THE CHARACTERISATION OF ANAS2 AND BIOFORTIFICATION … Hao Grace_Tan... · 2018-02-12 · through...

THE CHARACTERISATION OF CANAS2

AND BIOFORTIFICATION OF CHICKPEA

Grace Zi Hao TAN

BSc Applied Science (Biotechnology)

BSc Applied Science (Biotechnology). Hons

Principal supervisor:

Prof Sagadevan Mundree (QUT)

Associate supervisors:

Dr Brett Williams (QUT)

Dr Sudipta Das Bhowmik (QUT)

Dr Alex Johnson (University of Melbourne)

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Centre for Tropical Crops and Biocommodities

Science and Engineering Faculty

Queensland University of Technology

November, 2016

The Characterisation of CaNAS2 and Biofortification of Chickpea i

Keywords

Agrobacterium-mediated transformation, bioavailability, biofortification, chickpea,

ferritin, iron, iron deficiency, iron content, manganese, mineral accumulation,

nicotianamine synthase, transgenic, translocation, zinc.

ii The Characterisation of CaNAS2 and Biofortification of Chickpea

Abstract

Iron deficiency in humans is a significant global problem, contributing to the

bulk of global anaemia cases and afflicting both developed and developing nations.

Its effects are far-reaching, affecting people not just on a personal but also national

scale. Existing methods of alleviating this problem include supplementation, food

fortification and dietary diversification. Such measures however, are limited by the

economic status of the targeted demographics. An alternative and more sustainable

method is the enhancement of the inherent iron content and bioavailability in crops

through biofortification.

Iron biofortification through genetic modification has been done in several

important crops like rice and wheat, but there is no known precedent in legumes. The

crop of interest to this project is chickpea, the second most important pulse crop in

the world that is widely consumed where anaemia is prevalent. This study documents

the first known attempt at biofortifying chickpea via genetic modification. The genes

of interest are rice nicotianamine synthase 2 (NAS) and soybean ferritin (FER),

which have been successfully used to enhance plant iron content and bioavailability

in rice. In this project, the novel chickpea NAS2 gene was also characterised and

investigated for its potential in a cisgenic biofortification approach.

This research was conducted in three main stages. First, commercial chickpea

cultivars were surveyed to determine the existing iron content and identify the factors

influencing it. The desi cultivar, PBA HatTrick, was then used for further work.

Second, was the characterisation of CaNAS2 in chickpea and the model species,

tobacco. The NAS-GmFER transgenic approach was then applied to chickpea in the

third stage, and its effectiveness as a biofortification strategy assessed.

For the survey of commercial chickpea cultivars conducted in the first stage,

samples were obtained for six cultivars from five locations within Eastern Australia.

Trace element content was assessed via ICP-OES (inductively coupled plasma

optical emission spectroscopy). Iron content was in chickpea found to range from

3.36 to 5.2mg/100g. HatTrick, the cultivar of interest, had 3.97 to 4.37g/100g of iron,

most of which was stored in the cotyledons. Principal component analysis of the

mineral profiles indicated that seed iron content was influenced by both genotype

and the environment.

The Characterisation of CaNAS2 and Biofortification of Chickpea iii

In the second stage, chickpea (cv HatTrick) were grown under iron-sufficient

and iron-deficient conditions, and CaNAS2 expression measured via qPCR. The

results showed CaNAS2 to be systemically expressed under iron-sufficient

conditions, and downregulated under iron-deficiency. To determine the effect of

CaNAS2 on iron accumulation, the gene was then cloned and overexpressed in

tobacco alone or together with GmFER. The well-characterised OsNAS2 was used in

these gene combinations as a positive control. Transgene presence and expression

was confirmed via PCR and RT-PCR. A total of three CaNAS2, seven OsNAS2, ten

GmFER-CaNAS2 and six GmFER-OsNAS2 transgenic lines were generated and

grown to the T1 generation. Assessment of leaf iron content showed the transgenic

lines to be mostly similar to the vector control, with only one CaNAS2 and OsNAS2

line being significantly higher, with approximately 1.3-fold increase in iron content.

The NAS-GmFER gene combinations were then applied to chickpea in the

third stage of this project, where optimisation of the chickpea transformation

protocol allowed for up to three-fold increase in transformation efficiency. Several

transgenic lines were generated, of which a total of three OsNAS2-GmFER and two

CaNAS2-GmFER transgenic lines were carried down to the T3 and T4 generations.

Transgene presence, expression and copy number was confirmed via PCR, qPCR and

Southern analysis respectively. Glasshouse evaluation showed no significant

differences between the transgenic lines and non-transgenic control in terms of

morphology, biomass, harvest index, or yield. Seed iron concentrations of up to

9mg/100g were achieved; as with the transgenic tobacco however, only one

OsNAS2-GmFER lines was significantly higher than the control with a 1.3-fold

increase. Assessment of leaf iron content yielded similar results, despite preliminary

data showing enhanced iron accumulation.

