP had the highest activity with amylopectin

(928961255) which was approximately 24 greater than with glycogen

(665121356) Synthetic activities were statistically analyzed by Statistix 9

statistics analytical program at (Plt005) probability using by One-Way ANOVA

= Phosphorylation of the enzyme by ATP+plastidial protein kinase

133

followed by LSD analysis (F= 24766 P=000001 see appendix 09 for the

statistical analysis of the data) There was no significant difference between the

activity of the tetrameric form of SP between amylopectin and glycogen

substrates Both glycogen and amylopectin showed significant differences in

synthetic activity compared to maltoheptaose for given substrate

concentrations The activity was much lower for the dimeric form in amylopectin

(174710) and glycogen (1746914) compared to the activities of the

tetrameric form with these substrates However the dimeric form showed

slightly higher in synthetic activity (503017) compared to tetrameric form

(29711) with maltoheptaose Synthetic activity of the dimeric form of SP was

not significantly different for glycogen amylopectin or maltoheptaose substrates

(Fig 313) The tetrameric form of SP with amylopectin and glycogen showed

significantly higher phosphorylitic activity at 25 mgmL substrate concentration

compared to maltoheptaose but no significant difference was observed between

amylopectin and glycogen The variation of the activity of tetrameric SP from

synthetic direction to phosphorylitic direction was greater in maltoheptaose (147

fold) compared to amylopectin (20 fold) and glycogen (11) (Fig 314) (see

Appendix 09 for the analysis of ANOVA)

The Vmax and Km of the tetrameric form of recombinant SP was greater

with amylopectin and lower in maltoheptaose in phosphorylitic direction (Table

31) Vmax of the tetrameric form was approximately 142 times greater than

the dimeric form (Table 31) The Km value of the dimeric form was

approximately 112 times greater than the tetrameric form for amylopectin and

about 275 times greater for glycogen (Table 31)

134

Figure 314 Synthetic and degradative activities of the tetrameric and dimeric forms of recombinant SP in different glucan substrates The activities were

compared at 25 mgmL substrate concentration in glycogen amylopectin and maltoheptaose Significantly different means (at Plt005) are shown with similar symbols S= Synthetic direction P= Phosphorolytic direction


recombinant SP in the phosphorolytic direction

Glucan Substrate

Tetrameric form of

recombinant SP

Vmax Km (nmolmgmin) (mgmL)

Dimeric form of

recombinant SP


Glycogen

Amylopectin

Maltoheptaose

89429 0024

131648 0078

49711 00298

5952 0066

9786 873

- -

Tetrameric form of Recombinant

SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger

135

34 Discussion


The investigations presented in this chapter tested the hypothesis that

plastidial SP has a role in starch biosynthesis and it is regulated by protein-

protein interaction with other starch biosynthetic enzymes Previous studies

confirm the involvement of SP in protein complex formation with other major

starch biosynthetic enzymes SP was involved in the formation of heteromeric

protein complexes with SBEI and SBEIIb in a phosphorylation dependent

manner in wheat amyloplasts (Tetlow et al 2004) In the ae mutant which

lacks SBEIIb SP formed complexes with SBEI SSI SSIIa and SBEIIa (Liu et al

2009) However in the present study the interaction of SP with other starch

biosynthetic enzymes in maize amyloplast stroma was not detected by co-

immunoprecipitation since the native protein was not immunoprecipated by

peptide specific anti-SP antibodies bound to Protein-A sepharose beads (Chapter

2 section 236) Dimeric and tetrameric configurations of SP may reduce

accessibility of the SP antibodies to the epitopes thus preventing

immunoprecipitation of recombinant dimeric and tetrameric forms by SP-specific

antibodies and Protein-A sepharose beads (Fig 314) We therefore developed

a recombinant SP with an affinity ligand S-tag on the N-terminus of the protein

in order to detect protein-protein interactions involving SP

The complete mRNA sequence (2805 bp) of plastidial SP of wild-type

maize without the transit peptide (TP) sequence of 70 amino acids was directly

136

cloned into pET29a expression vector and the complete protein with 935 amino

acids was over expressed in Acrtic Express Ecoli system

The amino acid sequence alignment of plastidial SP of maize wild-type

endosperm (Zea mays) (SP1 Gene Bank ACF946921) with the TP Ipomoea

batatas tubers (GenBank AAA632711) Oryza sativa endosperm (Japonica

type GenBank BAG493281) Triticum aestivum endosperm (GenBank

ACC592011) Solanum tuberosum tuber (GenBank CAA520361) and the

cytosolic form of maize (SP2 Gene Bank ACF946911) showed that C-terminus

is highly conserved among the species tested It further confirmed the previous

sequence analysis of Yu et al (2001) that the peptide sequences of maize SP

showed higher identities to potato sweet potato and spinach but the N-terminus

sequence did not align with any other N-terminal sequences

The L-78 amino acid insertion located in the middle of plastidial SP is a

unique characteristic of plastidial SP and is not seen in the cytosolic form of SP

(Young et al 2006 Yu et al 2001 Mori et al 1993) Computational analysis

also found that the unique L-80 insertion of the plasitidial form is located at 510-

590 amino acids in maize (Fig 32) The exact role of this insertion is not well

documented in maize SP Phosphorylation site prediction analysis indicated that

the L-80 insertion consists of 7 serine (Ser) (out of 28) and 2 (out of 7) of the

threonine (Thr) residues These observations are similar to the finding of Young

et al (2006) indicating that there are 10 Ser and 5 Thr residues on L-78

insertion in the plastidial SP in sweet potato tubers and also the serine and

threonine residues are intensively involved in the phosphorylation of the enzyme

137

(Young et al 2006) L-78 insertion of plastidial SP also prevents affinity towards

higher molecular branched glucans such as starch and glycogen (Young et al

2006 Mori et al 1993) Recombinant form of plastidial SP developed by

replacing the L-78 insertion with a cytosolic SP sequence in potato showed the

activity of the chimeric protein was five times lesser than the parent type

isoform but its affinity for glycogen was much higher (Mori et al 1993) In

contrast a higher affinity of the SP to low molecular malto-oligosaccharides

(MOS) is recorded in maize (Yu et al 2001)

Qualitative analysis of the synthetic activity of the recombinant SP on

01 glycogen affinity zymogram (Fig 37A) showed catalytic activity of the

protein The lower activity shown in lane 5 (uninduced Ecoli cells with the

plasmid but without the insert) may be due to the endogenous glucan

phosphorylase present in Ecoli However no such activity was found in induced

Ecoli cells with the plasmid but without the insert (lane 4) and the activity level

is negligible when compared with the recombinant SP activity The immunoblot

of the zymogram probed with anti-SP specific antibody recognized the SP in

recombinant soluble fraction (Fig 37B) However the faint band on the lane 3 in

uninduced culture in the immunoblot is due to the leaky promoter since there

was no band observed in other samples (Fig 37B) Further the degradative

activity of the recombinant SP on zymogram indicates that the protein is also

active in degradative direction (Fig 38D)

138

342 Gel filtration chromatography of recombinant SP

Native SP exists as monomeric dimeric or tetrameric forms with identical

subunits in maize amyloplast stroma (Albrecht et al 1998 Mu et al 2001 Liu et

al 2009) These different molecular confirmations remain throughout the

development of the endosperm Immunoblot analysis of soluble fraction of the

recombinant SP eluted through the GPC column indicated that the fractions

contained monomeric and high molecular dimeric tetrameric and multimeric

forms The synthetic activity was detected in the dimeric tetrameric and

multimeric forms of GPC-fractionated recombinant SP Inactivity shown in the

monomeric form was due to the lack of activity of the monomeric form or

relatively lower levels of protein are present in the fractions that could not be

detected on western blots

Glycogen phosphorylase in animals and bacteria is homodimeric and each

subunit linked to a pyridoxal phosphate co-factor which is involved in enzyme

catalysis (Buchbinder et al 2001) Two plastidial phosphorylases (PhoA and

PhoB) in Chlamydomonas reinhardtii which produces starch are shown to

function as homodimers containing two 91-kDa (PhoA) subunits and two 110-

kDa (PhoB) subunits (Dauvilleacutee et al 2006) Both of the Chlamydomonas SPs

lack the L-80 amino-acid insertion found in higher plant plastidial forms PhoB is

exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for

malto-oligosaccharides PhoA is moderately sensitive to ADP-glucose inhibition

and has a high affinity for unbranched malto-oligosaccharides which is similar to

the observation in higher plants (Dauvilleacutee et al 2006 Mu et al 2001) Further

the mutation in STA4 gene in Chlamydomonas reinhardtii display a significant

139

decrease in amounts of starch with abnormally shaped granules (Dauvilleacutee et al

2006) Similarly sh4 (shrunken4) mutant of maize displays a strong reduction

in starch content and this reduction was identified as lack of the SP cofactor

pyridoxal-5-phosphate (Tsai and Nelson 1969) However external addition of


affect the activity (Yu et al 2001) The product of the sh4 gene is still unknown

and this gene may or may not control the supply of pyridoxal phosphate (Yanase

et al 2006 Dauvilleacutee et al 2006 Burr and Nelson 1973 Yu et al 2001)

Formation of multi-subunit configurations of SP and direct involvement of

pyridoxal phosphate in this process in higher plants is yet to be investigated

The recombinant SP developed in this study consisting of catalytically active

dimeric and tetrameric forms were useful in understanding the different

biochemical and regulatory mechanisms of these structures

343 Investigating protein-protein interactions using immobilized

recombinant SP on S-Protein Agarose beads

The SP in maize amyloplast lysates exist as different conformational

structures the tetrameric dimeric forms (Liu et al 2009 Mu et al 2001)

However the relative availability quantity or the regulatory mechanisms of

these identical subunits in developing maize endosperm are not known The S-

tagged GPC fractions of tetrameric dimeric and monomeric forms obtained from

GPC analysis were used to analyze the protein- protein interactions of SP with

other enzymes The fractions were separately immobilized to S-protein agarose

beads after the fractions were incubated amyloplast lysates Immunoblot

140

analysis revealed that the tetrameric and dimeric forms were more strongly

immobilized to the beads The monomeric form present in the fractions may be

less than the other two forms and not strongly immobilized to agarose beads

The tetrameric form of recombinant SP interacted with SSIIa and SBEI

when the amyloplast extract was treated with 1 mM ATP but not in the

untreated amyloplast lysates or APase treated samples suggesting a

phosphorylation-dependent interaction In contrast no interaction was detected

with SBEIIb and the tetrameric form ATP-treated SBEI and SBEIIb strongly

interact with the dimeric form Similar interactions were found in wheat

amyloplast lysates SBEI and SBEIIb interacted with SP in a phosphorylation-

dependent manner (Tetlow et al 2004) However the multimeric status of SP

involved in these interactions in wheat amyloplast lysates is not known SSIIa

interacted with the dimeric form only in the untreated amyloplast lysates

Further the interaction between SBEI and the dimeric form of SP was

independent of ATP treatment APase-treated samples did not show any

interaction with any of the enzymes tested The isoforms of the major enzymes

involved in starch biosynthesis are regulated by protein phosphorylation

protein-protein interaction in maize amyloplast stroma (Liu et al 2009 Hennen-

Bierwagen et al 2008) Experiments in which isolated maize endosperm

amyloplasts were incubated with [γ-32P]-ATP provide direct evidence for

phosphorylation of SP (Pollack 2009) The heteromeric complexes of starch

synthesis enzymes assembled in the amylose extender (ae) mutant (lacking

SBEIIb) in maize amyloplast stroma was found by Liu et al (2009) The complex

formed by SSI SSII with SBEIIb in wild-type was replaced by forming SBE1

141

combined with SP in ae mutant (Liu et al 2009) The assortment of different

multimeric forms in the wild-type stroma may be available in different

compositions that may prevent interactions or the level of interactions may be

weak and not detectable

Functional interactions observed between SP and SBE isoforms in rice

endosperm suggested the mutual capacities for chain elongation and chain

branching (Nakamura et al 2012) The activity of SP and SBE isoforms also

depended on the mutual availability of each group of enzyme and purified SP

from rice endosperm could synthesize glucans from G-1-P in the presence of

SBE even without any exogenous glucan primer (Nakamura et al 2012) In vitro

glucan production was higher when SBEI presence compared to SBEIIa or

SBEIIb (Nakamura et al 2012) Functional and physical interactions between

SBE isoforms and SP (Nakamura et al 2012 Tetlow et al 2004 Ball and Morell

2003) and the high affinity towards low molecular malto oligosaccharide (MOS)

(Mu et al 2001) suggested that SP acts on MOS which are liberated by the

activity of debranching enzymes (DBE) to produce linear maltodextrin (MD) of a

length sufficient for a subsequent branching reaction by starch branching

enzymes (SBE) (Nakamura et al 2012 Ball and Morell 2003) Therefore direct

interactions between SP and SBE isoforms different multimeric forms interact in

unique and selective manner and phosphorylation and dephosphorylation of

these multimeric forms may play a major role in starch biosynthesis by

controlling the catalytic activity and assembly of protein complexes

Reduced numbers of Less number of starch granules with increased

granule surface observed in ss4 and ss4sp mutants in Arabidopsis leaves

142

suggested that SP may be involved in granule initiation in starch biosynthesis

process via any kind of functional or physical interaction between SP and SSIV

(Roland et al 2008 Planchot et al 2008) Investigating possible interactions of

SP with SSIV was one of the major objectives of this study in order to elucidate

the regulation of SP ATP treated amyloplast lysates showed a weak interaction

between SSIV and SP (Chapter 2) Phosphorylation of SP may be a vital

requirement for this interaction since SSIV was not regulated by

phosphorylation However no strong interaction was detected between SSIV

and the dimeric and tetrameric forms of recombinant SP The reason may be

due to the small amounts of SSIV that were available in the assays or

recombinant SP forms were not sufficiently phosphorylated or these proteins do

not interact

Dimeric and tetrameric forms of SP showed higher activities in

amylopectin in both synthetic and degradative directions and degradative

activity was greater in phosphorylitic direction compared to synthetic direction

(Fig 313) Both multimeric forms show higher activity with highly branched

glucan substrates The Km of the tetrameric form in the phosphorylitic direction

was about 25 times greater with amylopectin compared to glycogen or

maltoheptaose indicating the lower affinity of the tetrameric form towards

highly branched large molecule substrates (Table 31) The affinity of dimeric SP

for amylopectin was smaller than the tetramer (Km was increased by 111 times

and by 3 times in glycogen compared to tetrameric form) indicating that

different multimeric forms have different affinity levels for similar substrates

The reaction of the dimeric form in the phosphorylitic direction was not detected

143

in the given range of substrate concentrations of maltoheptaose that were

tested

Previous work with purified SP from maize amyloplasts recorded that the



of SP in maize amyloplast lysates recorded in this study was approximately 7

and 58 times lower than with purified SP in phosphorolytic reaction (Mu et al

2001) Unlike the purified form of SP the activity of SP present in amyloplast

lysates is regulated by other starch biosynthetic enzymes and metabolites such

as Pi G-1-P and ADP-glucose present in the lysates (Tiessen et al 2011 Mu et

al 2001 Schupp and Ziegler 2004 Matheson and Richardson 1978) For


thought to control SP activity (Mu et al 2001 Schupp and Ziegler 2004)

However according to the findings of Hwang et al (2010) the incorporation of

[U14C]-G-1-P into starch was only partially affected by the concentration of Pi in

rice Even under physiological G-1-P substrate levels (02 mM) and a 50-fold

higher level of Pi in vitro than the normal physiological level the Pho1 from of

rice was able to carry out the biosynthetic reaction Partially purified

recombinant tetrameric and dimeric forms produced in this study had 115 times

greater and 088 times less Vmax compared to purified-SP by Mu et al (2001) in

phosphorolytic direction in amylopectin The activity of SP in recombinant

multimers may be changed by desalting of the extracts which was not carried

out in the experiments

144

The preference of SP for different α-glucans has been recorded in many

plant species (Schupp and Ziegler 2004 Mu et al 2001 Yu et al 2001 Young

et al 2006 Mori et al 1993) Differentiating from maize SP in sweet potato

tubers plastidial SP showed a low binding affinity toward starch and a high

affinity toward low molecular weight linear MOS (Young et al 2006) In contrast

the cytosolic isoform has a high affinity towards highly branched polyglucan

amylopectin (Young et al 2006) The synthetic activity of SP to amylopectin

(Km =013) is higher when compared to the highly branched glycogen

(Km=045) in maize (Yu et al 2001) in potato tubers (Liddle et al 1961) and in

spinach leaves (Shimomura et al 1982) In sweet potato tubers the unique L-

78 amino acid peptide insertion located in the middle of plastidial form of SP

appears to block the binding site of SP to high molecular weight α-glucans

(Young et al 2006) However it was not observed in maize amyloplast SP

whether the L-78 insertion influences the kinetics of the enzyme In fact there is

no clear evidence for cleavage of L-78 in plastidial isoforms of SP from cereals

In this chapter experiments were carried out to elucidate the regulation of

SP in maize amyloplasts SP is a stromal enzyme and remains active throughout

the endosperm development and exists as homodimeric or homotetrameric

configurations throughout the developmental stages This study suggested that

the tetrameric and dimeric forms have different catalytic activities and may be

involved in starch biosynthesis by being regulated differently from each other

The synthetic and phosphorolytic activity assays showed that the SP multimers

are variously active in both directions SP showed greater activities with

amylopectin compared to glycogen and maltoheptaose in both synthetic and

145

phosphorylitic directions ATP-treated SP showed higher activities in both

directions in amylopectin substrate indicating that ATP may be involved in

regulating the SP through protein phosphorylation However the protein-protein

interactions could not be detected by co-immunoprecipitation as the native SP

could not be immunoprecipitated by SP-antibodies and Protein-A sepharose

beads This may be due to lack of accessibility of the epitopes in the dimeric

and tetrameric configurations Therefore the development of S-tagged

recombinant SP was used for analyzing protein-protein interactions of SP

146

CHAPTER 4

147

Biochemical Investigation of the Regulation of Starch Synthase IV in Maize Endosperm

41 Introduction

The glucan synthases catalyze the elongation of glucan chains by

transferring a glucosyl moiety to the non-reducing end of an α-(1-4)-linked

glucan primer Glucan synthases are found in both eukaryotes and prokaryotes

but the most intensively studied enzyme in this class is glycogen synthase which

is responsible for α-glucan elongation of glycogen (Szydlowski et al 2011 Ball

and Morell 2003 Roach 2002 Cao et al 1999 Denyer et al 1995 Madsen

1991 Preiss 1988 Preiss 1984) Glycogen is a water-soluble polyglucan that is

produced in mammals fungi bacteria cyanobacteria and archaebacteria (Ball

and Morell 2003 Roach 2002 Furukawa et al 1993 Furukawa et al 1990) In

contrast starch is a water-insoluble polyglucan produce in plants green algae

and some cyanobacteria (Nakamura et al 2005 Ball and Morell 2003) One of

the principle differences between glycogen and starch synthesis is the nucleotide

sugar substrate of the glucan synthases involved in biosynthesis UDP-glucose is

the glucan donor for glycogen synthesis (Leloir et al 1961) and ADP-glucose is

the substrate of starch synthesis (Nakamura et al 2005 Ball and Morell 2003

Roach 2002) Structurally glycogen is distinct from starch in that glycogen is

water-soluble and possesses a higher degree of branching (10) and has a

more open structure that expands in a globular fashion (Ball and Morell 2003

Roach 2002) Starch is characterized by clustered branch points (about 6 of

branching frequency) creating a water-insoluble granule (Manners 1989)

Several isoforms of starch synthases and branching enzymes are found in plants

148

whereas glycogen synthase and glucan branching enzyme each exist as a single

isoforms (Fujita et al 2011 Ball and Morell 2003)

The starch synthesized in higher plants consists of two types of glucose

polymers amylose and the amylopectin Amylose is a sparsely branched linear

molecule found to be about 1000 to 50000 glucose units whereas highly

branched amylopectin has 105ndash106 glucose units Both amylose and amylopectin

are elongated by the starch synthases (SS) by transferring the α-D-glucose

units from ADP-glucose the precursor of the starch biosynthesis to the non

reducing end of the glucan chain (Leloir et al 1961) Five major isoforms of

starch synthases (SS) have been recorded in higher plants SSI SSII SSIII

SSIV and GBSS (Tetlow 2011 Ball and Morell 2003) GBSS is essential for

amylose synthesis and is exclusively bound to the starch granule (Nakamura et

al 1993 Sano 1984 Echt and Schwartz 1981) SSI SSII SSIII and SSIV

isoforms are thought to be responsible for amylopectin synthesis (Dian et al

2005 Denyer et al 1999 Gao et al 1998 Denyer et al 1995) Mutant analysis

and biochemical studies have shown that each class of SS has a distinct role in

the synthesis of amylopectin (Nakamura 2002 Fontaine et al 1993 Morell et

al 2003) SSI is responsible for producing DP= 8-12 glucan chains (Commuri

and Keeling 2001) SSII and SSIII respectively produce 12-25 (Zhang et al

2004 Morell et al 2003) and DP= 25-40 or greater glucan chains in

amylopectin (Tomlinson and Denyer 2003) However there is little information

available about the functionrole of SSIV The role and the regulation of the

SSIV in storage starch biosynthesis are largely unknown The different isoforms

of starch biosynthetic enzymes are differentially expressed at different stages of

149

endosperm development in cereals (Dian et al 2005) The SSIIa SSIII-1 and

SBEIIa are expressed in early stage and SSI SSII-3 SSIII-2 and SBEIIb are

expressed in the middle stage of the grain filling and GBBSSI SSIV and SBE1

are differentially expressed at the later stage of the grain filling in cereals (Liu et

al 2009 Fujita et al 2006 Dian et al 2005 Morell et al 1997 Gao et al

1996)

Sequence analysis revealed that there are some similarities and

differences of the SSIV in different plant species (Leterrier et al 2008 see

figure 41) The predicted protein sequence of SSIV in maize endosperm is 104

kDa in size and has a highly conserved C-terminal region compared with other

SSs The C-terminus contains the catalytic and the starch-binding domains of

SSs (Cao et al 1999) In common with other SSs (Fig 15 and Fig 41) the N-

terminal region of SSIV is different from other SS isoforms (Leterrier et al

2008) (Fig 42) In addition two coiled-coil domains were found in the SSIV-

specific region from amino acids 1ndash405 which are thought to be involved in

protein-protein interactions (Leterrier et al 2008 Jody et al 2004) (Fig 42)

14-3-3-protein recognition sites [RKXXpSXP and RKXXXpSP Muslin et al

(1996)] are also found in the N-terminal region of SSIV and may be involved in

the regulation of the enzyme [14-3-3 proteins are commonly linked to binding

with various signaling proteins such as kinases and phosphatases and act as

lsquoadaptor proteinsrsquo in various phosphorylation-dependent protein-protein

interactions (Comparot et al 2003)] (Fig 42) Expression of SSIV is tissue-

dependent and found to be highest in non-endosperm tissues such as in leaf

embryo and roots in wheat and the level of expression in the endosperm was

150

relatively low independent from the regulation of the circadian clock Therefore

the transcript accumulation levels did not coincide with the period of high carbon

flux to starch in the wheat endosperm (Leterrier et al 2008)

SSIV is exclusively localized in the amyloplast stroma (Leterrier et al

2008 Roldan et al 2007) Two different genes the OsSSIV-1 was expressed in

the endosperm and OsSSIV-2 was expressed in leaves in rice (Dian et al 2005)

In addition the cDNA sequence of SSIV expressed in developing wheat seedling

is similar to rice SSIV-2 and shares a similar exon-intron arrangement

(Leterrier et al 2008) These findings suggest that two different SSIV isoforms

may be responsible in transient and storage starch biosynthesis No such

different isoforms of SSIV have been identified in maize Further the SSIV

protein in Arabidopsis thaliana (11299 kDa in size Roldan et al 2007) shows

87 intron sequence identity to rice (OsSSIV-2 in leaves accession number

AY373258) (Leterrier et al 2008)

151


Ta -------MACS-------------AAAGVEATALLSPRCPAPSPPDGRSRRRLALASGTR 40

Os -------MAC--------------LAAGAEAAPLLFRRRLAPSPVAAR--RRLLVSCRAR 37

Zm PHPPRLPMSCS-------------AAAGAEATALLIR-SAAPSTIVGR--HRLAMSRRTS 90

At KGSPKPILSINSGLQSNNDEESDLENGSADSVPSLKSDAEKGSSIHGSIDMNHADENLEK 120

Ta HRSLRAAAQRPHKSATGAD--PLYNNRANVRSDEAS-------VSAEKERQRKYNDGDGI 91

Os RRGLRLVAQSAGSRGCGVVGAPGCDYWVNMQRDEAS-------VSSDKERQEKYGDENGI 90

Zm RRNLRTGVHPHQKSAPSAN----HRNRASIQRDRAS-------ASIDEEQKQMSEDENGL 139

At KDDIQTTEVTRRKSKTAKKKGESIHATIDIGHDDGKNLDNITVPEVAKALSLNKSEGEQI 180

Ta SNLKLEDLVGMIQNTEKNILLLNQARLQAMEHADKVLKEKEALQRKINILETRLSETDEQ 151

Os SNLQLEDLIQMIQNTEKNIMLLNQARLQALEHVETVLKEKEDLQRKLKILETRLSETDAR 150

Zm LDIQLEDLVGMIQNTQKNILLLNQARLQALERADKILKEKETLQQKINILEMKLSETGKQ 199

At SDGQFGELMTMIRSAEKNILRLDEARATALDDLNKILSDKEALQGEINVLEMKLSETDER 240

Ta HKLSSEGNFS----DS--------------------PLALELGILKEE--NILLKEDIEF 185

Os LKLSAEGQFGTEINDS--------------------LPVLELDDIKEENMETLLKDDIQF 190

Zm SVLSSEVKSD--------------------------EESLEFDVVKEE--NMLLKDEMNF 231

At IKTAAQEKAHVELLEEQLEKLRHEMISPIESDGYVLALSKELETLKLE--NLSLRNDIEM 298

Ta FKTKLIEVAEIEEGIFKLEKERALLDASLRELESRFIAAQADTMKLGPR----DAWWEKV 241

Os LKTMLIEVAETENSIFTLEKERALLDASLRELESRFIDAQADMLKSDPRQY--DAWWEKV 248

Zm LKGKLIEITETEESLFKLEKECALLNASLRELECTSTSAQSDVLKLGPLQQ--DAWWEKV 289

At LKSELDSVKDTGERVVVLEKECSGLESSVKDLESKLSVSQEDVSQLSTLKIECTDLWAKV 358

Ta EKLEDLLETTANQVEHAAVILDHNHDLQDRLDNLEASLQAANISKFSCS----LVDLLQQ 297

Os ENLGDLLETATNKVENAAMVLGRNHDLEDKVDKLEASLAEANISKFSCY----FVDLLQE 304

Zm ENLEDLLDSTANQVEHASLTLDGYRDFQDKVDKLKASLGTTNVSEFCLY----LVDILQQ 345

At ETLQLLLDRATKQAEQAVIVLQQNQDLRNKVDKIEESLKEANVYKESSEKIQQYNELMQH 418

Ta KVKLVEDRFQACNSEMHSQIELYEHSIVEFHDTLSKLIEESEKRSLENFTGNMPSELWSK 357

Os KIKSVEERFQVCNHEMHSQIELYENSIAEFHDILSKLVEETEKRSLEHSASSMPSELWSR 364

Zm RVKSVEERFQACNHEMHSQIELYEHSIVEFHGTLSKLINESEKKSMEHYAEGMPSEFWSR 405

At KVTLLEERLEKSDAEIFSYVQLYQESIKEFQETLESLKEESKKKSRDEPVDDMPWDYWSR 478

Ta ISLLIDGWLLEKKIAYNDASMLREMVRKRDSRLREAYLSYRGTENRDVMDSFLKMALPGT 417 Os ISLLIDGWLLEKRISYNDANTLREMVRKRDSCLREAYLSCRGMKDREIVDNFLKITLPGT 424 Zm ISLLIDGWSLEKKISINDASMLREMAWKRDNRLREAYLSSRGMEERELIDSFLKMALPGT 465

At LLLTVDGWLLEKKIASNDADLLRDMVWKKDRRIHDTYIDVKDKNERDAISAFLKLVSSPT 538

Ta SSGLHIAHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQVDQVSNLKV 477

Os SSGLHIIHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQNDQVNNLKV 484

Zm SSGLHIVHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQHNQINNLKV 525

At SSGLYVVHIAAEMAPVAKVGGLGDVVAGLGKALQRKGHLVEIILPKYDCMQYDRVRDLRA 598

Ta LDVLVQSYFEGNMFNNKIWTGTVEGLPVYFIEPQHPAMFFSRAQYYGEHDDFKRFSYFSR 537

Os LDVVVQSYFEGNLFNNKIWTGTVEGLPVYFIEPQHPAKFFWRAQYYGEHDDFKRFAYFSR 544

Zm LDVVVKSYFEGNMFANKIWTGTVEGLPVYFIEPQHPGKFFWRAQYYGEHDDFKRFSYFSR 585

At LDTVVESYFDGKLYKNKIWIGTVEGLPVHFIEPQHPSKFFWRGQFYGEQDDFRRFSYFSR 658

Ta AALELLYQSGKKVDIIHCHDWQTAFVAPLYWDVYANLGFNSARICFTCHNFEYQGTAPAR 597

Os AALELLYQSQKKIDIIHCHDWQTAFVAPLYWEAYANLGFNSARICFTCHNFEYQGAAPAQ 604

Zm VALELLYQSGKKVDIIHCHDWQTAFVAPLYWDVYANLGFNSARICFTCHNFEYQGIAPAQ 645

At AALELLLQSGKKPDIIHCHDWQTAFVAPLYWDLYAPKGLDSARICFTCHNFEYQGTASAS 718

CC

152

Ta DLAWCGLDVEHLDRPDRMRDNSHG-RINAVKGAIVYSNIVTTVSPTYALEVR-SEGGRGL 655

Os DLACCGLDVQQLDREDRMRDNSHG-RINVVKGAIVYSNIVTTVSPTYALEVR-SEGGRGL 662

Zm DLAYCGLDVDHLDRPDRMRDNSHG-RINVVKGAVVYSNIVTTVSPTYAQEVR-SEGGRGL 703

At ELGSCGLDVNQLNRPDRMQDHSSGDRVNPVKGAIIFSNIVTTVSPTYAQEVRTAEGGKGL 778

Ta QDTLKVHSRKFLGILNGIDTDTWNPSTDRYLKVQYNAKDLQGKAANKAALREQLNLASAY 715

Os QDSLKLHSRKFVGILNGIDTDTWNPSTDRHLKVQYNANDLQGKAANKAALRKQLNLSSTN 722

Zm QDTLKVHSKKFVGILNGIDTDTWNPSTDRFLKVQYSANDLYGKSANKAALRKQLKLASTQ 763

At HSTLNFHSKKFIGILNGIDTDSWNPATDPFLKAQFNAKDLQGKEENKHALRKQLGLSSAE 838

Ta PSQPLVGCITRLVAQKGVHLIRRAIYKTAELGGQFVLLGSSPVPEIQREFEGIADHFQNN 775

Os ASQPLVGCITRLVPQKGVHLIRHAIYKTAELGGQFVLLGSSPVPHIQREFEGIADHFQNN 782

Zm ASQPLVGCITRLVPQKGVHLIRHAIYKITELGGQFVLLGSSPVQHIQREFEGIADQFQNN 823

At SRRPLVGCITRLVPQKGVHLIRHAIYRTLELGGQFVLLGSSPVPHIQREFEGIEQQFKSH 898

Ta NNIRLILKYDDALSHCIYAASDMFVVPSIFEPCGLTQMIAMRYGSVPIVRKTGGLNDSVF 835

Os NNIRLLLKYDDSLSHWIYAASDMFIVPSMFEPCGLTQMIAMRYGSVPIVRKTGGLNDSVF 842

Zm NNVRLLLKYDDALAHMIFAASDMFIVPSMFEPCGLTQMVAMRYGSVPVVRRTGGLNDSVF 883

At DHVRLLLKYDEALSHTIYAASDLFIIPSIFEPCGLTQMIAMRYGSIPIARKTGGLNDSVF 958

Ta DFDDETIPMEVRNGFTFVKADEQGLSSAMERAFNCYTRKPEVWKQLVQKDMTIDFSWDTS 895

Os DFDDETIPKELRNGFTFVHPDEKALSGAMERAFNYYNRKPEVWKQLVQKDMRIDFSWASS 902

Zm DLDDETIPMEVRNGFTFLKADEQDFGNALERAFNYYHRKPEVWKQLVQKDMKIDFSWDTS 943

At DIDDDTIPTQFQNGFTFQTADEQGFNYALERAFNHYKKDEEKWMRLVEKVMSIDFSWGSS 1018

Ta ASQYEDIYQKAVARARAVA--- 914

Os ASQYEDIYQRAVARARAAA--- 921

Zm VSQYEEIYQKTATRARAAA--- 962

At ATQYEELYTRSVSRARAVPNRT 1040

Figure 41 Amino acid sequence alignment of SSIV in different plant species

Ta- Triticum asetivum (GenBank DQ4004161) At- Arabidipsis thaliana

(GenBank FW3015601) Os- Oryza sativa (GenBank FB7025731) Zm- Zea mays (GenBank AAC197339) The epitope for the peptide specific anti-SSIV antibodies of maize is highlighted in red The coiled-coil domain (CC) and the

conserved catalytic domains in the Cndashterminal region (K-V-G-G-L and K-T-G-G-K) are shown in blue boxes

153

Figure 42 A schematic diagram showing the major domains found within the predicted amino acid sequence of SSIV in wheat endosperm The starch catalytic domain (GT-5) and glycosyltranferase domain (GT-1) characteristic of the SS

family are shown Predicted 14-3-3 recognition sites and the coiled-coil domains (blue boxes and CC respectively) as well as the two highly conserved KVGGL

and KTGGL domains are also shown (Leterrier et al 2008)

Although the involvement of SSIV in glucan chain length elongation is not clear

the growth rate in the mutant alleles of ss4 in Arabidopsis thaliana was

decreased without changing total SS activity (Roldan et al 2007) Further the

starch content was deceased by 35-40 in the mutant lines while the size of

silique number of seeds per silique and germination ratios remained unchanged

(Roldan et al 2007) Interestingly the total activity of starch phosphorylase

(SP) was increased by 14ndash2-fold in both cytosolic and plastidial forms in

Arabidopsis ssiv mutants (Roldan et al 2007) More importantly the

amyloseamylopectin ratio or the structure of the starch were not altered in the

ss4 mutants the starch granule surface area was increased by 15 times and by

4 times in ss4sp double mutants indicating the increase in granule size

(Planchot et al 2008) In contrast the number of granules per chloroplast

14-3-3 14-3-3

154

decreased to 2-3 in ss4 single mutants where as the wild-type contains

contained 4ndash5 starch granules per chloroplast Interestingly the double mutants

of ssivsp had 1-2 granules per chloroplast (Planchot et al 2008) These

observations suggested that the SSIV potentially interacts (either functionally or

physically) with SP and both are involved in the priming of the starch granule

(Planchot et al 2008 Roldan et al 2007) The mechanism of starch granule

initiation is largely unknown (DrsquoHulst et al 2010 DrsquoHulst and Merida 2012)

The homologous double mutants of starch synthases produced in

Arabidopsis thaliana (ss1ss4 ss2ss4 and ss3ss4) are helpful in understanding

the interactive role of SS in starch biosynthesis (Szydlowski et al 2009) Starch

accumulation deceased in ss1ss4 and ss2ss4 double mutants equal to the sum

of the decreased starch levels in their respective single mutant lines However

starch accumulation in the single mutants of ss4 and ss3 were recorded as

122 (Zhang et al 2005) and 62 (Rolden et al 2007) respectively compared

to their wild- types at the end of 12h light period However the double mutant

of ss3ss4 did not accumulate any measurable amounts of starch irrespective of

light conditions (Szydlowski et al 2009) Therefore the presence of either SSIII

or SSIV appears to be a crucial requirement in transient starch biosynthesis

(Szydlowski et al 2009) In addition the significant increase in the activity of SP

in the ss3ss4 double mutants suggested the existing of alternative SP-mediated

starch biosynthetic pathway using hexose phosphates as glycosyl donors

(Szydlowski et al 2009 Fettke et al 2010)

The investigations discussed in this chapter tested the hypothesis that

SSIV is involved in storage starch biosynthesis in maize amyloplasts and that

155

the enzyme is regulated by protein phosphorylation and protein-protein

interactions The cellular localization and biochemical analyses were performed

to characterize and understand the regulatory mechanism of the enzyme

Recent evidence from Arabidopsis thaliana suggested that SP and SSIV may

physically andor functionally interact and may be involved in priming the starch

granule The possible interactions of SSIV specifically with SP and with other

starch biosynthetic enzymes were tested in maize amyloplast stroma

156


421 Analysis of the localization of SSIV in the plastid

To investigate the localization of SSIV in the amyloplast amyloplasts were

isolated and the soluble and granule bound proteins and plastid envelop

membrane proteins were separated from 22 DAA (days after anthesis) old maize

endosperms as described earlier in chapter 2 The presence of SSIV and other

SS isoforms SSI SSII and SSIII in the amyloplast stroma and the granule was

determined by running the protein extracts on 10 SDS gels and the

immunoblotted proteins were identified using peptide-specific anti-maize

antibodies The purified SSIV antibody generated using the synthetic peptide

ANHRNRASIQRDRASASI from the first bleed serum developed in rabbit was used

after dilution by 1800 in 15 BSA (antibodies were purified as described in

chapter 2) The procedures for SDS-PAGE and immunoblot analysis were as

described in chapter 2

422 Determination of the protein expression of SSIV in developing

endosperm

The equal amounts of proteins from the amyloplast lysates extracted from

the maize kernels at 12 15 17 22 DAA were run on 10 SDS gels Following

the electrophoresis the immunoblots were probed with peptide specific SSIV

antibodies

157

423 Determination of SSIV catalytic activity by zymogram analysis

Zymogram analysis was performed to estimate the activity of SSIV and

other SS isoforms of amyloplast stroma following incubation of the lysates with

ATP or APase to respectively phosphorylate and dephosphorylate amyloplast

proteins

SS zymograms were carried out according to the methods described by

(Tetlow et al 2004) 90 μg of proteins were run in a well after gels were

prepared as native 5 (wv) polyacrylamide gels in 375 mM TRIS-HCl pH 88

and 10 mg of the α-amylase inhibitor Acarbose (Bayer lsquoPrandasersquo) and 03

(wv) rabbit liver glycogen (type III Sigma-Aldrich) The gel was run using

025M Tris 192 mM glycine running buffer without SDS at 120V for 15hr in the

cold room After electrophoresis the gel was incubated for 48ndash72 h in a buffer

containing 50 mM glycylglycine pH 90 100 mM (NH4)2SO4 20 mM DTT 5 mM

MgCl2 05 mg mL-1 BSA and 4 mM ADP-glucose

424 Substrate-affinity electrophoresis

Affinity electrophoresis was carried out as described earlier by Commuri

and Keeling (2001) using different glucan substrates at various concentrations

amylopectin glycogen and maltoheptaose (at 0 5 10 25 mgmL

concentrations) in the native gels Amyloplast lysates (22 DAA) were run on the

gel at a protein content of 30 microgmL per well The migration distances of specific

enzyme were measured after immunoblotting Affinity electrophoresis served as

a means of measuring protein-glucan interactions and the dissociation

constants (Kd) were calculated from the retardation of the electrophoretic

158

mobility of enzymeprotein by the substrate contained in the supporting

medium


GPC analysis was performed as described in Chapter 2 section 22243

426 Co-Immunoprecipitation of SSIV

In order to identify protein-protein interactions of SSIV and other starch

biosynthetic enzymes co-imunoprecipitation was performed with amyloplast

lysates of 22 DAA using the methods previously described in Chapter 2 section

222574 using peptide specific anti-SSIV antibodies

427 Phosphorylation of SSIV using -32P-ATP

Phosphorylation of SSIV was investigated by incubating 400 μL of

amyloplast lysate with 05 uCi of -32P-ATP in a final concentration of 1 mM ATP

on a rotator for an hour at 250C and then the SSIV was immunoprecipitated by

using SSIV specific antibodies bound to Protein-A sepharose beads following the

procedure described in section 424 in Chapter 2 Non-specifically bound

proteins were removed by washing the remaining pellet for eight times each

with 1 mL phosphate buffered saline (PBS) followed by three similar washes

with 10 mM HEPESKOH pH 75 buffer (at 100 g 1 min centrifugation)