Based on these results, the NAS-GmFER biofortification approach appears to

be capable of enhancing iron in tobacco or chickpea, albeit not to the same

effectiveness as in rice. The precise mechanism behind this was unclear, though

differing physiologies, and limitations in plant uptake were suspected to be possible

causes. Such factors can be investigated in future studies.

Being the first known attempt at transgenic biofortification of a pulse crop, this

project provides insight which can serve to advise future attempts and pinpoint areas

for further development. In addition, the improvement of the chickpea transformation

iv The Characterisation of CaNAS2 and Biofortification of Chickpea

protocol in this study presents an opportunity for advancing both basic and applied

research, which has considerable implications for chickpea industry as a whole.

The Characterisation of CaNAS2 and Biofortification of Chickpea v

Table of Contents

Keywords .................................................................................................................................. i

Abstract .................................................................................................................................... ii

Table of Contents ......................................................................................................................v

List of Figures ....................................................................................................................... viii

List of Tables ............................................................................................................................x

List of Abbreviations ............................................................................................................. xii

Statement of Original Authorship ...........................................................................................xv

Acknowledgements ............................................................................................................... xvi

Chapter 1: Introduction ...................................................................................... 1

1.1 Aims and Objectives .......................................................................................................2

Chapter 2: Literature Review ............................................................................. 3

2.1 World population and malnutrition ................................................................................3 2.1.1 Iron deficiency anaemia .......................................................................................3

2.2 Biofortification ...............................................................................................................5 2.2.1 Approaches to biofortification ..............................................................................6 2.2.2 Target crops ..........................................................................................................7

2.3 Pulses as a vehicle for biofortification ............................................................................7 2.3.1 Pulse production and market ................................................................................8 2.3.2 Challenges ..........................................................................................................10 2.3.3 Chickpea .............................................................................................................13

2.4 Iron metabolism in plants .............................................................................................16 2.4.1 Strategy I – The reduction-based strategy ..........................................................19 2.4.2 Strategy II – The phytosiderophore chelation strategy .......................................21 2.4.3 Translocation ......................................................................................................23 2.4.4 Storage ................................................................................................................24 2.4.5 Regulation ..........................................................................................................27

2.5 Engineering for enhanced iron content .........................................................................28

2.6 Summary and Implications ...........................................................................................29

Chapter 3: General Materials and Methods ................................................... 31

3.1 General materials ..........................................................................................................31 3.1.1 Sources of specialised reagents ..........................................................................31 3.1.2 Iron metabolism genes ........................................................................................31 3.1.3 Bacterial strains ..................................................................................................31 3.1.4 Plant material ......................................................................................................32 3.1.5 General solutions: Abbreviations and composition ............................................32

3.2 General methods ...........................................................................................................34 3.2.1 General molecular techniques ............................................................................34 3.2.2 Bacterial transformation .....................................................................................35 3.2.3 Plant transformation ...........................................................................................36 3.2.4 Plant growth conditions ......................................................................................40 3.2.5 Verification and molecular characterisation of transgenic plants.......................40

vi The Characterisation of CaNAS2 and Biofortification of Chickpea

3.2.6 Trace element analysis ....................................................................................... 45

3.3 Data analysis ................................................................................................................ 47

Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick ................. 49

4.1 INTRODUCTION ....................................................................................................... 49

4.2 MATERIALS AND METHODS ................................................................................. 51 4.2.1 Seed material and locations ............................................................................... 51 4.2.2 Measurement of trace element distribution within the chickpea seed ............... 53 4.2.3 Statistical analysis .............................................................................................. 54

4.3 RESULTS .................................................................................................................... 54 4.3.1 Trace element composition of Australian-grown chickpea ............................... 54 4.3.2 Relationships between location and cultivar on seed trace elemental

composition ........................................................................................................ 58 4.3.3 Cotyledons the primary storage for iron in PBA HatTrick seeds ...................... 60

4.4 DISCUSSION .............................................................................................................. 63

Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes .. 67

5.1 INTRODUCTION ....................................................................................................... 67

5.2 MATERIALS AND METHODS ................................................................................. 69 5.2.1 Designation of chickpea NAS2 .......................................................................... 69 5.2.2 Assessment of NAS amino acid sequence and protein properties ..................... 69 5.2.3 Phylogenetic analysis of NAS proteins .............................................................. 70 5.2.4 Assessment of CaNAS2 expression ................................................................... 70 5.2.5 Isolation and cloning of chickpea NAS2 and other genes of interest ................ 72 5.2.6 Generation of expression plasmids .................................................................... 73 5.2.7 Generation and molecular characterisation of transgenic tobacco ..................... 76 5.2.8 Assessment of iron accumulation in transgenic tobacco leaf ............................ 76