Following washing the immunoprecipitated pellet was boiled in 2X SDS loading

buffer for 8 min and separated by SDS-PAGE Following electrophoresis proteins

in the gel were transferred to nitrocellulose membranes exposed to X-ray film

159

for two weeks at -800C The phosphorylation of SSIV was detected by alignment

of X-ray film with the developed immunoblot which was probed with anti-SSIV

specific antibodies

160

43 Results

431 Testing the specificity of peptide specific anti-SSIV antibodies

The SSIV isoform in maize is predicted to be 104 kDa based on its amino

acid sequence The SSIV-specific antibody (ANHRNRASIQRDRASASI) was

derived against amino acids located at position 55-72 at the N-terminal end of

full length amino acid sequence of maize SSIV (909 amino acids see figure 41)

(Accession number - EU990361) Immunoblots of the amyloplast lysates run on

SDS-PAGE were probed with purified SSIV antibodies and pre-immune serum to

detect the specificity of the purified antibodies in detecting SSIV (Fig 43A) The

purified anti-SSIV specific antibodies were subjected to a series of dilutions and

the optimal concentration of antibodies required to detect SSIV in amyloplast

lysate was 1800 dilution (Fig 43B)

432 Localization of SSIV

Localization of SSIV in maize amyloplast was investigated by

immunodetection using the peptide-specific antibodies to SSIV Analysis of the

proteins extracted from the wild-type amyloplast stroma and the loosely-bound

proteins from the starch granule at 22 DAA confirmed that SSIV is localized only

in the amyloplast stroma while SSI and SSII and in some cases SSIII can be

seen in both amyloplast stroma and as granule-associated proteins (Fig 44)

161

Figure 43 Immunoblots of amyloplst proteins probed with purified SSIV-specific antibodies (A) Purified anti-SSIV specific antibodies were diluted to

1800 11000 12000 and 15000 in 15 BSA to determine the optimal concentration of the antibodies required to detect SSIV (B)

MW MW MW MW

(A)

kDa

150

100

50

MW

(B)

162

Figure 44 Immunodetection of SSI SSII SSIII and SSIV in stroma and starch granules of wild-type maize amyloplasts at 22 DAA Amyloplast lysates (25 μg

proteins) were separated on 10 acrylamide gels electroblotted onto nitrocellulose membranes and developed with peptide-specific anti-maize antibodies The expected mass (predicted from the amino acid sequence) of

each protein is given below the respective immunoblot

433 Determination of the expression of SSIV in developing endosperm

Testing of equal amounts of proteins from the amyloplast lysates

extracted from the maize kernels at 12 15 17 22 DAA with the peptide specific

SSIV antibodies showed that the SSIV protein is expressed in the later stages of

endosperm development (Fig 45)

163

Figure 45 Immunodetection of SSIV at different stages of endosperm

development in maize wild-type amyloplasts Amyloplast lysates from 12 15 17 and 22 old endosperms were run (25 μg proteins per well) in SDS-PAGE and immunoblot was developed by the peptide specific anti-SSIV antibodies


different α-glucan substrates

The affinity of SSIV in amyloplast lysates for α-glucans was established by

affinity electrophoresis (Fig 46A) The amyloplast lysates (approximately 30 μg

proteins) were subjected to native PAGE in the presence of different

concentrations (0 05 1 25 mgmL) of amylopectin glycogen and

maltoheptaose (see Fig 46A) The relative migration (Rm) and then dissociation

constant (Kd) of the SSIV were calculated from the plot of the graph developed

by 1Rm vs substrate concentration as described by Commuri and Keeling

(2001) (Fig 46B) The SSIV showed a relatively higher Kd value in glycogen

(25 mgmL) followed by maltoheptaose (15 mgmL) and the amylopectin (10

mgmL) (Fig 54B) (Table 41)

164

Figure 46A A representative western blot of the native zymogram gel showing

the mobility of SIIV in different glucan substrates used to determine the relative mobility of the SSIV in amyloplast lysates The relative mobility of SSIV was determined by the transferring the native zymogram to nitrocellulose

membranes and probing with anti-SSIV antibodies The mean relative mobility (Rm) was determined as the ratio of the migration of the activity band and the

migration of the dye from three different experiments

(A)

Amylopectin (mgmL) Maltoheptaose (mgmL) Glycogen (mgmL)

0 05 1 25 0 05 1 25 0 05 1 25


0 05 1 25 0 05 1 25 0 05 1 25

165

Figure 46B Plots of the reciprocal of the relative mobility (1Rm) of maize SSIV against the concentration of different glucan substrates The dissociation constant (Kd) of SSIV is shown as the intersect at the X-axis

y = 04885x + 12983

000

100

200

300

-3 -25 -2 -15 -1 -05 0 05 1 15 2 25 3

1R

m

Substrate Concentration (mgmL)

Glycogen

y = 10576x + 11474

000

200

400

600

-25 -2 -15 -1 -05 0 05 1 15 2 25

1R

m


Amylopectin

y = 07772x + 11909

0

1

2

3

4

-25 -2 -15 -1 -05 0 05 1 15 2 25 3 35

1R

m


Maltoheptaose

(B)

166

Table 41 Comparison of Kd values of maize SSIV (from present study) with SSI SSIIa and SP in different glucan substrates SSI SSIIa and SP values were

estimated by Commuri and Keeling (2001)

Glucan

Substrate

Kd values (mgmL)

SSI

SSIIa

SP

SSIV

Amylopectin Starch Glycogen

Maltoheptaose

02004

049001 -

-

024001

049001 -

-

002001

008001 -

-

10001 -

25002

1507

435 Investigating the regulation of SSIV by phosphorylation using -

32P-ATP

Possible phosphorylation of SSIV was investigated by pre-incubating

amyloplast lysates with -32P-ATP immunoprecipitating SSIV with peptide-

specific antibodies and analyzing the immunoblots by autoradiography Figure

45 shows the developed nitrocellulose membrane of immunoprecipitated SSIV

after treatment of amyloplast lysates with -32P-ATP (47A) and the

autoradiograph developed from the same nitrocellulose membrane (47B) The

immunoblot developed by anti-SSIV antibodies showed that the SSIV was

successfully immunoprecipitated no SSIV band was detected in the remaining

supernatant after the SSIV was immunoprecipitated (Fig 47A) However the

autoradiograph did not show any radioactivity corresponding to SSIV indicating

that SSIV was not phosphorylated under these conditions (Fig 47B) (Apendix

06 shows the predicted phosphorylation sites of maize SSIV)

167


incubation of amyloplast lysates with -32P-ATP 400 μL of amyloplast lysates

(approximately 1 mg mL) were treated with final concentration of 05 uCi of -32P-ATP + 1 mM ATP mixture on a rotator for in hour SSIV was immunoprecipitated by using anti-SSIV specific antibodies After the non-

specifically bound proteins were removed the pellet was boiled in 1X SDS loading buffer for 8 min and run the SDS-PAGE The proteins in the gel were

transferred to nitrocellulose membranes and developed with anti-SSIV antibodies (A) and the autoradiograph was developed from the same membrane after the proteins were transferred to the X-ray film for two weeks at -800C (B)

The phosphorylation of SSIV was tested by aligning the X-ray film with the developed immunoblot with anti-SSIV specific antibodies Arrows indicate the

location of SSIV

(A) (B)

168

436 Determination of the activity of ATP or APase treated SSIV on

zymogram

ATP-treated or APase-treated SS activity was measured qualitatively by

an in-gel activity assay Maize amyloplast lysates were pre-incubated with 1mM

ATP and SS activity was detected on zymogram and compared with the

untreated amyloplast lysates (Fig 48A) The SS activity banding profile in

untreated amyloplast lysates was changed equally in ATP or ATP+PI

(PI=phosphatase inhibitor) treated samples PI was added to inhibit the activity

of endogenously available protein phosphatases No difference in the banding

pattern was observed between untreated PI treated and APase treated samples

Immunoblot analysis of the SS activity zymogram for SSI and SSII

activities showed that the samples treated with ATP (with or without PI) became

more mobile and therefore showed less affinity to the given glycogen

concentration (03) than in untreated PI treated and APase treated samples

However no clear band corresponding to SSI was seen in APase treated

samples (Fig 48A) The peptide-specific SSIII antibodies did not recognize the

SSIII in any treatment (Fig 48B) The synthetic activity corresponding to SSIV

was reduced when treated with APase and the activities were higher in ATP

treated samples when compared with untreated controls (Fig 48B) However

the mobility of SSIV indicated by immunoblots was similar in all treatments (Fig

48B) SSIV in amyloplast lysates was removed by immunoprecipitation with

anti-SSIV specific antibodies SSIV immunoprecipitation was used in conjugation

with zymogram analysis to understand the mobility of the enzyme and the

changing of overall SS activity profile in the absence of SSIV The zymogram

169

lacking SSIV showed loss of a major activity band (as indicated by the arrow) on

the zymogram (Fig 48C) in addition to at least two other minor (unidentified)

bands of SS activity

170

Figure 48 Zymogram analysis of SS activity in amyloplast lysates of wild-type

maize endosperm at 22 DAA Amyloplast lysates were separated (90 μg protein per well) on a native 5 acrylamide gels containing 03 (wv) glycogen and

developed for 48 h at in a buffer containing 4 mM ADP-glucose SS activities were visualized by staining with Lugolrsquos solution Identical gels were electroblotted onto nitrocellulose membranes and probed with peptide specific

anti-SSI SSIIa (A) SSIII and SSIV peptide specific antibodies (B)

(B)

(A)

171

Figure 48C The activity of SS in the amyloplast lysates in the absence of

SSIV The native SSIV was removed by immunoprecipitation with anti-SSIV specific antibodies bound to Protein A-sepharose beads and the remaining supernatant was run with amyloplast lysates to compare the relative position of

SSIV and to detect the change of SS activity profile of other starch synthases (C)

437 Gel filtration chromatography of SSIV

The amyloplast lysates at 22 DAA from wild-type maize were treated with

ATP and APase and separated through a Superdex 200 10300GL gel permeation

column The fractions were subjected to SDS-PAGE followed by immunoblot

analysis to identify the SSIV eluted fractions SSIV eluted in fraction numbers

2930 in all treatments of amyloplast lysates Approximate molecular weight of

the fraction that SSIV eluted was determined by eluting the standard proteins

with known molecular weights from the same column which is approximately at

100 kDa (Fig 49)

1 2

1 Amyloplast lysates after removal of SSIV by immunoprecipitation

2 Amyloplast lysates with SSIV

(C)

172

Figure 49 Gel filtration chromatography analysis of SSIV in amyloplast lysates 450 μg of total protein in a volume of 500 microL from each treatment was

separated by size exclusion chromatography (GPC) through a Superdex 200 10300GL gel permeation column The fraction numbers from 16 to 41 were run on SDS-PAGE followed by immunoblot analysis using peptide specific anti SSIV

antibodies The SSIV bands are shown by the arrows at 104 kDa The column was calibrated by protein standards with known molecular weights and predicted

molecular weights of the fractions are indicated in boxes L= protein marker C=amyloplast lysates before loaded in the column Arrows indicate the location of SSIV

L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L

440 kDa 232kDa

SSIV-Untreated

Control

SSIV- ATP

Treated

SSIV- APase

Treated

100kDa

kDa

150

100

75

150

100

75

150

100

75

Fraction Numbers

173

458 Detection of protein-protein interactions of SSIV by co-

immunoprecipitation

To investigate the protein-protein interaction of SSIV with other starch

biosynthetic enzymes the co-immunoprecipitation was performed with maize

wild-type amyloplast lysates at 22 DAA The SSIV antibodies (30 mgmL) were

used to immunoprecipitate the native SSIV protein from amyloplast lysates (1

mL) using Protein-A Sepharose beads Figure 410 shows immunoblots of

immunoprecipitated SSIV probed with SSIV (Fig 410A) and other peptide-

specific starch biosynthetic enzymes antibodies of SSI SSIIa SSIII SBEI

SBEIIb ISOI and SP (Fig 410B)

SSIV in amyloplast lysates was completely immunoprecipitated since no

SSIV was detected in the remaining supernatant (Fig 410A) There is no non-

specific binding to the beads and only the purified SSIV antibodies were bound

to the beads since no band was observed in the immunoprecipitation carried out

by using pre-immune serum (Fig 410A) When the immunoblots were incubated

with SSI SSII and SSIII no bands were detected from SSIV

immunoprecipitated beads (Lane 1 in Fig 410B) and the enzyme levels showed

in supernatants remained same after the pull down Similarly SSIV

immunoblots probed with SBEI SBEIIb and ISOI antibodies showed no bands

(Fig 410B) The SSIV immunoblot probed with anti-SP specific antibodies

showed no clear interaction of SSIV with SP (Fig 410B) The faint band

observed in SSIV-pulldown beads may be from non-specific bounding of SP to

the beads Therefore no clear protein-protein interactions were detected

recorded between SSIV and other starch biosynthetic enzymes tested under

174

these conditions (Fig 410) In addition co-immunoprecipitation experiments

were performed with amyloplast lysates treated with 1 mM ATP or 30U APase

No interactions between SSIV and other starch biosynthetic enzymes were

detected but a weak interaction was detected with SP when amyloplast lysates

were treated with ATP (Fig 411) (Appendix 07 shows the Co-

immunoprecipitation of stromal proteins from wild-type maize amyloplasts using

peptide specific anti-SBEIIb antibodies to investigate the protein-protein

interactions of SBEIIb with SSIV and SP No interaction was detected between

SBEIIa and SSIV or SBEIIa and SP)

175

Figure 410A Immunoprecipitation of stromal SSIV from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the

protein-protein interactions 1 ml amyloplast lysates (1 mgmL) prepared from wild-type maize endosperm at 22 DAA were incubated with peptide-specific anti-

SSIV antibodies (30 mgmL final concentration) at room temperature for 1 hr and then immunoprecipitated with Protein-A-Sepharose beads The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS-

loading buffer and 30 μL was loaded onto 10 SDS gels Immunoblotted membrane was developed with maize anti-SSIV antisera (A) Arrow indicates

the immunoprecipitation of SSIV using SSIV specific antibodies The IgG is approximately showed at 50 kDa as a large thick band

(A)

176

Figure 410B Co-Immunoprecipitation of of stromal proteins from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the

protein-protein interactions SSIV in maize amyloplast lysates was immunoprecipitated by peptide-specific anti-SSIV antibodies (30 mgmL final concentration) with Protein-A-Sepharose beads (Fig 410A) and the washed

Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS-loading buffer and 30 μL was loaded onto 10 SDS gels Immunoblotted

membranes were developed with various anti-maize antisera as shown Arrow indicates the expected position of different starch biosynthetic enzymes in the Protein-A-Sepharose-antibody-antigen complexes The MW of the enzymes are

SSI at 74 kDa SSIIa at 85 kDa SBEI at 80 kDa SBEIIb at 85 kDa SP at 112 kDa and Iso-1 at 80 kDa The IgG is approximately showed at 50 kDa as a

large thick band

(B)

177

Figure 411 Co-immunoprecipitation of ATP or APase treated stromal proteins

from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the protein-protein interactions of SSIV with other starch

biosynthetic enzymes 1 ml amyloplast lysates (1 mgmL) prepared from wild-type maize endosperm at 22 DAA were incubated by adding 1mM ATP and APase

(25 unitml) for 1 hr and incubated further with peptide-specific anti-SSIV antibodies (30 mgmL final concentration) at room temperature for 1 hr The SSIV was immunoprecipitated with Protein-A-Sepharose beads The washed


membranes were developed with various anti-maize antisera as shown Arrows indicate the immunoprecipitation of SSIV using SSIV specific antibodies and the enzymes at SSI at 74 kDa SSIIa at 85 kDa SBEI at 80 kDa SBEIIb at 85 kDa

and SP at 112 kDa The IgG is seen at 50 kDa as a large thick band

Anti- SSIV

Anti- SSI

Anti- SSII

Anti- SBEI

Anti- SBEIIb

Anti- SP

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

L Protein marker

1 SSIV Co-IP+ATP-treated amyloplast lysates in beads

2 SSIV Preimmune Co-IP+ATP in beads

3 SSIV Co-IP+ATP supernatant

4 SSIV Co-IP+APase-treated amyloplast lysates in beads

5 SSIV Preimmune Co-IP+APase in beads

6 ATP treated amyloplast lysates (No SSIV antibody) in beads

7 APase treated amyloplast lysates (No SSIV antibody) in beads

8 Amyloplast lysate

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4 4 Discussion

Maize SSIV (Accession number ndash EU5990361) is the most recently

identified isoform of starch synthases and 104 kDa in size (Yan et al 2008)

Among the isoforms of starch synthases (SS) GBSS is essential for amylose

synthesis and is exclusively bound to the starch granule where as SSI SSII and

SSIII isoforms are found to be responsible for amylopectin biosynthesis (Ball

and Morell 2003) All isoforms are unique and probably play a distinct role in

the synthesis of amylopectin SSI is responsible for producing DP 8-12 glucan

chains (Nakamura 2002) SSII and SSIII respectively produce DP=12-25

(Zhang et al 2004 Morell et al 2003) and DP 25-40 or greater glucan chains in

amylopectin (Tomlinson and Denyer 2003) However the biochemical role of

SSIV in storage starch biosynthesis remains unclear The investigations

discussed in Chapter 4 are based on the hypotheses that SSIV in maize

regulates starch synthesis through the interactions between other starch

synthesis related enzymes by the formation of phosphorylation-dependent

protein complexes This study also tested the hypothesis that the SSIV and SP

proteins interact To investigate the role and regulation of SSIV its localization

and various biochemical characterizations were carried out

Immunodetection of SSIV indicated that the enzyme is exclusively

localized in the amyloplast stroma of the wild-type maize endosperm as similarly

observed in wheat endosperm by Leterrier et al (2008) (Fig 44) SSIV was not

detected as a granule bound protein in the starch granules SSI and SSIIa

isoforms are found both in the stroma and as granule bound proteins (Grimaud

et al 2008 Mu-foster et al 1996 Rahman et al 1995) However the

179

antibodies were not able to detect the SSIII in the granule The granule

association of the SSIV was investigated mostly in the Arabidopsis thaliana

chloroplast where transient starch is synthesized (Roldan et al 2007 Szydlowski

et al 2009) SSIV is thought to be a vital requirement to determine the correct

number of starch granules found in chloroplasts of Arabidopsis leaves and has

been suggested to be involved in granule initiation (Szydlowski et al 2009

Planchot et al 2008 Roldan et al 2007 DrsquoHulst and Merida 2012) However

loss of SSIV does not completely prevent starch granule formation in

chloroplasts suggesting that other factors may be involved in initiation process

other than SSIV Mutation in both SSIV and SSIII completely prevent starch

biosynthesis indicating a mutual requirement for SSIII and SSIV in starch

biosynthesis in the chloroplasts (Szydlowski et al 2009) To investigate the

process of granule initiation by SSIV the specific localization of SSIV in the

chloroplast was investigated by the florescence microscopic techniques

(Szydlowski et al 2009) The results indicated that the SSIV has a plastidial

localization and is present among the grana in the central part of the

chloroplast as well as in the grana-free peripheral part of the chloroplast

Further SSIV is not uniformly distributed within the stroma but was mainly

located in specific regions at the boundary of starch granules (Szydlowski et al

2009) Immunoblot analysis indicated that no SSIV was found inside the

granules in Arabidopsis leaves (Szydlowski et al 2009)