5.3 RESULTS .................................................................................................................... 76 5.3.1 Designation and sequence analysis of CaNAS2 ................................................ 76 5.3.2 Phylogenetic analysis of CaNAS2 ..................................................................... 79 5.3.3 CaNAS2 expression is downregulated in response to iron deficiency .............. 82 5.3.4 Generation of expression plasmids .................................................................... 87 5.3.5 Generation and molecular characterisation of transgenic tobacco ..................... 88 5.3.6 Transgenic tobacco exhibit to significant increase in leaf iron or zinc

contents .............................................................................................................. 91

5.4 DISCUSSION .............................................................................................................. 93

Chapter 6: Generation and Characterisation of Transgenic Chickpea ........ 99

6.1 INTRODUCTION ....................................................................................................... 99

6.2 MATERIALS AND METHODS ............................................................................... 100 6.2.1 Generation of transgenic chickpea ................................................................... 100 6.2.2 Molecular characterisation of transgenic chickpea .......................................... 102 6.2.3 Glasshouse trial for T3, T4 plants .................................................................... 103 6.2.4 Assessment of agronomic parameters .............................................................. 104 6.2.5 Assessment of iron content in transgenic chickpea plants ............................... 104

6.3 RESULTS .................................................................................................................. 105 6.3.1 Optimisation of chickpea transformation procedure ........................................ 105 6.3.2 Generation and molecular characterisation of transgenic chickpea ................. 107 6.3.3 Morphology and agronomic properties of transgenic chickpea ....................... 112 6.3.4 Iron content in transgenic chickpea ................................................................. 115

The Characterisation of CaNAS2 and Biofortification of Chickpea vii

6.4 DISCUSSION .............................................................................................................117

Chapter 7: General Discussion ....................................................................... 122

7.1 The physiological role of CaNAS2 in the subcellualr and systemic context ..............123

7.2 NAS-FER transgene combination has limited effect on iron accumulation in tobacco

and chickpea..........................................................................................................................125

Chapter 8: Concluding remarks ..................................................................... 131

Appendices .............................................................................................................. 133

Bibliography ........................................................................................................... 141

viii The Characterisation of CaNAS2 and Biofortification of Chickpea

List of Figures

Figure 2.1. Global burden of anaemia across all ages .................................................. 5

Figure 2.2. Average pulse production by region (FAO, 2016a). ............................... 10

Figure 2.3. Fe acquisition strategies in higher plants: Strategy I in

nongraminaceous plants (left) and Strategy II in graminaceous plants

(right). .......................................................................................................... 18

Figure 2.4. Biosynthetic pathway of mugeneic acid (MA) family of

phytosiderophores (Sharma and Dietz, 2006). ............................................. 21

Figure 2.5. Possible rate-limiting steps for grain iron accumulation (Sperotto et

al., 2012). ..................................................................................................... 28

Figure 4.1. Chickpea cultivars used in this study. ...................................................... 52

Figure 4.2. PCA of trace element composition of chickpea grown in QLD and

NSW based on overall mineral composition. ............................................... 59

Figure 4.3. Clustering analysis of chickpea grown in QLD and NSW based on

mineral composition of samples. ................................................................. 60

Figure 4.4. Relative distribution of trace elements within PBA HatTrick seeds. ...... 61

Figure 5.1. Schematic diagram of the mini-hydroponics system. .............................. 71

Figure 5.2. Expression plasmids generated for plant transformation. ........................ 75

Figure 5.3. Predicted 3D structures of OsNAS and CaNAS2 proteins. ..................... 77

Figure 5.4. Predicted biochemical properties of CaNAS2. ........................................ 78

Figure 5.5. Amino acid sequence of CaNAS2. .......................................................... 79

Figure 5.6. Phylogenetic relationship between CaNAS2 and NAS proteins from

other plants. .................................................................................................. 81

Figure 5.7. Qualitative assessment of CaNAS2 expression in different chickpea

tissues via PCR. ............................................................................................ 82

Figure 5.8. Qualitative assessment of the expression of other CaNAS family

members in different chickpea tissues via PCR. .......................................... 83

Figure 5.9. Morphology of iron-sufficient (+Fe) and iron-deficient (-Fe)

chickpea grown under hydroponics conditions. ........................................... 84

Figure 5.10. Representative photos of verification of RNA and cDNA for

qPCR analysis. ............................................................................................. 85

Figure 5.11. Expression of CaNAS2 in different tissues under iron-sufficient

(+Fe) and iron-deficient conditions (-Fe)..................................................... 86

Figure 5.12. PCR detection of the genes of interest in the Agrobacterium

strains AGL1 and LBA4404 used for plant transformation work. .............. 87

Figure 5.13. Representative photos of T0 tobacco PCR screening with gene-

specific primers. ........................................................................................... 89