In this study SSIV was shown to be expressed at later stages of

endosperm development (Fig 45) Immunodetection of proteins from

amyloplast lysates extracted from maize kernels at 12 15 17 22 DAA showed

180

that SSIV is expressed at greater levels in the later stage of endosperm

development (Fig 45) In rice endosperm a greater level of SSIV-1 expression

was found after 14 DAP (Dian et al 2005) SSIIa and SSIII are expressed in

early stage (8 DAA) (Dian et al 2005) and SSI in maize is expressed in the

middle stage (16-20 DAA) of endosperm development (Cao et al 1999) and

studies in other plants indicate that different SS isoforms are expressed at

different developmental stages (Dian et al 2005) In chapter 2 it was reported

that SP is actively expressed thoughout the various developmental stages in

maize endosperm Since the later stage of endosperm development shows

higher levels of SSIV expression SSIV may have other catalytic andor

regulatory roles in starch biosynthesis other than the proposed function of

granule initiation This idea is supported by the sequence analysis of SSIV in

maize endosperm The highly conserved C-terminal region of SSs has the

catalytic and the starch-binding domains (Cao et al 1999) (Fig 15) The C-

terminal domain of SSIV is conserved with other SS isoforms but the N-terminal

domain of SSIV protein is unique in cereal endosperms (Fig 15) SSIV in wheat

(at 1-405 specific region) has two coiled-coil domains which are thought to be

involved in protein-protein interactions (Leterrier et al 2008 Jody et al 2004)

The 14-3-3-proteins are commonly linked to binding with various signaling

proteins such as kinases and phosphatases (Comparot et al 2003) and the N-

terminus of SSIV in wheat has recognition sites for 14-3-3 proteins (Leterrier et

al 2008) which are conserved in other SS isoforms in barley (Alexander and

Morris 2006) and Arabidopsis (Sehnke et al 2001) Arabidopsis chloroplast

SSIII contains a conserved phosphoserine binding motif (RYGSIP) identifying it

181

as a putative target for binding with 14-3-3 proteins (Sehnke et al 2001)

Moreover GBSSI SSI SSII and SBEIIa were shown to interact with 14-3-3

proteins in wheat amyloplasts (Alexander and Morris 2006)

Determination of the affinity of the SSs in amyloplast lysates for different

α-glucan substrates was important characteristic to discriminate between the

different SS isoforms (Commuri and Keeling 2001) The substrate-enzyme

dissociation constant (Kd) is inversely proportional to the affinity of the enzyme

to the substrate The affinity of SSIV towards different glucan substrates in

different concentrations was evaluated in terms of relative migration (Rm) and

the dissociation constant (Kd) (Fig 46AB) The results indicated that the SSIV

has relatively higher affinity to amylopectin (Kd=10 mgml) compared to

glycogen (Kd=25 mgml) and maltoheptaose (Kd=15 mgml) (Fig 46B) In

previous studies the affinity of SSI and SSII was found to be higher in

amylopectin compared to starch (Kd=02 and 049 mgml respectively)

(Commuri and Keeling 2001) (Table 41) Deletion of the N-terminal arm of

maize SSI did not affect the Kd value suggesting that the starch-affnity domain

of SSI is probably associated with or close to the catalytic domain at the C-

terminus (Cao et al 1999 Leterrier et al 2008) SSI and SSII elongate different

lengths of glucan chains but share similar affinities towards amylopectin (Cao et

al 1999) Affinity of SSIII (Km=428 mM) and SSIV (Km=096 mM) for ADP-

glucose was varied in Arabidopsis chloroplasts suggesting that the substrate

binding capacity may be different in different isoforms of SSs (Valdez et al

2008) Phylogenic analysis showed that the SSI SSII and GBSSI have distinct

evolutionary origins compared with SSIIIs and SSIV which have similar

182

evolutionary origins (Leterrier et al 2008) However the valine residue in the

common K-V-G-G-L substrate binding domain in evolutionary related SSIII and

SSIV may be different in primersubstrate binding capacities than the other SS

isoforms (Leterrier et al 2008) A slight reduction in the number of shorter

glucan chains (DP 7-10) in the starch of Arabidopsis SSIV mutants compared to

the wild-type indicated that SSIV may involved in producing shorter glucan

chains (Roldan et al 2007)

Protein phosphorylation has been shown to play an important role in the

regulation of enzymes involved in storage starch biosynthesis (Liu et al 2009

Hennen ndashBierwagen et al 2008 Tetlow et al 2008 Tetlow et al 2004) The

effect of protein phosphorylation on the activity of SSIV was investigated by

native affinity zymograms (Fig 48AB) Zymogram analysis of the activity of

SSIV indicated that the mobility of the protein was not altered following

treatment with ATP or APase However the activity of SSIV (based on

zymogram analysis) was reduced when treated with APase compared to ATP-

treated and untreated samples (Fig 48B) SSIV was not found to be

phosphorylated when the protein was tested with pre-incubated amyloplast

lysates with -32P-ATP (Fig 47) Therefore the reduction in the activity by APase

treatment may be due to the indirect effect of dephosphorylation of another

protein(s) that causes alterations in protein folding or has other regulatory

effects on SSIV Protein phosphorylation was identified as a mechanism for

regulating starch biosynthesis in developing wheat endosperm by Tetlow et al

(2004) and starch branching enzymes SBEI SBEIIa and SBEIIb and also SP in

amyloplast stroma were phosphorylated and further involved in protein-protein

183

interactions forming phosphorylation-dependent multi sub-unit complexes In

wheat endosperm amyloplasts protein phosphorylation enhanced the activity of

SBEIIb whereas dephosphorylation using alkaline phosphatase (APase) reduced

its catalytic activity (Tetlow et al 2004) The granule bound SS isoform of SSIIa

was also found to be phosphorylated (Tetlow et al 2004) In maize amyloplast

stroma two functional protein complexes one containing SSI SSII and another

containing SSII SSIII SBEIIa and SBEIIb were identified confirming the

phosphorylation-dependent physical interactions between SSs and SBEs

(Hennen ndashBierwagen et al 2008 Liu et al 2009) In zymogram analysis (Fig

48AB) SSI and SSII had less mobility in the gel than SSIV and formed dense

activity band in ATP-treated samples which could be due to the formation of

protein complexes In wheat and maize endosperms SSIIa can be

phosphorylated and a high molecular weight functional protein complex

consisting of SS isoforms (SSI SSIIa SSIII) and SBEs (SBEIIa and SBEIIb)

formed which showed higher affinity towards glucan substrate than the

respective monomers (Liu et al 2009 Hennen-Bierwagen et al 2008 Tetlow et

al 2008) GPC analysis showed no difference in the elution of SSIV when

amyloplast lysates were treated with ATP or APase (compared to untreated

samples) and SSIV eluted in fractions suggestive of a monomeric protein (Fig

49)

Mutant analysis in Arabidopsis suggests that SSIV in chloroplasts is

involved in starch granule formation since distruption of this enzyme resulted in

lower numbers of starch granules per chloroplast with increased granule sizes

(Roland et al 2008 Planchot et al 2008) This work suggested a possibility of a

184

functional or physical interaction between SP and SSIV (Roland et al 2008

Planchot et al 2008) In co-immunoprecipitation experiments amyloplast

lysates treated with 1 mM ATP detected interaction between SSIV and SP (Fig

411) Since SSIV was not phosphorylated by ATP under given experimental

conditions (Fig 47) phosphorylation of SP may be required to drive this

interaction (Fig 411) No protein-protein interaction was found between SSIV

and SSI SSIIa SSIII SBEI SBEIIb or ISOI in amyloplast lysates under these

experimental conditions (Fig 410B)

Recent work on the investigation of transient starch biosynthesis in

Arabidopsis thaliana suggests that SP and SSIV may interact and may be

involved in priming the starch granule (Roldan et al 2007 Planchot et al

2008 (Szydlowski et al 2009 Zhang et al 2005) The presence of either SSIII

or SSIV is recognized as a vital requirement in producing transient starches

(Szydlowski et al 2009) However given the expression of SSIV throughout

endosperm development it is possible that SSIV also plays a role in starch

biosynthesis despite its lack of interaction with other major SSs or SBEs SSIV

may be involved in protein-protein interactions with proteins which we could not

detect by co-immunoprecipitaion

185

General Discussion

The aim of the investigations presented in this thesis were to test the

hypothesis that the plastidial form of starch phosphorylase (SP) in cereal

endosperm is involved in starch synthesis by its direct interaction with other

enzymes of the pathway This study also tested the hypothesis that these

protein-protein interactions are regulated by protein phosphorylation A

biochemical approach was employed in order to address these questions In this

study maize was used as an example of a cereal maize is a widely grown crop

in OntarioNorth America and its endosperm produces high yields of starch

Maize has been used as a model plant in many starch biosynthetic studies and

efficient extraction procedures of amyloplasts and peptide-specific antibodies of

major starch biosynthetic enzymes of maize have been developed by our

laboratory

SP is the second most abundant enzyme present in maize amyloplasts

next to SBEIIb (Yu et al 2001) In the localization analysis SP is found only in

the amyloplast stroma of the wild-type maize endosperm (section 231 in the

thesis and Yu et al 2001) which was similarly observed in wild-type wheat

(Tetlow et al 2004) and rice (Satoh et al 2008) amyloplasts Interestingly the

ae- mutant of maize endosperm lacking SBEIIb in the plastid SP is not only

found in the stroma but also in the starch granule as a granule-bound protein

thought to be a result of its association within a multi sub-unit protein complex

formed by interaction with other starch biosynthetic enzymes (Liu et al 2009

Grimaud et al 2008) Further in the amyloplast stroma of the same ae- mutant

SP was shown to form larger multi sub-unit complexes with SBEI SBEIIa SSI

186

and SSIIa (Liu et al 2009) In the ae- mutant lacking SBEIIb increased

amounts of granule bound SBEI SBEIIa SSIII and SP are found without

affecting SSI or SSIIa (Grimaud et al 2008) These observations suggest a

functional role for SP in starch biosynthesis at least in the absence of SBEIIb It

was suggested that the presence of SP in the amyloplast stroma could

compensate for the activity of missing isoforms of major starch biosynthetic

enzyme (SBEIIb) in the ae- mutant (Liu et al 2009) In wild-type maize SP is

not bound to the granule but closely associated with the granule surfaces as are

some other enzymes eg SSI SBEI and SBEIIb (Fig 22) In contrast SSII was

comparatively abundant at the granule surface and very little of this enzyme is

detected in the stroma Recent evidence suggests that SSIIa is a central

component of the trimeric protein complex involved in amylopectin cluster

synthesis and directs it self and SSI and SBEIIb into the starch granule (Liu et

al 2012) As Tickle et al (2009) suggested in wheat amyloplasts SP could act

directly on the surface of the starch granule in a degradative manner where SP

modifies the granule structure in a phosphorolytic manner to produce G-1-P

which can be recycled back to produce starch via AGPase However more

investigations are required to analyze this hypothesis

In the wild-type maize amyloplast SP remains active in the synthetic

direction throughout the various developmental stages observed from 12 to 24

DAA (days after anthesis) in maize amyloplast (Chapter 2 section 232) This

observation was parallel to the observations in other storage starch producing

plants such as in wheat rice and in potato tubers suggesting that the SP has a

certain involvement in the starch synthesis process in plants (Tickle et al 2009

187

Satoh et al 2008 Schupp and Ziegler 2004 Yu et al 2001 Brisson et al

1989 Tsai and Nelson 1968) despite earlier suggestions that SP had a

primarily degradative role (Preiss 1982 Preiss 1984) Early studies of starch

synthesis suggested that SP was the enzyme responsible for glucan elongation

(Leloir 1964) However the fact that α-glucan phosphorylase (EC 2411)

found in animals fungi and prokaryotes plays a major role in glucan catabolism

(Alonso-Casajuacutes et al 2006 Ball and Morell 2003 Newgard et al 1989 Preiss

1984) led many researchers to believe that SP had an essentially degradative

role in plant cells In rice endosperm zymogram analysis of mutants lacking SP

showed no change in the activities of DBE isofoms (isoamylase and pullulanase)

SBE isoforms (SBEI SBEIIa and SBEIIb) and SS isoforms (SSI and SSIIIa) but

a reduction in total starch content was observed in the rice endosperm forming a

shrunken phenotype (Satoh et al 2008) The starch content per grain in

mutants lacking SP was even less than in the shrunken 2 mutants of rice

lacking the AGPase large subunit (Satoh et al 2008) Mutants of rice

endosperms lacking SP grown at 300C produced about 6 of the shrunken

phenotypes the starch content was similar in the wild-type Percentages of

shrunken phenotype was increased in SP mutant plants grown at 250C and 200C

by 35-39 and 66 respectively with a severe reduction in starch

accumulation suggesting that SP may play an important role in starch

biosynthesis at fluctuating andor adverse temperature conditions (Satoh et al

2008) Further the reduced starch content produced by mutants lacking SP and

the fact that SP is actively expressed in early stages of endosperm development

188

in rice endosperm suggest that SP is essential for the early steps of starch

biosynthesis in rice endosperm (Satoh et al 2008)

Peptide specific anti-SP antibodies recognized the plastidial SP in

amyloplasts but not in chloroplasts This may be due to reduced levels of SP in

chloroplasts or the chloroplastic SP may have different amino acid sequences in

the region where epitopes were designed (Chapter 2 Fig 21) In rice mutants

lacking plastidial SP the endosperm had severely reduced levels of starch and

had a shrunken phenotype (Satoh et al 2008) and in Arabidopsis leaves lacking

SP in chloroplasts no significant change in the total accumulation of starch was

observed compared to their wild-types (Zeeman et al 2004) suggesting a

divergent role of plastidial SP present in storage starch biosynthesis compared

to transient starch biosynthesis in chloroplasts

Recent research confirmed that SP in cereal endosperms is regulated by

protein phosphorylation as similarly observed in some other isoforms of the

major starch biosynthetic enzymes (Liu et al 2009 Pollack 2009 Hennen-

Bierwagen et al 2008 Grimaud et al 2008 Tetlow et al 2008 Tetlow et al

2004) Phosphorylation of SP may promote the formation of protein-protein

interactions (Liu et al 2009 Hennen-Bierwagen et al 2008 Grimaud et al

2008 Tetlow et al 2004) SP in wheat endosperm was shown to be involved in

the formation of protein complexes with SBEI and SBEIIb (Tetlow et al 2004)

From the research presented in this thesis the mobility of SP on phosphate

affinity SDS-PAGE using Phos-TagTM ligand-acrylamide gel showed no alteration

following treatment in ATP compared to untreated SP in the SDS-PAGE gel

(Chapter 2 Fig 26) Treatment of amyloplasts with 1 mM ATP [under

189

conditions previously determined to cause phosphorylation of SP by Pollack

(2009)] or APase (known to cause non-specific protein dephosphorylation) did

not alter the catalytic activity of SP (Chapter 2 Fig 25) Phosphorylation of SP

therefore may have a role in complex formation either with other enzymes of

starch synthesis (Chapter 3 section 334 and Chapter 4 section 458) or in the

formation of SP multimers but does not appear to play a role in regulating its

catalytic activity

In the present study GPC analysis confirmed that the SP exists in the

amyloplast stroma mainly as tetrameric and dimeric forms throughout the

developmental stages (both multimeric states were observed at 15-35 DAA) in

maize endosperm (Fig 28) These conformational structures of SP are found to

be as a natural molecular characteristic of SP which has previously been

observed in higher plants (Liu et al 2009 Mu et al 2001 Brisson et al 1989)

and the dimeric forms are observed in bacteria (Dauvillee et al 2006) and yeast

(Tanabe et al 1987) In the GPC analysis the elution profile of the ATP-treated

and APase treated native SP did not drastically change from the untreated

amyloplast lysates (Fig 28) suggesting that the formation of the homodimeric

or homotetrameric forms of SP is probably not controlled by protein

phosphorylation (Chapter 2 Fig 28)

It was previously reported that SP from wheat endosperm amyloplasts

formed protein complexes with SBEI and SBEIIb in a phosphorylation-dependent

manner (Tetlow et al 2004) In the maize ae- mutant lacking SBEIIb SP was

shown to interact with different proteins The complex in ae- contained SSI

SSIIa and SBEI and SP In this complex it was suggested SBEI and SP in some

190

way compliment the loss of SBEIIb in the mutant (Liu et al 2009) In addition

SP which is part of the novel protein complex was found as a granule-bound

protein reinforcing the fact that protein complex components become granule

bound by an as yet unknown mechanism (Liu et al 2009 Grimaud et al 2008)

The multimeric status of the SP in the wild-type wheat endosperm complex and

the complex in ae- mutant endosperm is not known

In this study peptide specific anti-maize SP antibodies were used to

immunoprecipitate the native SP from the wild-type maize amyloplast stroma

using Protein-A Sepharose beads to investigate possible protein-protein

interactions (Chapter 2 section 236) It was not possible to immunoprecipitate

the native SP using the Protein-A Sepharose beads (Fig 210) and consequently

we were unable to employ the antibodies in immunoprecipitation and co-

immunoprecipitation experiments The reason for the inability of the peptide-

specific antibodies to recognize the native protein is unclear but it is possible

that the native SP in someway shields the epitope irrespective of the multimeric

state of the protein Therefore an S-tagged recombinant SP was developed by

over expressing the full length mRNA sequence (3053 bp) of plastidial maize SP

in Artic Express Ecoli cells after cloning in pET29a expression vectors (Chapter

3)

GPC was a useful tool for separating the amyloplast lysates or cell

extracts and in identification of major starch biosynthetic enzymes as monomers

or in complexes in fractionated extracts with predicted molecular weights

(Hennen-Bierwagen et al 2008 Tetlow et al 2208 Liu et al 2009)

Fractionation of recombinant SP extracts by GPC partially purified the

191

recombinant SP and enabled us to identify different multimeric forms of

recombinant SP (Fig 39A) Greater amounts of recombinant SP was aggregated

(2000 kDa) and found to be active including tetrameric dimeric and

monomeric forms of SP (Fig 39BC) Dimeric and tetrameric forms of active

recombinant S-tagged SP separated by GPC were immobilized by S-Protein

Agarose beads and used as affinity ligands to isolate and detect amyloplast

proteins which interact with SP (Fig 310) The various pull down assays that

were carried out with recombinant SP and amyloplast lysates indicated that

certain starch biosynthetic enzymes specifically interacted with the dimeric and

tetrameric forms of SP in a phosphorylation-dependent manner (Figs 312

313) Many of the protein-protein interactions previously observed in cereal

endosperm amyloplasts have also been shown to be phosphorylation dependent

(Liu et al 2009 Grimaud et al 2008 Hennen-Bierwagen et al 2008 Tetlow et

al 2008 Tetlow et al 2004) SBEI directly interacted with both tetrameric and

dimeric forms of SP and the SBEIIb interacted only with the dimeric forms of SP

when plastid lysates were pre-treated with 1 mM ATP Weak interactions

between SSIIa and SP were observed unlike the SBE-SP interactions no

interactions between SP and SSIIa have been observed previously Unlike the

SP-SBE interactiions previous experiments involving immunoprecipitation of

SSIIa have not detected SP as an interacting partner The ATP-dependence of

some of the protein-protein interactions suggest a phosphorylation dependent

mechanism of complex assembly In other complexes studied some of the

components are directly phosphorylated (Liu et al 2009) Other than the SP

previous research had already confirmed that SSIIa SBEI and SBEIIb are

192

regulated by protein phosphorylation (Liu et al 2009 Tetlow et al 2008 Tetlow

et al 2004)