The Characterisation of CaNAS2 and Biofortification of Chickpea ix

Figure 5.14. Detection of transgene expression in GM tobacco lines via PCR. ........ 90

Figure 5.15. Average A) iron and B) zinc content in non-transgenic (n=10) and

transgenic tobacco leaves (n=4 to 7). ........................................................... 92

Figure 6.1. Overview of the chickpea transformation process. ............................... 101

Figure 6.2. Morphology of emerging putative transgenic shoots. ........................... 106

Figure 6.3. Representative photo of PCR screening of T0 plants. ........................... 108

Figure 6.4. Detection of transgene expression in transgenic chickpea lines via

PCR. ........................................................................................................... 109

Figure 6.5. Relative expression of transgenes in transgenic chickpea. .................... 110

Figure 6.6. Southern analysis of transgenic chickpea lines used in the

glasshouse trial. .......................................................................................... 111

Figure 6.7. Morphology of 9 week old transgenic chickpea at the

flowering/pod-filling stage. ........................................................................ 113

Figure 6.8. Agronomic properties of transgenic chickpea under glasshouse

conditions. .................................................................................................. 114

Figure 6.9. Preliminary study on leaf iron, zinc and manganese contents in 7

week old transgenic chickpea at the T1 generation. .................................. 116

Figure 6.10. Leaf iron, zinc and manganese contents in 7 week old transgenic

chickpea. .................................................................................................... 116

Figure 6.11. Iron, zinc and manganese contents in transgenic chickpea seeds. ....... 117

Figure 8.1. Representative photos of T1 tobacco screening via PCR using gene-

specific primers. ......................................................................................... 137

Figure 8.2. Concentrations of A) iron, and B) zinc in T1 transgenic tobacco

leaves.......................................................................................................... 138

Figure 8.3. GUS staining of transiently transformed chickpea ................................ 139

Figure 8.4. Iron, zinc, manganese and phosphorus content of GM chickpea

seeds. .......................................................................................................... 140

x The Characterisation of CaNAS2 and Biofortification of Chickpea

List of Tables

Table 2.1. Recommended Dietary Allowances (RDAs) for iron (Trumbo et al.,

2001). ............................................................................................................. 4

Table 2.2. Growth in production of major pulse producers (FAO, 2014, 2016b). ..... 10

Table 2.3. Factors affecting bioavailability of some trace elements (House,

1999). ........................................................................................................... 12

Table 2.4. Nutritional values per 100g of chickpea and important staple crops

(USDA, 2013). ............................................................................................. 14

Table 2.5. Information and assumptions used to set target levels for iron

biofortification in chickpea. ......................................................................... 16

Table 4.1. Description of chickpea cultivars used in this study. ................................ 52

Table 4.2. Locations from which seed samples were obtained. ................................. 53

Table 4.3. Cultivation conditions for each location. .................................................. 53

Table 4.4. Summary of Fe, Zn and P concentrations in kabuli and desi cultivars

grown at different locations. ........................................................................ 56

Table 4.5. Pearson’s correlation coefficient between the different trace

elements in PBA HatTrick. .......................................................................... 57

Table 4.6. Representation quality of a variable for each axis. ................................... 59

Table 4.7. Relative mass distribution within chickpea seeds of PBA HatTrick. ....... 61

Table 4.8. Concentrations of macro-elements in the different chickpea parts. .......... 62

Table 4.9. Concentrations of micro-elements in the different chickpea parts. ........... 62

Table 5.1. List of characterisation studies done on NAS from selected species. ....... 68

Table 5.2. List of primers used in qPCR. ................................................................... 72

Table 5.3. List of cloning primers. ............................................................................. 73

Table 5.4. List of primers used for screening. ............................................................ 76

Table 5.5. Similarity between the CaNAS and OsNAS amino acid sequences. ........ 77

Table 5.6. Summary of transgenic tobacco lines generated and progressing to

the T1 generation. ........................................................................................ 90

Table 6.1. Summary of modifications made to the original protocol. ..................... 102

Table 6.2. List of primers used for qPCR. ............................................................... 103

Table 6.3. Summary of transgenic lines generated. ................................................. 108

Table 8.1. Profile of ferrosol soil from Kingaroy (Chauhan, 2015). ........................ 133

Table 8.2. Concentration of macro-elements in dry chickpea seed. ........................ 134

Table 8.3. Concentration of micro-elements in dry chickpea seeds. ........................ 135

Table 8.4. List of proteins used in the phylogenetic analysis. ................................. 136

The Characterisation of CaNAS2 and Biofortification of Chickpea xi

Table 8.5. Germination rates and segregation of transgenic chickpea lines. ........... 139

xii The Characterisation of CaNAS2 and Biofortification of Chickpea

List of Abbreviations

Abbreviations

aa = Amino acids

BAP = 6-benzylaminopurine

BLAST = Basic Logical Alignment Tool

bp = Base pairs

Ca = Cicer arietinum (chickpea)

CaMV = Cauliflower mosaic virus

cDNA = Complementary DNA

CTAB = Cety trimethyl ammonium bromide

C-terminal = Carboxyl- terminal

DEPC = diethylpyrocarbonate

dH2O = Distilled water

DIG = Digoxygenin

DMSO = Dimethyl sulphoxide

dNTPs = Deoxyribonucleotide triphosphates

DTT = 1, 4-dithiothreitol

2, 4,-D = 2, 4-dichlorophenoxyacetic acid

EDTA = Ethylenediaminetetraacetic acid

E.coli = Escherichia coli

GUS = Β-glucoronidase

HDPE = High-density polyethylene

ICP-OES = Inductively coupled plasma atomic emission

spectroscopy

IPTG = Iso-propyl-β-D-thiogalatopyranoside

LA-ICP-MS = Laser-abalation inductively coupled plasma mass

spectroscopy

LB = Luria-Bertani

MES = 2-(N-morpholino)ethanesulfonic acid

MS = Murashige and Skooge media

NAA = α-napthalene acetic acid

NCBI = National Centre for Biotechnology Information

nos = Nopaline synthase

N-terminal = Amino terminal

The Characterisation of CaNAS2 and Biofortification of Chickpea xiii

ORF = Open reading frame

Os = Oryza sativa (rice)

PCR = Polymerase chain reaction

PP = Polypropylene

qPCR = Quantitative real-time polymerase chain reaction

RNase = Ribonuclease

RT-PCR = Reverse transcription polymerase chain reaction

SDS = Sodium dodecyl sulphate

SSC = Saline sodium citrate buffer

TAE = Tris acetate EDTA

Tris = Tris(hydroxymethyl)aminomethane

TPS = Tris-phosphate buffer

Tween20 = Polyoxyethylene (20) sorbitan monolaurate

UC = University of California

uidA = Reporter gene encoding β-glucuronidase

X-gal = 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

xiv The Characterisation of CaNAS2 and Biofortification of Chickpea

Units

°C = Degrees Celcius

d = days

Da = Daltons(s)

g = Gram(s)

g = Relative centrifugal force in units of gravity

h = Hour(s)

L = Litre(s)

M = Molar

m = Metre(s)

MW = Molecular weight

min = Minute(s)

mol = Mole(s)

rpm = Revolutions per minute

s = Second(s)

V = Volt(s)

vol = Volume(s)

v/v = Volume per volume

W = Watt

w/v = Weight per volume

Prefixes

G = Giga (109)

M = Mega (106)

k = Kilo (103)

c = Centi (10-2)

m = Milli (10-3)

µ = Micro (10-6)

n = Nano (10-9)

p = Pico (10-12)

The Characterisation of CaNAS2 and Biofortification of Chickpea xv

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made.

Signature:

Date:

QUT Verified Signature

January 2018

xvi The Characterisation of CaNAS2 and Biofortification of Chickpea

Acknowledgements

Alas, it is time. It has been a long, tumultuous journey in so many ways, and to

have come thus far would have been impossible alone. All glory to my Lord and

Saviour, Jesus Christ, who has brought me to where I am despite my constant

failings and flaws, who never gave up on me even when I gave up on myself. Thanks

be to Him too, for the many people with whom these past years have been shared.

For all its difficulties and challenges, it has been a time most precious and blessed.

To the Fantastic Four (aka my wonderful supervisory team): Saga, Brett,

Sudipta and Alex – words can scarce express my gratitude. One could not ask for

better mentors or role models, and my growth as a scientist would not have been

possible without all of you. Beyond the science, you all have also taught me more

about being a better person. Thank you for this chance to do this PhD, for seeing in

me more than I saw in myself, and for challenging me to reach new heights which I’d

otherwise never dare aspire to. I would not have come so far without your

immeasurable patience or constant encouragement and guidance.

To the Tropical Pulses group and the greater Abiotic Stress group as a whole,

thank you for being not just an amazing team, but also a family away from home. To

my friends and colleagues in the CTCB, thank you too for your guidance, friendship

and support around the lab. You guys are an incredible bunch, and it’s been an

honour to be able to work alongside all of you.

I would also like to thank following people for their invaluable contributions in

the various components of this project.

Dr Alex Johnson, for providing the genes used in this study.

Hao, for his guidance and help with the molecular work.

TJ Higgins, for his guidance and mentorship in the chickpea transformation

process, as well as getting our presentations up to shape.

Sudipta and Alam, for their guidance and assistance in the tissue culture and

glasshouse work.