Glucan phosphorylases found in both prokaryotic and eukaryotic systems

exist as dimers or tetramers of identical subunits (Dauvillee et al 2006 Mu et

al 2001 Brisson et al 1989 Tanabe et al 1987) Both dimeric and tetrameric

configurations of SP have been observed in maize amyloplasts lysates (Mu et al

2201 Liu et al 2009) In addition to SP SBEIIa and SBEIIb have been found to

be associated as homodimers (Tetlow et al 2008) However based on the

elution profiles from GPC analysis it was not clear that the interactions found

between the homodimeric forms of SP were with monomers or homodimeric

forms of SBEIIb Although the precise roles of the various protein-protein

interactions in amyloplasts is not clear it is possible that some of the

interactions with SP and other enzymes regulate SP activity by controlling the

multimeric status of the protein Different multimeric states of SP may have

variable affinities for other proteins which may be controlled and regulated by

protein phosphorylation The relative competition of different multimeric forms

of SP and other proteins for each other is an area for future study For example

homodimeric forms of SBEIIb interacting with SP may prevent the interactions

between the tetrameric forms of SP

The protein-protein interactions is the fact that may enzymes of the

pathway are differentially expressed throughout endosperm development In

maize endosperm SSIIa SSIII and SBEIIa are expressed in early stages of

development (approximately 8-15 DAA) and SSI SSIIb and SBEIIb are

expressed in the middle stage (approximately 16-24 DAA) and GBBSSI SSIV

193

and SBE1 are expressed at the later stage (over 24 DAA) of the grain filling (Liu

et al 2009 Zhang et al 2004 Mu et al 2001 Mu-Forster et al 1996)

As discussed in previous studies SP has various potential functions in

starch biosynthesis SP showed a higher capacity to synthesize longer linear

glucans from small MOS than SSIIa (Satoh et al 2008) A possible function of

SP was suggested by Nakamura et al (2012) and Satoh et al (2008) based on

the lsquostarch trimming modelrsquo (Ball and Morell 2003) whereby small malto

dextrins produced by the activity of DBE provide a substrate for SP to produce

linear glucan chains which in turn serve as the substrates for SBE to form

branched glucans in the starch initiation process


rice endosperm Purified SP from rice endosperm synthesized glucans from G-1-

P in the presence of SBE without any exogenous glucan primer and glucan

production was higher when SBEI was present compared to SBEIIa or SBEIIb

(Nakamura et al 2012) Activities of SP and SBE were dependent on the mutual

availability SP and SBE and showed mutual capacities for chain elongation and

chain branching (Nakamura et al 2012) These observations further support the

function of SP proposed by Satoh et al (2008) In contrast according to the

proposed functions of SP suggested by Tickle et al (2009) SP may play a

degradative role by directly acting on the starch granule to produce G-1-P or

may degrade the MOS which are produced by DBE reaction to produce G-1-P

and supplying the substrate for AGPase for starch biosynthesis The presence of

catalytically active SP thoughout the grain filling period of maize endosperm

and the interaction of different multimeric forms of SP with SBE insoforms

194

support a synthetic role for SP in starch biosynthesis in maize endosperm as

suggested by Satoh et al (2008) and Nakamura et al (2012) in rice Low G-1-P

concentrations and high PiG-1-P ratios are considered as the controlling

mechanism of SP activity in glucan synthesis (Tiessen et al 2011 Schupp and

Ziegler 2004 Mu et al 2001 Matheson and Richardson 1978) Plastidial and

cytosolic SP activities in degradative direction were reduced by 80 and 20

respectively when Pi was added in vitro (Mu et al 2001) suggesting that Pi

regulates degradative activity of plastidial SP more than cytosolic SP Low levels

of G-1-P and a 50-fold excess of Pi in vitro were able to sustain the SP

biosynthetic reaction (Hwang et al 2010) suggesting that plastidial SP

preferentially carries out starch biosynthesis over degradation of starch

The leaves of Arabidopsis ss4 mutants (where transient starch is

synthesized) showed reductions in granule number and increased granule size

(14-2 fold) (Roldan et al 2007) and the double mutants of ss4 and sp further

increased the granule size by 4-fold (Planchot et al 2008) compared with the

wild-type plants suggesting the possibility that SSIV and SP may form

functional protein-protein interactions and are in some way involved in granule

initiation in chloroplasts One of the major hypotheses tested in the study was to

investigate the possible interactions between SSIV and SP In co-

immunoprecipitation experiments conducted by using peptide-specific anti-SSIV

antibodies in ATP-treated amyloplasts lysates SP weakly interacted with SSIV

(Chapter 4 section 4 section 48) Since there was no evidence for SSIV

phosphorylation (Chapter 4 section 45) the ATP-dependent interaction

observed may be due to phosphorylation of SP or other as yet unidentified

195

factors Since the reciprocal interactions using S-tagged recombinant SP did not

show any interactions with SSIV the results with the SSIV co-

immunoprecipitation experiment should be treated with caution It is possible

that SP and SSIV interact weakly andor transiently in vivo and under these

experimental conditions the interaction is not observed consistently In the S-

tagged SP studies the total protein (05 mgmL) of the amyloplast lysates were

comparatively lower than in the co-immunoprecipitation analysis (10 mgmL)

so that the amount of available SSIV may be limited and below detectable

levels in these interactions Also the recombinant forms of SP may not be

phosphorylated as efficiently as the native form leading to less stable

interactions The phosphorylation status of the recombinant SP following ATP-

treatment of amyloplast lysates was not examined The interaction found in the

study between SP and SSIV may have significance in relation to our

understanding of the initiation of the starch granule In addition SP was the

only protein which interacted with SSIV indicating a high specificity towards SP

Activity andor the affinity of the SSIV required to initiate the priming of granule

initiation may be regulated by the interactions with SP

To elucidate both the synthetic and the degradative activities of the

recombinant tetrameric and dimeric forms of SP they were tested in glucan

substrates of maltoheptaose glycogen and amylopectin and at 25 mgmL

concentration both multimeric states are active in both synthetic and

phosphorylitic directions (Fig 313) The higher activities of both multimeric

forms of SP with high molecular weight amylopectin followed by glycogen and

maltoheptaose were observed in both synthetic and phosphorolytic direction

196

and was similar to previous findings in maize (Yu et al 2001) potato (Liddle et

al 1961) and spinach leaves (Shimomura et al 1982) Bacterial SP has a

tetrameric configuration and also shows a higher activity in starch than in

maltopentaose in both directions (Weinhaumlusel et al 1997) The Km values

indicate the affinity level of SP towards different glucan substrates in

phosphorolytic direction (Table 31) In tetrameric SP the higher Vmax showed

with amylopectin also showed a higher Km (lower affinity) compared to

maltoheptaose which had a lower Vmax but a lower Km (higher affinity) which

was similarly observed in both synthetic and degradative directions by Mu et al

(2001) and suggests higher affinity of enzyme to the substrate not essentially

increased the activity of SP (Table 31)

The variation in the activity of tetrameric SP from synthetic direction to

phosphorylitic direction was greater in maltoheptaose (147 fold) compared to

amylopectin (21 fold) and glycogen (11 fold) (Table 31) indicating the

preference of SP for low molecular MOS in degradative directions This has also

been observed by Mu et al (2001) However the higher activities of SP forms

with highly branched amylopectin conflicts with the proposed function of SP in

the suggested model proposed by Satoh et al (2008) and Nakamura et al

(2012) In the model during discontinuous synthesis of starch granules the

short glucan chains released from pre-amylopectin by the action of debranching

enzymes are converted to longer glucan chains by SP

In potato tuber (plastidial SP) and leaf (cytosolic SP) were defined as low

affinity (SP-L) and high affinity (SP-H) isoforms respectively according to the

197

affinities showed to both amylopectin and glycogen in synthetic direction (Mori

et al 1993) (Table 1) The proposed function of the L-78 insertion located in the

middle of the plastidial SP which was not observed in cytosolic SP (Yu et al

2001 Albrecht et al 1998 Nakano and Fukui 1986) is to obstruct the binding

affinity of plastidial SP to large highly branched starch compared to glycogen

(Young et al 2006 Albrecht et al 1998) Very little is known about the

regulatory mechanism of SP-specific L-78 insertion existing in the plastidial form

of SP and no evidence for L-78 cleavage or the function of the insertion is

available for maize In the sweet potato tuber enzyme serine residues located in

L-78 insertion are phosphorylated and are thought to then target the L-78

peptide for proteolytic cleavage (Young et al 2006)

The results presented in this thesis demonstrate that SP is catalytically

active in dimeric and tetrameric forms throughout the endosperm development

and is involved in protein-protein interactions with the major starch biosynthetic

enzymes Some of the interactions were enhanced by pre-treatment with ATP

and SP has previously been shown to be phosphorylated (Pollock 2009 Liu et

al 2009 Grimaud et al 2008 Tetlow et al 2004) suggesting phosphorylation

of SP may control in some as yet unknown manner protein-protein

interactions For future directions investigating the glucan priming and glucan

synthesizing capacities of different dimeric and tetrameric forms and their

regulation by G-1-P or Pi in vitro would be essential in further understanding the

function of SP Fig 51 illustrates the proposed functions dimeric and tetrameric

isoforms of SP in starch biosynthesis phosphorylation of SP and SBE enzymes

facilitate the formation of protein-protein interactions between these enzymes

198

and between SP and SSIV Interaction between SP and SBE may regulate and

activate SBE to in turn facilitate interactions with starch synthases in the

amyloplast Another potential function for SP is in starch granule initiation by

interacting with SSIV (Fig 411)

Figure 51 Schematic diagram illustrating the proposed functions of dimeric and tetrameric forms of plastidial SP Phosphorylation of SP and SBE facilitate

the formation of protein-protein interactions phosphorylated SBEI interacts with both dimeric and tetrameric forms of SP while phosphorylated SSIIa interacts with the tetrameric form of SP and phosphorylated SBEIIb interacts with dimeric

SP forms and may regulate and activate the branching enzymes to facilitate interactions with starch synthases in the amyloplast while SP remains in the

stroma A second function of SP may be in starch granule initiation by interacting with SSIV Phosphorylated proteins are denoted by the P symbol

199

This research provides further insight into our growing understanding of the

coordinated activities of different enzymes associated in starch synthesis

through protein-protein interactions and complex formation in developing maize

endosperm The protein-protein protein interactions and the complexes formed

in amyloplasts are suggested to be a vital requirement in synthesizing starches

with different morphological characteristics by modulating granule fine structure

Understanding the basis of these modulations is essential for rational

manipulation of starch in crops Application of starch in food and non-food

industries depends on different structural and functional properties of starch

which can be modified with the knowledge of its genetic manipulations This

research provides information to understand the basics of starch biosynthesis to

develop models in developing modify polymer structures of starch

200

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218

Appendixes Appendix 01

Synthetic activity of plastidial SP in amyloplast lysates (22 DAA) was

slightly reduced with the absence of SSIIa Protein-protein interactions between

both dimeric and tetrameric forms of SP with SSIIa may have affected for the

activity of SP There was no different in the synthetic activity of SP when SSIV

was removed

Synthetic activity of plastidial SP in amyloplast lysates (22 DAA) in the absence

of SSIIa (A) and SSIV (C) was tested in non-denaturing affinity native zymogram containing 01 glycogen in the gel Immunoblot of the zymogram

gels (A and C) were probed by peptide specific anti-SP (B and D) antibodies SSIIa and SSIV in amyloplast lysates were removed by immunoprecipitating the proteins using anti-SSIIa and anti-SSIV antibodies bound to Protein-A sepharose

beads The supernatants obtained after immunoprecipitation of stromal SSIIa and SSIV were used (90 microgmL per well) in zymogram analysis

(A) (B)

(C) (D)

219

Appendix 02

Immunoprecipitation of stromal SP from maize amyloplasts (22 DAA) in the

absence of SSIIa was tested using peptide specific anti-SP antibodies following immunoprecipitation with anti-SSIIa antibodies 1 ml amyloplast lysates (1 mgmL) were incubated with peptide-specific anti-SSIIa (15 mgmL final

concentration) at room temperature for 1 hr and then immunoprecipitated with Protein-A-Sepharose beads The supernatants were obtained after the beads

bound to SSIIa were centrifuged at 13000 rpm for 5min at 40C Supernatant was used (1 mgmL) immunoprecipitate SP by anti-SP antibodies (15 mgmL final concentration) Washed Protein-A-Sepharose-antibody-antigen complexes

were boiled in 200 μL 1XSDS-loading buffer and 30 μL was loaded onto 10 SDS gels Immunoblotted membranes were developed with SSIIa (A) and SP (B)

anti-maize antisera

The results showed that SP was not immunoprecipitated by anti-SP

antibodies bound to Protein A-sepharose beads after removing the SSIIa present

in the amyloplast lysates suggesting that the SSIIa is not preventing the

binding of SP to anti-SP antibodies bound to Protein A-sepharose beads

(A) (B)

220

Appendix 03

Primers used in PCR to isolate the complete coding sequence of plastidial SP of maize endosperm SP-F1 and SP-R1 primers were designed with the part of

transit peptide sequence and 15 bp overhangs from pET29a vector are underlined in SP-F2 and SP-R2 primers

Primer

Name

Primer sequence Annealing

temperature SP-F1

SP-R1

5rsquo GCGGAGGTGGGGTTCTCCT 3rsquo

5rsquo GCGAAAGAACCTGATATCCAC 3rsquo

640C

620C

SP-F2

SP-R2

5rsquoGGTTCCATGGCTGATTCAGCGCGCAGCG 3rsquo

5rsquoGAATTCGGATCCGATCTAGGGAAGGATGGC 3rsquo

460C

480C

Appendix 04

Primers used in sequence analysis of the isolated plastidial SP sequenced cloned

into pET29a vector In addition to SP-F2 AND SP-R2 primers (see appendix 03) T7 promoter and T7 terminator universal primers and F1 F2 F3 primeres were

used The mRNA complete coding sequence of plastidial SP of maize endosperm from NCBI was used to design the primers

Primer Name

Primer sequence Annealing temperature

Location in original

sequence T7-

promoter

T7-

terminator

F1

F2

F3

5prime TAA TAC GAC TCA CTA TAG GG 3prime

5rsquo GCTAGTTATTGCTCAGCGG 3rsquo 5rsquo GGAACCAGATGCTGCCCTG 3rsquo

5rsquo GTTGCAGTGCAGATGAATGAC 3rsquo

5rsquo GGTGTAGCTGAAATTCACAGTG 3rsquo

480C

760C

620C

680C

680C

-

- 393-411 bp

1006-1026 bp

1636-1657 bp

221

Appendix 05

Following is the alignment comparison of the predicted amino acid

sequence of plastidial maize SP obtained from NCBI with the amino acid

sequence of the recombinant SP produced in the study Amino acid sequence of

recombinant SP was derived from the mRNA sequence of PCR product of the full

length sequence (2805 bp) of SP (except transit peptide) Arrow shows thw

change in amino acid sequence of recombinant SP from the predicted sequence

(httpwwwchembnetorgsoftwareLALIGN_formhtml)

(A) wwwtmp251331seq predicted SP (NCBI) 849 bp - 849 aa

(B) wwwtmp251332seq Recombinant SP 724 bp - 724 aa

using matrix file BL50 (15-5) gap-openext -14-4 E(limit) 005

996 identity in 706 aa overlap (73-7781-706) score 4614 E(10000) 0

80 90 100 110 120 130

Predicted TLNYPAWGYGLRYEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVE

Recombinant TLNYPAWGYGLRYEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVE

10 20 30 40 50 60

140 150 160 170 180 190

Predicted GTDGRKHWIGGENIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAY

Recombinant GTDGRKHWIGGENIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAY

70 80 90 100 110 120

200 210 220 230 240 250

Predicted EAHLNAKKICHILYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLNWEDFPS

Recombinant EAHLNAKKICHILYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLNWEDFPS

130 140 150 160 170 180

260 270 280 290 300 310

Predicted KVAVQMNDTHPTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDI

Recombinant KVAVQMNDTHPTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDI

190 200 210 220 230 240

320 330 340 350 360 370

Predicted MQKLLPRHVEIIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVK

Recombinant MQKLLPRHVEIIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVK

250 260 270 280 290 300

222

380 390 400 410 420 430

Predicted PKDKKESPAKSKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSE

Recombinant PKDKKESPAKSKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSE

310 320 330 340 350 360

440 450 460 470 480 490

Predicted DELDPFVKSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNK

Recombinant DELDPFVKSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNK

370 380 390 400 410 420

500 510 520 530 540 550

Predicted TNGVTPRRWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKAN

Recombinant TNGVTPXRWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKAN

430 440 450 460 470 480

560 570 580 590 600 610

Predicted KMKVVSLIREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKS

Recombinant KMKVVSLIREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKS

490 500 510 520 530 540

620 630 640 650 660 670

Predicted FVPRVCIFGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALI

Recombinant FVPRVCIFGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALI

550 560 570 580 590 600

680 690 700 710 720 730

Predicted PASELSQHISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHE

Recombinant PASELSQHISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHE

610 620 630 640 650 660

740 750 760 770

Predicted IAGLRKERAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELMGSLEGN

Recombinant IAGLRKERAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELXGSLXGN 670 680 690 700

223

Appendix 06

Predicted phosphorylation sites of maize SSIV was analyzed by NetPhos 20

server

Phosphorylation sites predicted

Ser 37 Thr 7 Tyr 9

Serine predictions


_________________________v_________________

Sequence 21 PAHTSTPLF 0010

Sequence 38 DAAASSSTP 0520 S

Sequence 39 AAASSSTPF 0193

Sequence 40 AASSSTPFT 0213

Sequence 55 RLPMSCSAA 0580 S

Sequence 57 PMSCSAAAG 0003

Sequence 71 LLIRSAAPS 0007

Sequence 75 SAAPSTIVG 0979 S

Sequence 86 RLAMSRRTS 0840 S

Sequence 90 SRRTSRRNL 0998 S

Sequence 104 PHQKSAPSA 0010

Sequence 107 KSAPSANHR 0013

Sequence 115 RNRASIQRD 0883 S

Sequence 122 RDRASASID 0940 S

Sequence 124 RASASIDEE 0947 S

Sequence 133 QKQMSEDEN 0995 S

Sequence 194 EMKLSETGK 0027

Sequence 200 TGKQSVLSS 0622 S

Sequence 203 QSVLSSEVK 0974 S

Sequence 204 SVLSSEVKS 0687 S

Sequence 208 SEVKSDEES 0997 S

Sequence 212 SDEESLEFD 0987 S

Sequence 245 ETEESLFKL 0603 S

Sequence 259 LLNASLREL 0964 S

Sequence 267 LECTSTSAQ 0420

Sequence 269 CTSTSAQSD 0096

Sequence 272 TSAQSDVLK 0116

Sequence 298 DLLDSTANQ 0421

Sequence 307 VEHASLTLD 0007

Sequence 326 KLKASLGTT 0228

Sequence 333 TTNVSEFCL 0132

Sequence 349 QRVKSVEER 0997 S

Sequence 363 HEMHSQIEL 0947 S

Sequence 371 LYEHSIVEF 0115

Sequence 380 HGTLSKLIN 0028

Sequence 386 LINESEKKS 0953 S

Sequence 390 SEKKSMEHY 0988 S

Sequence 400 EGMPSEFWS 0540 S

Sequence 404 SEFWSRISL 0038

Sequence 407 WSRISLLID 0040

Sequence 414 IDGWSLEKK 0047

Sequence 420 EKKISINDA 0966 S

Sequence 425 INDASMLRE 0275

Sequence 444 EAYLSSRGM 0981 S

Sequence 445 AYLSSRGME 0033

Sequence 456 ELIDSFLKM 0024

Sequence 466 LPGTSSGLH 0043

Sequence 467 PGTSSGLHI 0007

Sequence 492 ADVISGLGK 0005

Sequence 532 VVVKSYFEG 0915 S

Sequence 581 FKRFSYFSR 0944 S

Sequence 584 FSYFSRVAL 0778 S

Sequence 594 LLYQSGKKV 0610 S

Sequence 626 LGFNSARIC 0004

Sequence 667 MRDNSHGRI 0987 S

Sequence 681 AVVYSNIVT 0025

Sequence 688 VTTVSPTYA 0545 S

Sequence 697 QEVRSEGGR 0658 S

Sequence 711 LKVHSKKFV 0981 S

Sequence 729 TWNPSTDRF 0293

Sequence 739 KVQYSANDL 0758 S

Sequence 747 LYGKSANKA 0009

Sequence 761 LKLASTQAS 0017

Sequence 765 STQASQPLV 0009

Sequence 803 VLLGSSPVQ 0009

Sequence 804 LLGSSPVQH 0231

Sequence 844 IFAASDMFI 0179

Sequence 851 FIVPSMFEP 0541 S

Sequence 868 MRYGSVPVV 0245

Sequence 881 GLNDSVFDL 0978 S

Sequence 939 KIDFSWDTS 0886 S

Sequence 943 SWDTSVSQY 0862 S

Sequence 945 DTSVSQYEE 0904 S

_________________________^_________________

224



_________________________v_________________

Sequence 9 RPRPTARAR 0972 T

Sequence 20 DPAHTSTPL 0593 T

Sequence 22 AHTSTPLFP 0060

Sequence 27 PLFPTAAHA 0027

Sequence 41 ASSSTPFTL 0159

Sequence 44 STPFTLQPH 0041

Sequence 65 GAEATALLI 0022

Sequence 76 AAPSTIVGR 0375

Sequence 89 MSRRTSRRN 0960 T

Sequence 96 RNLRTGVHP 0035

Sequence 154 MIQNTQKNI 0269

Sequence 181 KEKETLQQK 0067

Sequence 196 KLSETGKQS 0274

Sequence 240 LIEITETEE 0376

Sequence 242 EITETEESL 0204

Sequence 266 ELECTSTSA 0177

Sequence 268 ECTSTSAQS 0127

Sequence 299 LLDSTANQV 0017

Sequence 309 HASLTLDGY 0440

Sequence 329 ASLGTTNVS 0061

Sequence 330 SLGTTNVSE 0134

Sequence 378 EFHGTLSKL 0481

Sequence 465 ALPGTSSGL 0103

Sequence 545 NKIWTGTVE 0134

Sequence 547 IWTGTVEGL 0564 T

Sequence 608 HDWQTAFVA 0583 T

Sequence 632 RICFTCHNF 0035

Sequence 685 SNIVTTVSP 0101

Sequence 686 NIVTTVSPT 0803 T

Sequence 690 TVSPTYAQE 0013

Sequence 706 GLQDTLKVH 0697 T

Sequence 723 NGIDTDTWN 0228

Sequence 725 IDTDTWNPS 0239

Sequence 730 WNPSTDRFL 0182

Sequence 762 KLASTQASQ 0027

Sequence 773 VGCITRLVP 0032

Sequence 792 IYKITELGG 0021

Sequence 859 PCGLTQMVA 0406

Sequence 875 VVRRTGGLN 0109

Sequence 889 LDDETIPME 0037

Sequence 899 RNGFTFLKA 0025

Sequence 942 FSWDTSVSQ 0423

Sequence 954 IYQKTATRA 0148

Sequence 956 QKTATRARA 0066

_________________________^_________________



_________________________v_________________

Sequence 313 TLDGYRDFQ 0547 Y

Sequence 338 EFCLYLVDI 0017

Sequence 368 QIELYEHSI 0100

Sequence 394 SMEHYAEGM 0964 Y

Sequence 442 LREAYLSSR 0287

Sequence 512 ILPKYDCMQ 0513 Y

Sequence 533 VVKSYFEGN 0026

Sequence 554 GLPVYFIEP 0046

Sequence 570 WRAQYYGEH 0012

Sequence 571 RAQYYGEHD 0409

Sequence 582 KRFSYFSRV 0045

Sequence 592 LELLYQSGK 0494

Sequence 615 VAPLYWDVY 0886 Y

Sequence 619 YWDVYANLG 0973 Y

Sequence 638 HNFEYQGIA 0701 Y

Sequence 649 QDLAYCGLD 0208

Sequence 680 GAVVYSNIV 0136

Sequence 691 VSPTYAQEV 0467

Sequence 738 LKVQYSAND 0261

Sequence 744 ANDLYGKSA 0941 Y

Sequence 789 RHAIYKITE 0229

Sequence 832 LLLKYDDAL 0081

Sequence 866 VAMRYGSVP 0123

Sequence 918 RAFNYYHRK 0028

Sequence 919 AFNYYHRKP 0320

Sequence 947 SVSQYEEIY 0904 Y

Sequence 951 YEEIYQKTA 0983 Y

_________________________^_________________

225

Appendix 07

Co-immunoprecipitation of stromal proteins from wild-type maize amyloplasts

using peptide specific anti-SBEIIb antibodies to investigate the protein-protein interactions between SBEIIb SSIV and SP 1 ml amyloplast lysates (1 mgmL) prepared from wild-type maize endosperm at 22 DAA were incubated with

peptide-specific anti-SSIV antibodies (15 mgmL final concentration) at room temperature for 1 hr and then immunoprecipitated with Protein-A-Sepharose

beads The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS-loading buffer and 30 μL loaded onto 10 SDS gels Immunoblotted membranes were developed with anti-maize SBEIIb SSIV and

SP antisera

SBEIIb Co-IP

Probed with anti-SBEIIb

kDa

150

100

75

50

kDa

150

100

75

50

kDa

150

100

75

50

SBEIIb Co-IP

Probed with anti-SP

SBEIIb Co-IP

Probed with anti-SSIV

L Protein marker

1 SBEIIb Co-IP in protein A-Sepharose beads

2 SBEIIb Co-IP Pre Immune in protein A-Sepharose beads

3 SBEIIb Co-IP-supernatant

4 SBEIIb Co-IP- Pre Immune supernatant

5 Protein A-Sepharose beads + amyloplast lysates


L 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6

226

Appendix 08

All the data were analysed using Statistix 09 statistical program

ONE-WAY ANOVA of the synthetic activity of SP of amyloplast lysates in

different glucans

One-Way AOV for V001 V002 V003 V004 V005 V006 V007 V008 V009

Source DF SS MS F P

Between 8 395428 494286 8274 0028

Within 18 10753 5974

Total 26 406182

Grand Mean 76055 CV 1016

Homogeneity of Variances F P

Levenes Test 128 03152

OBriens Test 057 07911

Brown and Forsythe Test 030 09570

Welchs Test for Mean Differences

Source DF F P

Between 80 9327 00008

Within 74

Component of variance for between groups 162771

Effective cell size 30

Variable Mean

V001 10053 (Glycogen-Untreated)

V002 11445 (Glycogen-ATP-treated)

V003 3743 (Glycogen-APase-treated)

V004 6908 (Maltoheptaose-Untreated)

V005 6001 (Maltoheptaose-ATP-treated)

V006 2735 (Maltoheptaose-APase-treated)

V007 9943 (Amylopectin-Untreated)

V008 14334 (Amylopectin-ATP-treated)

V009 3288 (Amylopectin-APase-treated)

Observations per Mean 3

Standard Error of a Mean 44624

Std Error (Diff of 2 Means) 63108

LSD All-Pairwise Comparisons Test

Variable Mean Homogeneous Groups

V008 14334 A

V002 11445 B

V001 10053 C

V007 99427 C

V004 69077 D

V005 60013 D

V003 37430 E

V009 32877 E

V006 27353 E

Alpha 005 Standard Error for Comparison 63108

227

Critical T Value 2101 Critical Value for Comparison 13259

There are 5 groups (A B etc) in which the means

are not significantly different from one another

Statistix

ONE-WAY ANOVA of the phosphorolytic activity of SP of amyloplast lysates in

different glucans

One-Way AOV for V001 V002 V003 V004 V005 V006

Source DF SS MS F P

Between 5 367208 734416 3557 00004

Within 12 24776 20647

Total 17 391984







Source DF F P

Between 50 2605 00008

Within 55



Variable Mean












V005 58873 A

V004 46913 B

V006 33433 C

V002 27040 CD

V001 24493 DE

V003 16640 E





228

Statistix

ONE-WAY ANOVA of the synthetic and phosphorolytic activity of recomb

tetrameric form of SP in different glucans


Source DF SS MS F P

Between 5 5359282 1071856 24766 00000

Within 12 51935 4328

Total 17 5411217







Source DF F P

Between 50 79369 00011

Within 47



Variable Mean

V001 66511 (Glycogen-Tetrameric form)

V002 7621 (Glycogen-Dimeric form)

V003 9289 (Amylopectin-Tetrameric form)

V004 17967 (Amylopectin-Dimeric form)

V005 30 (Maltoheptaose-Tetrameric form)

V006 4417 (Maltoheptaose-Dimeric form)






V004 17967 A

V003 92890 B

V002 76208 C

V001 66513 C

V006 44168 D

V005 29500 E





229

Appendix 09

1 Chemical composition of the phosphotase inhibitor cocktail (PI G-

Biosciences trade name Phosphatase ArrestTM Catalog number 788-

450)

Phosphotase inhibitor cocktail (G-Bioscience) has five phosphatase

inhibitors target serinethreonine specific and tyrosine specific and dual

specificity phosphatises The solution is 100x strength containing NaF Na

orthovanadate Na pyrophosphate beta glycerophosphate and Na molybdate

10uL per mL of the amyloplast sample (with total protein concgt1mgmL) was

used in the experiments

2 Chemical composition of the protease inhibitor cocktail (PI G-

Biosciences trade name Photease ArrestTM Catalog number 786-322)

ProteCEASEtrade is a superior general protease inhibitor cocktail that is

suitable for purification from mammalian plant bacteria and yeast samples The

cocktail contains both irreversible and reversible protease inhibitors to inhibit

serine cysteine and other proteases EDTA is an optional component for

inhibiting metalloproteases ProteCEASEtrade has been specifically developed for

large scale preparative applications

230

Appendix 10

Representative graph illustrating the elution profile of amyloplast lysates

ran on Superdex 200 10300GL gel permeation column (Protein

Content= 10 mgmL Loaded Volume 05 mL)

AP 22DAYS RENUKA00110_UV AP 22DAYS RENUKA00110_Fractions AP 22DAYS RENUKA00110_Logbook

-20

00

20

40

60

80

100

mAU

60 70 80 90 100 110 120 ml

A10 A11 A12 A13 A14 A15 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11

Fraction Number

231

Representative graph illustrating the elution profile of recombinant SP ran

on Superdex 200 10300GL gel permeation column

(Protein Content= 675 mgmL Loaded Volume 05 mL)

Fraction Number

sp rrecomb16may201210_UV sp rrecomb16may201210_Fractions sp rrecomb16may201210_Logbook

0

20

40

60

80

100

120

140

mAU

00 50 100 150 ml

A1 A3 A5 A7 A9 A11 A13 A15 B2 B4 B6 B8 B10 B12 B14 C1 C3 C5 C7C8

ABSTRACT

ROLE AND REGULATION OF PLASTIDIAL STARCH PHOSPHORYLASE AND

STARCH SYNTHASE IV IN STARCH BIOSYNTHESIS IN MAIZE

ENDOSPERM AMYLOPLASTS

Renuka M Subasinghe Advisor University of Guelph 2013 Dr Ian Tetlow

Storage starch is synthesized in sub-cellular organelles called amyloplasts

in higher plants The synthesis of the starch granule is a result of the

coordinated activity of several groups of starch biosynthetic enzymes There are

four major groups of these enzymes ADP-glucose pyrophosphorylase (AGPase)

starch synthases (SS) starch branching enzymes (SBE) and starch debranching

enzymes (SDE) Starch phosphorylase (SP) exists as both dimeric and

tetrameric forms in plastids in developing cereal endosperm and catalyses the

reversible transfer of glucosyl units from glucose-1-phosphate to the non-

reducing end of α-1-4 linked glucan chains although the precise role in the

pathway remains unclear The present study was conducted to investigate the

role and regulation of SP and SSIV in starch biosynthesis in developing maize

endosperm The results of this study showed that the tetrameric form of SP

accounts for the majority of measurable catalytic activity with the dimeric form

being barely active and the monomer catalytically inactive A catalytically active

recombinant maize SP was heterologously expressed and used as an affinity

ligand with amyloplast lysates to test protein-protein interactions in vitro

Results showed that the different multimeric status of SP influenced interactions

with other enzymes of starch synthesis Tetrameric SP interacted with SBEI and










endosperm

iv

Acknowledgements





the program















program



v



vi

Table of Contents

Title Page

Abstract


Dedicationv





Page

CHAPTER 11













vii






















viii
























ix





















x




















xi























xii







7 Appendixes218

xiii

List of Figures

CHAPTER 1








CHAPTER 2










surface68



xiv









APase72













xv

CHAPTER 3




















SP126




xvi






beads131






CHAPTER 4


Species151152









xvii












Absence of SSIV171













xviii

List of Tables

CHAPTER 1



CHAPTER 2











substrates84



CHAPTER 3



xix

CHAPTER 4




xx








AP - amyloplasts

















xxi

























xxii





Ser - serine




Thr - threonine






1

CHAPTER 1

2

























3
























4









2002)

5

(A)

(B)




6



7













8
























9

























10

























11

























12

























13











(Geigenberger 2011)













14

























15














16













and Keeling 2001)











17
























18

























19























20
























21
























22

























23

























24




















2005 Gao et al 1997)

25






















26






















27


32168)



















mutually exclusive




28





















29





following equation


















30



higher plants





















31






















al 2006)

32
























33

























34




2001)



Plant Tissue

Substrate

Vmax

(umolminmg)

Km

(mgml)

Reference

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

058 (s)

063 (s)

013 (s)

045 (s)

Yu et al

2001

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

Maltoheptaose

73 (s) 111 (p)

716 (s) 1180(p)

78 (s) 1993 (p)

0017 (s) 0028(p)

025 (s) 094(p)

008 (s) 01 (p)

Mu et al

2001

Sweet potato

tuber crude

extract

Starch

G-1-P

Pi

0077 (s)

0115 (p)

1052 (s) 1498(p)

Young et al

2006

Potato tubers

Recombinant

proteins of

SP-L and SP-

H types

Maltopentaose

SP- L type

SP- H type

Glycogen

SP- L

SP- H

Amylopectin

SP- L

SP- H

Amylose

DP=30

SP- L

SP- H

396 (s) 165 (p)

961 (s) 368 (p)

83 (p)

94 (p)

79 (p)

83 (p)

139 (P)

182 (P)

013 (s)

112 (s)

10400 (p)

98 (p)

82 (p)

36 (p)

76 (P)

87 (P)

Mori et al

1993

35

























36

























37

























38

























39





















(Zeeman et al 2004)

40
























41
























42

























43












was



protein complexes








44

















45

CHAPTER 2

46



21 Introduction

















47
























48










49






















(Fig 22)



50

























51
















52


221 Materials







2212 Chemicals


222 Methods








2009




53
























54















immunoblotting








55






















56

22252 Enzyme assays








C]-G1P was











C]-G-





57













unitsμL-1


4 as the

source of Pi










58









(SDS-PAGE)









for 15hr

59




ProtoGelreg




TEMED 0008 001

















60






TEMED 10 μL 4 μL
















61














Solutions






62
















follows (Table 24)

63




















64













Blotreg











65




















66

23 Results























67









(A) (B)

68





immunoblot

(A)

(B)

69













70




SSIV (Appendix 01)













Days After Anthesis

12 15 19 22 12 15 19 22

A B


12 15 19 22 12 15 19 22

A B


71














20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


72








73








only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2



only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2

74












75
























76




0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number

Lo

gM

W

77





(A)

78







(B)

79




column

(C)

80






(D)

81





(E)

82




glucan substrates












20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29


20 21 22 23 24 25 26 27 28 29



20 21 22 23 24 25 26 27 28 29


83
























ANOVA F= 3557 P= 00004)

84















Substrate

Pre-treatment


Synthetic

Activity

Maltoheptaose

Amylopectin

Glycogen

69060

99453

100526

60056

143352Dagger

114410dagger

60250

90450

73312

59156

75874

74208

27238

32845Dagger

37420dagger

Phosphorolytic

Activity

Maltoheptaose

Amylopectin

24615

46913

27014

58840

- -

- -

16634

33418

85




Glucan Substrate

Treatment

Km (mgmL)

Vmax

(nmolmgmin)

Amylopectin

Untreated

ATP

APase

18plusmn002

31plusmn001

13plusmn005

483plusmn02

654plusmn006

337plusmn02

Maltoheptaose

Untreated

ATP

APase

33plusmn002

67plusmn0001

23plusmn0001

279plusmn001

339plusmn0003

176plusmn002












86





87

24 Discussion























88

























89







configuration


















90




Pi levels


endosperm


















91








phosphorylation
















92












with ATP (Fig 211)













93

























94


substrates






















activity of SP

95
























96

























97












98

CHAPTER 3

99


biosynthesis

31 Introduction













1996)








100

























101
























102
























103





















104
























ngmL-1


105









100 ngmL-1










106






Forward Primers




Reverse Primers



SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1



15bp overhang from




Forward Primers




Reverse Primers




SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1


SP-F1



15bp overhang from




107

























108



















the study)

109

















and pulldown assay






110






















111












112

3 3 Results





















113







SP2 ------------------------------------------------------------











































114












SP2 ------------------------------------------------------------











































115






116

Serine predictions


_________________________v_________________















































































117

_________________________^________________



_________________________v_________________















































_________________________^_________________



_________________________v_________________



































_________________________^_________________


118


3321 PCR















119



ethidium bromide









2805bp

5000

3000

2000

bp

2805bp

5000

3000

2000

bp

120

























121






122





(A) (B)

kDa

150

100

75

50

L 1 2 3 4 5 6 L 1 2 3 4 5 6



2 Uninduced control




123






(B)

(A)

124






(A) (B) (C) (D)

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

125













(Fig 39B)

126






(A)

(B)

(C)

127












310)













128






129














L 1 2 3 4 5 6 7 8

kDa

150

100

75

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8

kDa

150

100

75

Anti-SP

Anti-S-Tag

130






131



132














133

























134





Glucan Substrate

Tetrameric form of

recombinant SP


Dimeric form of

recombinant SP


Glycogen

Amylopectin

Maltoheptaose

89429 0024

131648 0078

49711 00298

5952 0066

9786 873

- -


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger

135

34 Discussion






















136
























137






















138

























139
























140

























141

























142












not interact













143


tested






















144

























145









146

CHAPTER 4

147


41 Introduction






















148

























149






1996)



















150















151












































CC

152





























153
















14-3-3 14-3-3

154

























155








156
















endosperm




antibodies

157





proteins



















158


medium




















159



specific antibodies

160

43 Results



















161



MW MW MW MW

(A)

kDa

150

100

50

MW

(B)

162









163















164





(A)


0 05 1 25 0 05 1 25 0 05 1 25


0 05 1 25 0 05 1 25 0 05 1 25

165


y = 04885x + 12983

000

100

200

300

-3 -25 -2 -15 -1 -05 0 05 1 15 2 25 3

1R

m


Glycogen

y = 10576x + 11474

000

200

400

600

-25 -2 -15 -1 -05 0 05 1 15 2 25

1R

m


Amylopectin

y = 07772x + 11909

0

1

2

3

4

-25 -2 -15 -1 -05 0 05 1 15 2 25 3 35

1R

m


Maltoheptaose

(B)

166



Glucan

Substrate

Kd values (mgmL)

SSI

SSIIa

SP

SSIV


Maltoheptaose

02004

049001 -

-

024001

049001 -

-

002001

008001 -

-

10001 -

25002

1507


32P-ATP













167







location of SSIV

(A) (B)

168


zymogram























169




170





(B)

(A)

171












100 kDa (Fig 49)

1 2



(C)