The Characterisation of CaNAS2 and Biofortification of Chickpea xvii

Charlotte, Karine, Aarshi and Sunny, for their advice, assistance and support

in the elemental analysis. Special shout-out to Bulu as well, for teaching and

watching over me during extraction process.

Tom, Col, Rex and Yash, for provision of the chickpea seeds and

environmental data used in Chapter 4. Many thanks for humouring me in

this spontaneous endeavour that somehow evolved into a chapter of its own.

Julien, for the crash course on Geneious and phylogenetics.

Rob and JY, for sharpening up my writing and presentation skills during

Honours, and setting the foundation for my PhD life.

Associate Prof Terry Walsh and Professor Martin Sillence, for reviewing

my major milestones in this PhD. Their feedback at the different stages of

this PhD was most constructive and invaluable.

Dani, the ever reliable manager of the CTCB labs, for her meticulous work

in managing the day-to-day business in the lab. Thank you also for teaching

me how to be more organised, a trait I sorely needed improvement in. Your

teachings have saved my hide more times than I care to count.

I would also like to thank the Queensland Government for their funding for the

Tropical Pulses for Queensland project, without which this PhD would not exist.

To my family and friends, thank you for being there for me. A lot of things

have happened over these years and your support and encouragement has kept me

afloat in the storms. Though geography separates us, you all are ever in my heart and

prayers.

To all of you who have walked with me one way or another, thank you. Thank

you for your love and kindness, for your patience and constant guidance. Thank you

for putting up with me even as I fumbled through life trying to be a better human

than I was yesterday. This would’ve not been possible without you. God bless.

Chapter 1: Introduction 1

Chapter 1: Introduction

Despite food production keeping up with the burgeoning global population, the

problem of micronutrient deficiency has yet to be eradicated. Iron deficiency in

humans in particular, is a worldwide problem in both developed and developing

nations, affecting approximately 30% of the global population. The shifting focus

from quantity to quality and the development of management and food processing

methods aim to enhance the nutritional value of food. However, while effective, such

measures are not always feasible and may be limited by the economic status of the

targeted demographics.

Breeding for self-fortifying plants, also known as biofortification is a

sustainable means for the delivery of deficient nutrients; the one-time cost of

development is negated by the long-term benefits. Biofortification can be achieved

through conventional breeding or genetic modification. Conventional breeding is

extremely time-consuming as the lack of specificity may result in the loss of

desirable traits over successive generations, requiring many years and generations of

plants before a product is ready for use. The degree of nutritional enhancement is

also dependent on the available gene pool, and this can be particularly problematic in

plants with low fertility and diversity. In contrast, genetic modification is highly

specific, allowing for the addition of desired traits without loss of existing agronomic

qualities and effectively reducing the time required for product development.

Additionally, versatility of the techniques used allows for the expansion of the range

of available traits by tapping into the genetic resources of other species.

This project focused on the iron biofortification of the important leguminous

crop, chickpea, through genetic modification. Two components involved in iron

homeostasis, nicotianamine synthase (NAS) and ferritin, have been effectively used

to enhance plant iron content and bioavailability in other crop species. The effect on

legumes remains unknown. This project is the first known attempt at the iron

biofortification of legumes, specifically chickpea, through genetic modification. It is

hypothesized that transformation with NAS and ferritin will enhance the iron content

and bioavailability of chickpea, and thus provide a sustainable and affordable means

to alleviate global iron deficiency.

2 Chapter 1: Introduction

1.1 AIMS AND OBJECTIVES

The project aimed for the biofortification of chickpea for enhanced iron content

through genetic engineering. Considering that chickpea naturally has higher iron

content than rice, from which most existing iron biofortification genes have been

sourced, there exists a possibility that more efficient components can be found in

chickpea. As such, this project included a study of the currently uncharacterised

NAS2 homologue in chickpea with the purpose of assessing its effectiveness in

biofortification strategies in comparison to the well-characterised OsNAS2.

The aims of this project were accomplished through the following objectives:

1. Molecular characterisation of chickpea NAS2

2. Design and construction of vectors for plant transformation

3. Generation of transgenic chickpea expressing OsNas2, CaNas2 and

soybean ferritin

The outline of the project is as illustrated below:

Chapter 2: Literature Review 3

Chapter 2: Literature Review

2.1 WORLD POPULATION AND MALNUTRITION

The current world population stands at an estimated 7.3 billion (United

Nations, 2015) and is projected to increase by 2 billion over the next four decades.

Concomitant to this growth is the challenge of providing sustenance amidst

dwindling resources. Currently food production is adequate at approximately four

billion metric tonnes per annum, yet in spite of this, about 870 million people still

suffer from chronic malnutrition due to factors like unequal distribution, wastage and

poor diets (FAO, 2012; IMECHE, 2013).