172





L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L

440 kDa 232kDa

SSIV-Untreated

Control

SSIV- ATP

Treated

SSIV- APase

Treated

100kDa

kDa

150

100

75

150

100

75

150

100

75

Fraction Numbers

173


immunoprecipitation























174










175






(A)

176






large thick band

(B)

177








Anti- SSIV

Anti- SSI

Anti- SSII

Anti- SBEI

Anti- SBEIIb

Anti- SP

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

L Protein marker








8 Amyloplast lysate

178

4 4 Discussion
























179

























180

























181

























182

























183




















49)





184



















185

General Discussion












laboratory












186

























187
























188

























189







catalytic activity


















190



















3)






191

























192


et al 2004)























193

























194

























195

























196
























197

























198









199













200

References


















201


300









1321-1332









202





















203






412


653








Phytologist 10(1111) 1469-8137






204




275-284













Chem 273 25045-25052





205










30 1223ndash1232











10 1295-1306

206




















207





















208








Physiol 120 1147-1156











209









187











210



Genetics 31 75-86


Physiol 53(5) 869-878













24 69-99




211























212





















213










1252


717




215-221





4547-59

214






Research 21 5-32














215




Bot Res 40 1-61















184-195

216





















217














54 865-879

218






was removed





(A) (B)

(C) (D)

219

Appendix 02






anti-maize antisera





(A) (B)

220

Appendix 03



Primer

Name


temperature SP-F1

SP-R1



640C

620C

SP-F2

SP-R2



460C

480C

Appendix 04




Primer Name



sequence T7-

promoter

T7-

terminator

F1

F2

F3





480C

760C

620C

680C

680C

-

- 393-411 bp

1006-1026 bp

1636-1657 bp

221

Appendix 05












80 90 100 110 120 130



10 20 30 40 50 60

140 150 160 170 180 190



70 80 90 100 110 120

200 210 220 230 240 250



130 140 150 160 170 180

260 270 280 290 300 310



190 200 210 220 230 240

320 330 340 350 360 370



250 260 270 280 290 300

222

380 390 400 410 420 430



310 320 330 340 350 360

440 450 460 470 480 490



370 380 390 400 410 420

500 510 520 530 540 550



430 440 450 460 470 480

560 570 580 590 600 610



490 500 510 520 530 540

620 630 640 650 660 670



550 560 570 580 590 600

680 690 700 710 720 730



610 620 630 640 650 660

740 750 760 770



223

Appendix 06


server


Ser 37 Thr 7 Tyr 9

Serine predictions


_________________________v_________________










































































_________________________^_________________

224



_________________________v_________________













































_________________________^_________________



_________________________v_________________




























_________________________^_________________

225

Appendix 07





SP antisera

SBEIIb Co-IP


kDa

150

100

75

50

kDa

150

100

75

50

kDa

150

100

75

50

SBEIIb Co-IP

Probed with anti-SP

SBEIIb Co-IP


L Protein marker







L 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6

226

Appendix 08



different glucans


Source DF SS MS F P

Between 8 395428 494286 8274 0028

Within 18 10753 5974

Total 26 406182







Source DF F P

Between 80 9327 00008

Within 74



Variable Mean















V008 14334 A

V002 11445 B

V001 10053 C

V007 99427 C

V004 69077 D

V005 60013 D

V003 37430 E

V009 32877 E

V006 27353 E


227




Statistix


different glucans


Source DF SS MS F P

Between 5 367208 734416 3557 00004

Within 12 24776 20647

Total 17 391984







Source DF F P

Between 50 2605 00008

Within 55



Variable Mean












V005 58873 A

V004 46913 B

V006 33433 C

V002 27040 CD

V001 24493 DE

V003 16640 E





228

Statistix




Source DF SS MS F P

Between 5 5359282 1071856 24766 00000

Within 12 51935 4328

Total 17 5411217







Source DF F P

Between 50 79369 00011

Within 47



Variable Mean












V004 17967 A

V003 92890 B

V002 76208 C

V001 66513 C

V006 44168 D

V005 29500 E





229

Appendix 09



450)















230

Appendix 10





-20

00

20

40

60

80

100

mAU

60 70 80 90 100 110 120 ml


Fraction Number

231




Fraction Number


0

20

40

60

80

100

120

140

mAU

00 50 100 150 ml











endosperm

iv

Acknowledgements





the program















program



v



vi

Table of Contents

Title Page

Abstract


Dedicationv





Page

CHAPTER 11













vii






















viii
























ix





















x




















xi























xii







7 Appendixes218

xiii

List of Figures

CHAPTER 1








CHAPTER 2










surface68



xiv









APase72













xv

CHAPTER 3




















SP126




xvi






beads131






CHAPTER 4


Species151152









xvii












Absence of SSIV171













xviii

List of Tables

CHAPTER 1



CHAPTER 2











substrates84



CHAPTER 3



xix

CHAPTER 4




xx








AP - amyloplasts

















xxi

























xxii





Ser - serine




Thr - threonine






1

CHAPTER 1

2

























3
























4









2002)

5

(A)

(B)




6



7













8
























9

























10

























11

























12

























13











(Geigenberger 2011)













14

























15














16













and Keeling 2001)











17
























18

























19























20
























21
























22

























23

























24




















2005 Gao et al 1997)

25






















26






















27


32168)



















mutually exclusive




28





















29





following equation


















30



higher plants





















31






















al 2006)

32
























33

























34




2001)



Plant Tissue

Substrate

Vmax

(umolminmg)

Km

(mgml)

Reference

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

058 (s)

063 (s)

013 (s)

045 (s)

Yu et al

2001

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

Maltoheptaose

73 (s) 111 (p)

716 (s) 1180(p)

78 (s) 1993 (p)

0017 (s) 0028(p)

025 (s) 094(p)

008 (s) 01 (p)

Mu et al

2001

Sweet potato

tuber crude

extract

Starch

G-1-P

Pi

0077 (s)

0115 (p)

1052 (s) 1498(p)

Young et al

2006

Potato tubers

Recombinant

proteins of

SP-L and SP-

H types

Maltopentaose

SP- L type

SP- H type

Glycogen

SP- L

SP- H

Amylopectin

SP- L

SP- H

Amylose

DP=30

SP- L

SP- H

396 (s) 165 (p)

961 (s) 368 (p)

83 (p)

94 (p)

79 (p)

83 (p)

139 (P)

182 (P)

013 (s)

112 (s)

10400 (p)

98 (p)

82 (p)

36 (p)

76 (P)

87 (P)

Mori et al

1993

35

























36

























37

























38

























39





















(Zeeman et al 2004)

40
























41
























42

























43












was



protein complexes








44

















45

CHAPTER 2

46



21 Introduction

















47
























48










49






















(Fig 22)



50

























51
















52


221 Materials







2212 Chemicals


222 Methods








2009




53
























54















immunoblotting








55






















56

22252 Enzyme assays








C]-G1P was











C]-G-





57













unitsμL-1


4 as the

source of Pi










58









(SDS-PAGE)









for 15hr

59




ProtoGelreg




TEMED 0008 001

















60






TEMED 10 μL 4 μL
















61














Solutions






62
















follows (Table 24)

63




















64













Blotreg











65




















66

23 Results























67









(A) (B)

68





immunoblot

(A)

(B)

69













70




SSIV (Appendix 01)













Days After Anthesis

12 15 19 22 12 15 19 22

A B


12 15 19 22 12 15 19 22

A B


71














20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


72








73








only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2



only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2

74












75
























76




0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number

Lo

gM

W

77





(A)

78







(B)

79




column

(C)

80






(D)

81





(E)

82




glucan substrates












20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29


20 21 22 23 24 25 26 27 28 29



20 21 22 23 24 25 26 27 28 29


83
























ANOVA F= 3557 P= 00004)

84















Substrate

Pre-treatment


Synthetic

Activity

Maltoheptaose

Amylopectin

Glycogen

69060

99453

100526

60056

143352Dagger

114410dagger

60250

90450

73312

59156

75874

74208

27238

32845Dagger

37420dagger

Phosphorolytic

Activity

Maltoheptaose

Amylopectin

24615

46913

27014

58840

- -

- -

16634

33418

85




Glucan Substrate

Treatment

Km (mgmL)

Vmax

(nmolmgmin)

Amylopectin

Untreated

ATP

APase

18plusmn002

31plusmn001

13plusmn005

483plusmn02

654plusmn006

337plusmn02

Maltoheptaose

Untreated

ATP

APase

33plusmn002

67plusmn0001

23plusmn0001

279plusmn001

339plusmn0003

176plusmn002












86





87

24 Discussion























88

























89







configuration


















90




Pi levels


endosperm


















91








phosphorylation
















92












with ATP (Fig 211)













93

























94


substrates






















activity of SP

95
























96

























97












98

CHAPTER 3

99


biosynthesis

31 Introduction













1996)








100

























101
























102
























103





















104
























ngmL-1


105









100 ngmL-1










106






Forward Primers




Reverse Primers



SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1



15bp overhang from




Forward Primers




Reverse Primers




SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1


SP-F1



15bp overhang from




107

























108



















the study)

109

















and pulldown assay






110






















111












112

3 3 Results





















113







SP2 ------------------------------------------------------------











































114












SP2 ------------------------------------------------------------











































115






116

Serine predictions


_________________________v_________________















































































117

_________________________^________________



_________________________v_________________















































_________________________^_________________



_________________________v_________________



































_________________________^_________________


118


3321 PCR















119



ethidium bromide









2805bp

5000

3000

2000

bp

2805bp

5000

3000

2000

bp

120

























121






122





(A) (B)

kDa

150

100

75

50

L 1 2 3 4 5 6 L 1 2 3 4 5 6



2 Uninduced control




123






(B)

(A)

124






(A) (B) (C) (D)

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

125













(Fig 39B)

126






(A)

(B)

(C)

127












310)













128






129














L 1 2 3 4 5 6 7 8

kDa

150

100

75

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8

kDa

150

100

75

Anti-SP

Anti-S-Tag

130






131



132














133

























134





Glucan Substrate

Tetrameric form of

recombinant SP


Dimeric form of

recombinant SP


Glycogen

Amylopectin

Maltoheptaose

89429 0024

131648 0078

49711 00298

5952 0066

9786 873

- -


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger

135

34 Discussion






















136
























137






















138

























139
























140

























141

























142












not interact













143


tested






















144

























145









146

CHAPTER 4

147


41 Introduction






















148

























149






1996)



















150















151












































CC

152





























153
















14-3-3 14-3-3

154

























155








156
















endosperm




antibodies

157





proteins



















158


medium




















159



specific antibodies

160

43 Results



















161



MW MW MW MW

(A)

kDa

150

100

50

MW

(B)

162









163















164





(A)


0 05 1 25 0 05 1 25 0 05 1 25


0 05 1 25 0 05 1 25 0 05 1 25

165


y = 04885x + 12983

000

100

200

300

-3 -25 -2 -15 -1 -05 0 05 1 15 2 25 3

1R

m


Glycogen

y = 10576x + 11474

000

200

400

600

-25 -2 -15 -1 -05 0 05 1 15 2 25

1R

m


Amylopectin

y = 07772x + 11909

0

1

2

3

4

-25 -2 -15 -1 -05 0 05 1 15 2 25 3 35

1R

m


Maltoheptaose

(B)

166



Glucan

Substrate

Kd values (mgmL)

SSI

SSIIa

SP

SSIV


Maltoheptaose

02004

049001 -

-

024001

049001 -

-

002001

008001 -

-

10001 -

25002

1507


32P-ATP













167







location of SSIV

(A) (B)

168


zymogram























169




170





(B)

(A)

171












100 kDa (Fig 49)

1 2



(C)

172





L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L

440 kDa 232kDa

SSIV-Untreated

Control

SSIV- ATP

Treated

SSIV- APase

Treated

100kDa

kDa

150

100

75

150

100

75

150

100

75

Fraction Numbers

173


immunoprecipitation























174










175






(A)

176






large thick band

(B)

177








Anti- SSIV

Anti- SSI

Anti- SSII

Anti- SBEI

Anti- SBEIIb

Anti- SP

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

L Protein marker








8 Amyloplast lysate

178

4 4 Discussion
























179

























180

























181

























182

























183




















49)





184



















185

General Discussion












laboratory












186

























187
























188

























189







catalytic activity


















190



















3)






191

























192


et al 2004)























193

























194

























195

























196
























197

























198









199













200

References


















201


300









1321-1332









202





















203






412


653








Phytologist 10(1111) 1469-8137






204




275-284













Chem 273 25045-25052





205










30 1223ndash1232











10 1295-1306

206




















207





















208








Physiol 120 1147-1156











209









187











210



Genetics 31 75-86


Physiol 53(5) 869-878













24 69-99




211























212





















213










1252


717




215-221





4547-59

214






Research 21 5-32














215




Bot Res 40 1-61















184-195

216





















217














54 865-879

218






was removed





(A) (B)

(C) (D)

219

Appendix 02






anti-maize antisera





(A) (B)

220

Appendix 03



Primer

Name


temperature SP-F1

SP-R1



640C

620C

SP-F2

SP-R2



460C

480C

Appendix 04




Primer Name



sequence T7-

promoter

T7-

terminator

F1

F2

F3





480C

760C

620C

680C

680C

-

- 393-411 bp

1006-1026 bp

1636-1657 bp

221

Appendix 05












80 90 100 110 120 130



10 20 30 40 50 60

140 150 160 170 180 190



70 80 90 100 110 120

200 210 220 230 240 250



130 140 150 160 170 180

260 270 280 290 300 310



190 200 210 220 230 240

320 330 340 350 360 370



250 260 270 280 290 300

222

380 390 400 410 420 430



310 320 330 340 350 360

440 450 460 470 480 490



370 380 390 400 410 420

500 510 520 530 540 550



430 440 450 460 470 480

560 570 580 590 600 610



490 500 510 520 530 540

620 630 640 650 660 670



550 560 570 580 590 600

680 690 700 710 720 730



610 620 630 640 650 660

740 750 760 770



223

Appendix 06


server


Ser 37 Thr 7 Tyr 9

Serine predictions


_________________________v_________________










































































_________________________^_________________

224



_________________________v_________________













































_________________________^_________________



_________________________v_________________




























_________________________^_________________

225

Appendix 07





SP antisera

SBEIIb Co-IP


kDa

150

100

75

50

kDa

150

100

75

50

kDa

150

100

75

50

SBEIIb Co-IP

Probed with anti-SP

SBEIIb Co-IP


L Protein marker







L 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6

226

Appendix 08



different glucans


Source DF SS MS F P

Between 8 395428 494286 8274 0028

Within 18 10753 5974

Total 26 406182







Source DF F P

Between 80 9327 00008

Within 74



Variable Mean















V008 14334 A

V002 11445 B

V001 10053 C

V007 99427 C

V004 69077 D

V005 60013 D

V003 37430 E

V009 32877 E

V006 27353 E


227




Statistix


different glucans


Source DF SS MS F P

Between 5 367208 734416 3557 00004

Within 12 24776 20647

Total 17 391984







Source DF F P

Between 50 2605 00008

Within 55



Variable Mean












V005 58873 A

V004 46913 B

V006 33433 C

V002 27040 CD

V001 24493 DE

V003 16640 E





228

Statistix




Source DF SS MS F P

Between 5 5359282 1071856 24766 00000

Within 12 51935 4328

Total 17 5411217







Source DF F P

Between 50 79369 00011

Within 47



Variable Mean












V004 17967 A

V003 92890 B

V002 76208 C

V001 66513 C

V006 44168 D

V005 29500 E





229

Appendix 09



450)















230

Appendix 10





-20

00

20

40

60

80

100

mAU

60 70 80 90 100 110 120 ml


Fraction Number

231




Fraction Number


0

20

40

60

80

100

120

140

mAU

00 50 100 150 ml


iv

Acknowledgements





the program















program



v



vi

Table of Contents

Title Page

Abstract


Dedicationv





Page

CHAPTER 11













vii






















viii
























ix





















x




















xi























xii







7 Appendixes218

xiii

List of Figures

CHAPTER 1








CHAPTER 2










surface68



xiv









APase72













xv

CHAPTER 3




















SP126




xvi






beads131






CHAPTER 4


Species151152









xvii












Absence of SSIV171













xviii

List of Tables

CHAPTER 1



CHAPTER 2











substrates84



CHAPTER 3



xix

CHAPTER 4




xx








AP - amyloplasts

















xxi

























xxii





Ser - serine




Thr - threonine






1

CHAPTER 1

2

























3
























4









2002)

5

(A)

(B)




6



7













8
























9

























10

























11

























12

























13











(Geigenberger 2011)













14

























15














16













and Keeling 2001)











17
























18

























19























20
























21
























22

























23

























24




















2005 Gao et al 1997)

25






















26






















27


32168)



















mutually exclusive




28





















29





following equation


















30



higher plants





















31






















al 2006)

32
























33

























34




2001)



Plant Tissue

Substrate

Vmax

(umolminmg)

Km

(mgml)

Reference

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

058 (s)

063 (s)

013 (s)

045 (s)

Yu et al

2001

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

Maltoheptaose

73 (s) 111 (p)

716 (s) 1180(p)

78 (s) 1993 (p)

0017 (s) 0028(p)

025 (s) 094(p)

008 (s) 01 (p)

Mu et al

2001

Sweet potato

tuber crude

extract

Starch

G-1-P

Pi

0077 (s)

0115 (p)

1052 (s) 1498(p)

Young et al

2006

Potato tubers

Recombinant

proteins of

SP-L and SP-

H types

Maltopentaose

SP- L type

SP- H type

Glycogen

SP- L

SP- H

Amylopectin

SP- L

SP- H

Amylose

DP=30

SP- L

SP- H

396 (s) 165 (p)

961 (s) 368 (p)

83 (p)

94 (p)

79 (p)

83 (p)

139 (P)

182 (P)

013 (s)

112 (s)

10400 (p)

98 (p)

82 (p)

36 (p)

76 (P)

87 (P)

Mori et al

1993

35

























36

























37

























38

























39





















(Zeeman et al 2004)

40
























41
























42

























43












was



protein complexes








44

















45

CHAPTER 2

46



21 Introduction

















47
























48










49






















(Fig 22)



50

























51
















52


221 Materials







2212 Chemicals


222 Methods








2009




53
























54















immunoblotting








55






















56

22252 Enzyme assays








C]-G1P was











C]-G-





57













unitsμL-1


4 as the

source of Pi










58









(SDS-PAGE)









for 15hr

59




ProtoGelreg




TEMED 0008 001

















60






TEMED 10 μL 4 μL
















61














Solutions






62
















follows (Table 24)

63




















64













Blotreg











65




















66

23 Results























67









(A) (B)

68





immunoblot

(A)

(B)

69













70




SSIV (Appendix 01)













Days After Anthesis

12 15 19 22 12 15 19 22

A B


12 15 19 22 12 15 19 22

A B


71














20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


72








73








only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2



only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2

74












75
























76




0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number

Lo

gM

W

77





(A)

78







(B)

79




column

(C)

80






(D)

81





(E)

82




glucan substrates












20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29


20 21 22 23 24 25 26 27 28 29



20 21 22 23 24 25 26 27 28 29


83
























ANOVA F= 3557 P= 00004)

84















Substrate

Pre-treatment


Synthetic

Activity

Maltoheptaose

Amylopectin

Glycogen

69060

99453

100526

60056

143352Dagger

114410dagger

60250

90450

73312

59156

75874

74208

27238

32845Dagger

37420dagger

Phosphorolytic

Activity

Maltoheptaose

Amylopectin

24615

46913

27014

58840

- -

- -

16634

33418

85




Glucan Substrate

Treatment

Km (mgmL)

Vmax

(nmolmgmin)

Amylopectin

Untreated

ATP

APase

18plusmn002

31plusmn001

13plusmn005

483plusmn02

654plusmn006

337plusmn02

Maltoheptaose

Untreated

ATP

APase

33plusmn002

67plusmn0001

23plusmn0001

279plusmn001

339plusmn0003

176plusmn002












86





87

24 Discussion























88

























89







configuration


















90




Pi levels


endosperm


















91








phosphorylation
















92












with ATP (Fig 211)