Malnutrition, as defined by the World Health Organization (WHO), is “the

cellular disparity amid the supply of energy, nutrients and the body’s demand for

them to ascertain maintenance, growth and specific functions” (Batool et al., 2013).

It refers to both the insufficient and excessive intake of nutrients (both macro and

micro) and as such covers not only food shortage but also obesity. Undernourishment

can be classified categories: protein-energy malnutrition and micronutrient

deficiency. As the names suggest, the former refers to inadequate calorie or protein

intake while the latter to the lack of essential micronutrients such as vitamin A,

iodine, zinc and iron (Batool et al., 2013).

While both pose significant risks to health and negatively affect overall

productivity and quality of life, micronutrient deficiency, also known “hidden

hunger”, is perhaps the more pervasive and lethal due to the lack of visible effects. It

is consequently more difficult to identify and tackle, and afflicts both developing and

developed nations

2.1.1 Iron deficiency anaemia

Among the various kinds of micronutrient deficiencies in humans, iron

deficiency is the most prevalent, afflicting more than two billion individuals

worldwide (WHO, 2008). It has been identified as the greatest contributor to

anaemia, accounting for 66.2% of cases globally (Alvarez-Uria et al., 2014). The

extent of its impact is such that the terms are used interchangeably and the

prevalence of anaemia is used as a measure for the more specific iron deficiency

4 Chapter 2: Literature Review

anaemia (IDA) (WHO, 2001). IDA can be attributed to three main factors –

increased iron requirement (e.g. growth and pregnancy), poor absorption, and

inadequate dietary intake. The recommended values for daily iron intake for human

adults are 8 mg for males and 18 mg for females (Trumbo et al., 2001), and

insufficient intake impedes the formation of biologically important compounds, most

notably haeme, resulting in anaemia. Symptoms include fatigue, loss of energy, and

dizziness, all of which diminish the work capacity of the individual. Iron deficiency

also results in poor pregnancy outcomes and impediment of physical and cognitive

development, thereby increasing the risk of morbidity in children (WHO, 2008).

Table 2.1. Recommended Dietary Allowances (RDAs) for iron (Trumbo et al.,

2001).

mg/day

Age Male Female Pregnancy Lactation

Birth to 6 months 0.27 0.27

7–12 months 11 11

1–3 years 7 7

4–8 years 10 10

9–13 years 8 8

14–18 years 11 15 27 10

19–50 years 8 18 27 9

51+ years 8 8

This presents a problem of great economic and social significance, particularly

in developing countries where approximately 50% of pregnant women and 40% of

preschool children suffer from IDA (WHO, 2008). The consequences of this

manifest not only in the form of lives lost, but also in a rising generation of

individuals afflicted with developmental complications. While considerable progress

to reduce IDA has been made in several countries, it remains a significant a problem

given that a prevalence rate below 10% has yet to be seen in any country (see Figure

2.1).

Out of the three major risk factors contributing to IDA, the issue of dietary

intake is the most feasible to address on a large scale. Efforts to remedy the problem

include food-based strategies like dietary diversification, food fortification and


supplementation. However, while such measures have proven to be effective in

alleviating the problem of iron deficiency, they also incur a recurring cost and the

beneficiaries are limited to those who can afford it, namely those in developed

countries. Consequently such measures are unfeasible for the low-income

demographics that, incidentally, have the greatest need. The challenge then is to

develop a cost-effective means to deliver the required nutrients to the vulnerable

parties.

Figure 2.1. Global burden of anaemia across all ages in A) 1990 and B) 2013

(Kassebaum, 2016).

2.2 BIOFORTIFICATION

One such means of nutrient delivery is biofortification, which can generally be

defined as the enhancement of nutritional quality in the edible portions of food crops

during plant growth (HarvestPlus, 2015b; WHO, 2016). The precise definition of the

term “biofortification” may vary depending on the scope of the means (HarvestPlus,

2015b; WHO, 2016); for the purpose of this review it shall be used to refer solely to

the generation of self-fortifying plants, to the exclusion of agronomic interventions

(e.g. fertiliser application, management practices etc.).


Biofortification emerged within the last two decades as an approach to combat

micronutrient deficiency. While it cannot be considered a cure-all to micronutrient

deficiency, it alleviates the problem by complementing existing strategies like the

aforementioned ones of dietary diversification, fortification and supplementation.

With the one-time cost of development negated by the long term benefits,

biofortification presents a sustainable means of delivering the needed micronutrients

across large spatial and temporal scales (Nestel et al., 2006; Horton et al., 2008; De

Moura et al., 2014; HarvestPlus, 2015b).

Currently there exists two means of generating biofortified crops. The first is

conventional breeding, in which the desired traits are selected for and traditionally

bred into successive generations. The second is genetic modification (GM), in which

the genetic material of the host is altered in a manner that does not occur naturally.