93

























94


substrates






















activity of SP

95
























96

























97












98

CHAPTER 3

99


biosynthesis

31 Introduction













1996)








100

























101
























102
























103





















104
























ngmL-1


105









100 ngmL-1










106






Forward Primers




Reverse Primers



SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1



15bp overhang from




Forward Primers




Reverse Primers




SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1


SP-F1



15bp overhang from




107

























108



















the study)

109

















and pulldown assay






110






















111












112

3 3 Results





















113







SP2 ------------------------------------------------------------











































114












SP2 ------------------------------------------------------------











































115






116

Serine predictions


_________________________v_________________















































































117

_________________________^________________



_________________________v_________________















































_________________________^_________________



_________________________v_________________



































_________________________^_________________


118


3321 PCR















119



ethidium bromide









2805bp

5000

3000

2000

bp

2805bp

5000

3000

2000

bp

120

























121






122





(A) (B)

kDa

150

100

75

50

L 1 2 3 4 5 6 L 1 2 3 4 5 6



2 Uninduced control




123






(B)

(A)

124






(A) (B) (C) (D)

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

125













(Fig 39B)

126






(A)

(B)

(C)

127












310)













128






129














L 1 2 3 4 5 6 7 8

kDa

150

100

75

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8

kDa

150

100

75

Anti-SP

Anti-S-Tag

130






131



132














133

























134





Glucan Substrate

Tetrameric form of

recombinant SP


Dimeric form of

recombinant SP


Glycogen

Amylopectin

Maltoheptaose

89429 0024

131648 0078

49711 00298

5952 0066

9786 873

- -


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger

135

34 Discussion






















136
























137






















138

























139
























140

























141

























142












not interact













143


tested






















144

























145









146

CHAPTER 4

147


41 Introduction






















148

























149






1996)



















150















151












































CC

152





























153
















14-3-3 14-3-3

154

























155








156
















endosperm




antibodies

157





proteins



















158


medium




















159



specific antibodies

160

43 Results



















161



MW MW MW MW

(A)

kDa

150

100

50

MW

(B)

162









163















164





(A)


0 05 1 25 0 05 1 25 0 05 1 25


0 05 1 25 0 05 1 25 0 05 1 25

165


y = 04885x + 12983

000

100

200

300

-3 -25 -2 -15 -1 -05 0 05 1 15 2 25 3

1R

m


Glycogen

y = 10576x + 11474

000

200

400

600

-25 -2 -15 -1 -05 0 05 1 15 2 25

1R

m


Amylopectin

y = 07772x + 11909

0

1

2

3

4

-25 -2 -15 -1 -05 0 05 1 15 2 25 3 35

1R

m


Maltoheptaose

(B)

166



Glucan

Substrate

Kd values (mgmL)

SSI

SSIIa

SP

SSIV


Maltoheptaose

02004

049001 -

-

024001

049001 -

-

002001

008001 -

-

10001 -

25002

1507


32P-ATP













167







location of SSIV

(A) (B)

168


zymogram























169




170





(B)

(A)

171












100 kDa (Fig 49)

1 2



(C)

172





L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L

440 kDa 232kDa

SSIV-Untreated

Control

SSIV- ATP

Treated

SSIV- APase

Treated

100kDa

kDa

150

100

75

150

100

75

150

100

75

Fraction Numbers

173


immunoprecipitation























174










175






(A)

176






large thick band

(B)

177








Anti- SSIV

Anti- SSI

Anti- SSII

Anti- SBEI

Anti- SBEIIb

Anti- SP

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

L Protein marker








8 Amyloplast lysate

178

4 4 Discussion
























179

























180

























181

























182

























183




















49)





184



















185

General Discussion












laboratory












186

























187
























188

























189







catalytic activity


















190



















3)






191

























192


et al 2004)























193

























194

























195

























196
























197

























198









199













200

References


















201


300









1321-1332









202





















203






412


653








Phytologist 10(1111) 1469-8137






204




275-284













Chem 273 25045-25052





205










30 1223ndash1232











10 1295-1306

206




















207





















208








Physiol 120 1147-1156











209









187











210



Genetics 31 75-86


Physiol 53(5) 869-878













24 69-99




211























212





















213










1252


717




215-221





4547-59

214






Research 21 5-32














215




Bot Res 40 1-61















184-195

216





















217














54 865-879

218






was removed





(A) (B)

(C) (D)

219

Appendix 02






anti-maize antisera





(A) (B)

220

Appendix 03



Primer

Name


temperature SP-F1

SP-R1



640C

620C

SP-F2

SP-R2



460C

480C

Appendix 04




Primer Name



sequence T7-

promoter

T7-

terminator

F1

F2

F3





480C

760C

620C

680C

680C

-

- 393-411 bp

1006-1026 bp

1636-1657 bp

221

Appendix 05












80 90 100 110 120 130



10 20 30 40 50 60

140 150 160 170 180 190



70 80 90 100 110 120

200 210 220 230 240 250



130 140 150 160 170 180

260 270 280 290 300 310



190 200 210 220 230 240

320 330 340 350 360 370



250 260 270 280 290 300

222

380 390 400 410 420 430



310 320 330 340 350 360

440 450 460 470 480 490



370 380 390 400 410 420

500 510 520 530 540 550



430 440 450 460 470 480

560 570 580 590 600 610



490 500 510 520 530 540

620 630 640 650 660 670



550 560 570 580 590 600

680 690 700 710 720 730



610 620 630 640 650 660

740 750 760 770



223

Appendix 06


server


Ser 37 Thr 7 Tyr 9

Serine predictions


_________________________v_________________










































































_________________________^_________________

224



_________________________v_________________













































_________________________^_________________



_________________________v_________________




























_________________________^_________________

225

Appendix 07





SP antisera

SBEIIb Co-IP


kDa

150

100

75

50

kDa

150

100

75

50

kDa

150

100

75

50

SBEIIb Co-IP

Probed with anti-SP

SBEIIb Co-IP


L Protein marker







L 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6

226

Appendix 08



different glucans


Source DF SS MS F P

Between 8 395428 494286 8274 0028

Within 18 10753 5974

Total 26 406182







Source DF F P

Between 80 9327 00008

Within 74



Variable Mean















V008 14334 A

V002 11445 B

V001 10053 C

V007 99427 C

V004 69077 D

V005 60013 D

V003 37430 E

V009 32877 E

V006 27353 E


227




Statistix


different glucans


Source DF SS MS F P

Between 5 367208 734416 3557 00004

Within 12 24776 20647

Total 17 391984







Source DF F P

Between 50 2605 00008

Within 55



Variable Mean












V005 58873 A

V004 46913 B

V006 33433 C

V002 27040 CD

V001 24493 DE

V003 16640 E





228

Statistix




Source DF SS MS F P

Between 5 5359282 1071856 24766 00000

Within 12 51935 4328

Total 17 5411217







Source DF F P

Between 50 79369 00011

Within 47



Variable Mean












V004 17967 A

V003 92890 B

V002 76208 C

V001 66513 C

V006 44168 D

V005 29500 E





229

Appendix 09



450)















230

Appendix 10





-20

00

20

40

60

80

100

mAU

60 70 80 90 100 110 120 ml


Fraction Number

231




Fraction Number


0

20

40

60

80

100

120

140

mAU

00 50 100 150 ml


v



vi

Table of Contents

Title Page

Abstract


Dedicationv





Page

CHAPTER 11













vii






















viii
























ix





















x




















xi























xii







7 Appendixes218

xiii

List of Figures

CHAPTER 1








CHAPTER 2










surface68



xiv









APase72













xv

CHAPTER 3




















SP126




xvi






beads131






CHAPTER 4


Species151152









xvii












Absence of SSIV171













xviii

List of Tables

CHAPTER 1



CHAPTER 2











substrates84



CHAPTER 3



xix

CHAPTER 4




xx








AP - amyloplasts

















xxi

























xxii





Ser - serine




Thr - threonine






1

CHAPTER 1

2

























3
























4









2002)

5

(A)

(B)




6



7













8
























9

























10

























11

























12

























13











(Geigenberger 2011)













14

























15














16













and Keeling 2001)











17
























18

























19























20
























21
























22

























23

























24




















2005 Gao et al 1997)

25






















26






















27


32168)



















mutually exclusive




28





















29





following equation


















30



higher plants





















31






















al 2006)

32
























33

























34




2001)



Plant Tissue

Substrate

Vmax

(umolminmg)

Km

(mgml)

Reference

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

058 (s)

063 (s)

013 (s)

045 (s)

Yu et al

2001

Purified SP

from maize

amyloplast

stroma

Amylopectin

Glycogen

Maltoheptaose

73 (s) 111 (p)

716 (s) 1180(p)

78 (s) 1993 (p)

0017 (s) 0028(p)

025 (s) 094(p)

008 (s) 01 (p)

Mu et al

2001

Sweet potato

tuber crude

extract

Starch

G-1-P

Pi

0077 (s)

0115 (p)

1052 (s) 1498(p)

Young et al

2006

Potato tubers

Recombinant

proteins of

SP-L and SP-

H types

Maltopentaose

SP- L type

SP- H type

Glycogen

SP- L

SP- H

Amylopectin

SP- L

SP- H

Amylose

DP=30

SP- L

SP- H

396 (s) 165 (p)

961 (s) 368 (p)

83 (p)

94 (p)

79 (p)

83 (p)

139 (P)

182 (P)

013 (s)

112 (s)

10400 (p)

98 (p)

82 (p)

36 (p)

76 (P)

87 (P)

Mori et al

1993

35

























36

























37

























38

























39





















(Zeeman et al 2004)

40
























41
























42

























43












was



protein complexes








44

















45

CHAPTER 2

46



21 Introduction

















47
























48










49






















(Fig 22)



50

























51
















52


221 Materials







2212 Chemicals


222 Methods








2009




53
























54















immunoblotting








55






















56

22252 Enzyme assays








C]-G1P was











C]-G-





57













unitsμL-1


4 as the

source of Pi










58









(SDS-PAGE)









for 15hr

59




ProtoGelreg




TEMED 0008 001

















60






TEMED 10 μL 4 μL
















61














Solutions






62
















follows (Table 24)

63




















64













Blotreg











65




















66

23 Results























67









(A) (B)

68





immunoblot

(A)

(B)

69













70




SSIV (Appendix 01)













Days After Anthesis

12 15 19 22 12 15 19 22

A B


12 15 19 22 12 15 19 22

A B


71














20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


20 10 5 2 1

mM mM mM mM mM

20 10 5 2 1

mM mM mM mM mM

G-1-P Na2HPO4


72








73








only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2



only

A1 A2

B1 B2

C1 C2

Pho1

Pho2

Pho1

Pho1Pho2

74












75
























76




0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number

Lo

gM

W

77





(A)

78







(B)

79




column

(C)

80






(D)

81





(E)

82




glucan substrates












20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29


20 21 22 23 24 25 26 27 28 29



20 21 22 23 24 25 26 27 28 29


83
























ANOVA F= 3557 P= 00004)

84















Substrate

Pre-treatment


Synthetic

Activity

Maltoheptaose

Amylopectin

Glycogen

69060

99453

100526

60056

143352Dagger

114410dagger

60250

90450

73312

59156

75874

74208

27238

32845Dagger

37420dagger

Phosphorolytic

Activity

Maltoheptaose

Amylopectin

24615

46913

27014

58840

- -

- -

16634

33418

85




Glucan Substrate

Treatment

Km (mgmL)

Vmax

(nmolmgmin)

Amylopectin

Untreated

ATP

APase

18plusmn002

31plusmn001

13plusmn005

483plusmn02

654plusmn006

337plusmn02

Maltoheptaose

Untreated

ATP

APase

33plusmn002

67plusmn0001

23plusmn0001

279plusmn001

339plusmn0003

176plusmn002












86





87

24 Discussion























88

























89







configuration


















90




Pi levels


endosperm


















91








phosphorylation
















92












with ATP (Fig 211)













93

























94


substrates






















activity of SP

95
























96

























97












98

CHAPTER 3

99


biosynthesis

31 Introduction













1996)








100

























101
























102
























103





















104
























ngmL-1


105









100 ngmL-1










106






Forward Primers




Reverse Primers



SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1



15bp overhang from




Forward Primers




Reverse Primers




SP-R2-3005bp


pET29a vector

3030bp--3010bp


SP-F2SP-F1


SP-F1



15bp overhang from




107

























108



















the study)

109

















and pulldown assay






110






















111












112

3 3 Results





















113







SP2 ------------------------------------------------------------











































114












SP2 ------------------------------------------------------------











































115






116

Serine predictions


_________________________v_________________















































































117

_________________________^________________



_________________________v_________________















































_________________________^_________________



_________________________v_________________



































_________________________^_________________


118


3321 PCR















119



ethidium bromide









2805bp

5000

3000

2000

bp

2805bp

5000

3000

2000

bp

120

























121






122





(A) (B)

kDa

150

100

75

50

L 1 2 3 4 5 6 L 1 2 3 4 5 6



2 Uninduced control




123






(B)

(A)

124






(A) (B) (C) (D)

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP synthetic


synthetic zymogram

probed with anti-SP

antibodies

Immunoblot of SP

synthetic zymogram

probed with anti-S-

tag antibodies

SP degradative

activity zymogram

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

SP

Recom

bin

ant

Am

ylo

pla

stly

sate

s

125













(Fig 39B)

126






(A)

(B)

(C)

127












310)













128






129














L 1 2 3 4 5 6 7 8

kDa

150

100

75

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8

kDa

150

100

75

Anti-SP

Anti-S-Tag

130






131



132














133

























134





Glucan Substrate

Tetrameric form of

recombinant SP


Dimeric form of

recombinant SP


Glycogen

Amylopectin

Maltoheptaose

89429 0024

131648 0078

49711 00298

5952 0066

9786 873

- -


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger


SP

0250500750

1000

1250150017502000

S P S P S P

Gly Amy Malto

En

zym

e A

ctivity (

nm

olm

gm

in)

Dimeric form of

Recombinant SP

0

20

40

60

80

100

120

S P S P S P

Gly Amy MaltoE

nzym

e A

ctivity (

nm

olm

gm

in)

+

+

Dagger

Dagger

Dagger

135

34 Discussion






















136
























137






















138

























139
























140

























141

























142












not interact













143


tested






















144

























145









146

CHAPTER 4

147


41 Introduction






















148

























149






1996)



















150















151












































CC

152





























153
















14-3-3 14-3-3

154

























155








156
















endosperm




antibodies

157





proteins



















158


medium




















159



specific antibodies

160

43 Results



















161



MW MW MW MW

(A)

kDa

150

100

50

MW

(B)

162









163















164





(A)


0 05 1 25 0 05 1 25 0 05 1 25


0 05 1 25 0 05 1 25 0 05 1 25

165


y = 04885x + 12983

000

100

200

300

-3 -25 -2 -15 -1 -05 0 05 1 15 2 25 3

1R

m


Glycogen

y = 10576x + 11474

000

200

400

600

-25 -2 -15 -1 -05 0 05 1 15 2 25

1R

m


Amylopectin

y = 07772x + 11909

0

1

2

3

4

-25 -2 -15 -1 -05 0 05 1 15 2 25 3 35

1R

m


Maltoheptaose

(B)

166



Glucan

Substrate

Kd values (mgmL)

SSI

SSIIa

SP

SSIV


Maltoheptaose

02004

049001 -

-

024001

049001 -

-

002001

008001 -

-

10001 -

25002

1507


32P-ATP













167







location of SSIV

(A) (B)

168


zymogram























169




170





(B)

(A)

171












100 kDa (Fig 49)

1 2



(C)

172





L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L

440 kDa 232kDa

SSIV-Untreated

Control

SSIV- ATP

Treated

SSIV- APase

Treated

100kDa

kDa

150

100

75

150

100

75

150

100

75

Fraction Numbers

173


immunoprecipitation























174










175






(A)

176






large thick band

(B)

177








Anti- SSIV

Anti- SSI

Anti- SSII

Anti- SBEI

Anti- SBEIIb

Anti- SP

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8

L 1 2 3 4 5 6 7 8 kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

kDa150

100

75

L Protein marker








8 Amyloplast lysate

178

4 4 Discussion
























179

























180

























181

























182

























183




















49)





184



















185

General Discussion












laboratory












186

























187
























188

























189







catalytic activity


















190



















3)






191

























192


et al 2004)























193

























194

























195

























196
























197

























198









199













200

References


















201


300









1321-1332









202





















203






412


653








Phytologist 10(1111) 1469-8137






204




275-284













Chem 273 25045-25052





205










30 1223ndash1232











10 1295-1306

206




















207





















208








Physiol 120 1147-1156











209









187











210



Genetics 31 75-86


Physiol 53(5) 869-878













24 69-99




211























212





















213










1252


717




215-221





4547-59

214






Research 21 5-32














215




Bot Res 40 1-61















184-195

216





















217














54 865-879

218






was removed





(A) (B)

(C) (D)

219

Appendix 02






anti-maize antisera





(A) (B)

220

Appendix 03



Primer

Name


temperature SP-F1

SP-R1



640C

620C

SP-F2

SP-R2



460C

480C

Appendix 04




Primer Name



sequence T7-

promoter

T7-

terminator

F1

F2

F3





480C

760C

620C

680C

680C

-

- 393-411 bp

1006-1026 bp

1636-1657 bp

221

Appendix 05












80 90 100 110 120 130



10 20 30 40 50 60

140 150 160 170 180 190



70 80 90 100 110 120

200 210 220 230 240 250



130 140 150 160 170 180

260 270 280 290 300 310



190 200 210 220 230 240

320 330 340 350 360 370



250 260 270 280 290 300

222

380 390 400 410 420 430



310 320 330 340 350 360

440 450 460 470 480 490



370 380 390 400 410 420

500 510 520 530 540 550



430 440 450 460 470 480

560 570 580 590 600 610



490 500 510 520 530 540

620 630 640 650 660 670



550 560 570 580 590 600

680 690 700 710 720 730



610 620 630 640 650 660

740 750 760 770



223

Appendix 06


server


Ser 37 Thr 7 Tyr 9

Serine predictions


_________________________v_________________










































































_________________________^_________________

224



_________________________v_________________













































_________________________^_________________



_________________________v_________________




























_________________________^_________________

225

Appendix 07





SP antisera

SBEIIb Co-IP


kDa

150

100

75

50

kDa

150

100

75

50

kDa

150

100

75

50

SBEIIb Co-IP

Probed with anti-SP

SBEIIb Co-IP


L Protein marker







L 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6

226

Appendix 08



different glucans


Source DF SS MS F P

Between 8 395428 494286 8274 0028

Within 18 10753 5974

Total 26 406182







Source DF F P

Between 80 9327 00008

Within 74



Variable Mean















V008 14334 A

V002 11445 B

V001 10053 C

V007 99427 C

V004 69077 D

V005 60013 D

V003 37430 E

V009 32877 E

V006 27353 E


227




Statistix


different glucans


Source DF SS MS F P

Between 5 367208 734416 3557 00004

Within 12 24776 20647

Total 17 391984







Source DF F P

Between 50 2605 00008

Within 55



Variable Mean












V005 58873 A

V004 46913 B

V006 33433 C

V002 27040 CD

V001 24493 DE

V003 16640 E





228

Statistix




Source DF SS MS F P

Between 5 5359282 1071856 24766 00000

Within 12 51935 4328

Total 17 5411217







Source DF F P

Between 50 79369 00011

Within 47



Variable Mean












V004 17967 A

V003 92890 B

V002 76208 C

V001 66513 C

V006 44168 D

V005 29500 E





229

Appendix 09



450)















230

Appendix 10





-20

00

20

40

60

80

100

mAU

60 70 80 90 100 110 120 ml


Fraction Number

231




Fraction Number


0

20

40

60

80

100

120

140

mAU

00 50 100 150 ml


Role and Regulation of Starch Phosphorylase and Starch ...

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