Each method has its own advantages and disadvantages which will be discussed in a

later section. Irrespective of the means however, is the underlying principle of

manipulating the associated metabolic pathways which, in this case, is iron. Some

measure of understanding in that aspect is therefore required for effective

biofortification. With regards to that, a wealth of information has been gleaned and

reviewed extensively over the last 30 years in particular, fuelled by advances in

technology and analytical methods and growing interest in biofortification and

bioremediation (e.g. Briat et al., 1995; Hell and Stephan, 2003; Kim and Guerinot,

2007; Jeong and Guerinot, 2009; Thomine and Lanquar, 2011; Hindt and Guerinot,

2012; Kobayashi and Nishizawa, 2012).

2.2.1 Approaches to biofortification

Generation of biofortified crops can be done through two ways: conventional

breeding and/or genetic modification. Selection of desired traits by conventional

breeding is a practice that has existed since the advent of agriculture. Traditionally a

long-term process requiring much investment of time and effort, advances in

technology and molecular biology has since shortened the process and increased its

precision when targeting specific traits. Several quantitative trait loci (QTLs) for iron

accumulation has been identified in rice (Norton et al., 2010; Anuradha et al., 2012),

wheat (Xu et al., 2012), maize, (Jin et al., 2013) and bean (Blair et al., 2009; Blair et

al., 2010). Already, several crops have been developed through conventional

breeding under the HarvestPlus program and their success has been demonstrated in


several feeding trials. Consumption of biofortified pearl millet improved iron

adsorption and iron stores in women and children (Cercamondi et al., 2013; Kodkany

et al., 2013; Finkelstein et al., 2015), while biofortified rice have been found to help

maintain the iron stores of non-anaemic women (Haas et al., 2005).

Despite its effectiveness, the extent to which biofortification can be done

through conventional breeding is limited to the diversity in the gene pool and fertility

of the species. In cases where such limitations prevail, genetic modification provides

an alternative pathway.

2.2.2 Target crops

To date, starchy staples that contain little micronutrients like cereals, root

crops, and banana have the primary targets for iron biofortification (Namanya, 2011;

HarvestPlus, 2015a; Banana21, 2016). The advantages of such targets is that they

form the bulk of local diets and given proper processing, have a long shelf-life,

allowing for efficient delivery of the biofortified micronutrient over a large spatial

and temporal scale. A wealth of information has been generated concerning these

crops as a consequence of extensive focus. Biofortification works using genetic

modification in particular, have largely concentrated on major graminaceous crops

like rice, wheat and maize. In contrast, aside from banana (Matovu, 2016) and lettuce

(Goto et al., 2000), existing studies in non-graminaceous plants were conducted

mainly in model species like tobacco or Arabidopsis for characterisation purposes.

Given the physiological differences between the non-graminaceous and

graminaceous plants, it is difficult to extrapolate the effectiveness of iron

biofortification approaches in the latter to the former. As it stands, there remains

much to be explored in terms of iron biofortification of non-graminaceous crops.

2.3 PULSES AS A VEHICLE FOR BIOFORTIFICATION

Aside from the aforementioned starchy staples, another group of crops have

been targeted for biofortification, albeit to a lesser degree. Pulses, as defined by the

FAO (1994), are leguminous crops harvested for solely for dry grain. Like cereals,

they have a long history of cultivation and have been a significant constituent in

human diets since around 10, 000 BC (Fuller et al., 2001; Caracuta et al., 2015). As a

crop, pulses present two main benefits, both of which are complementary to cereals.

The first is their agronomic characteristics. By virtue of their nitrogen fixing


properties, pulses are often grown as an intercrop or as a mixed crop to replenish soil

nitrogen levels, thereby reducing the need for fertilisers. Cultivation with pulse crops

have also been shown to increase the uptake of nitrogen, sulphur and phosphorus by

cereals, resulting in an enhanced yield and grain quality (Li et al., 2003; Li et al.,

2004b; Agegnehu et al., 2006; Banik et al., 2006; Gooding et al., 2007). Yield

stability in also increased (Rao and Willey, 1980).

The second benefit of pulses is their nutritional density. Pulses are a rich source

of carbohydrates and fiber. Their most prominent feature however, is their high

protein content of 21–26% and an amino acid profile complementary to that of

cereals, being rich in lysine, leucine and arginine (Phillips, 1993; Iqbal et al., 2006;

Pulse Canada, 2016). Its excellence as a vegetarian source of protein and

affordability in contrast to livestock products has earned it the famous moniker of

‘poor man’s meat’. Pulses are also rich in micronutrients like folate, thiamine,

riboflavin, niacin, calcium, magnesium, iron and zinc (Phillips, 1993; Iqbal et al.,

2006; Jukanti et al., 2012). Other than contributing to the macro- and

micronutritional needs, several health benefits have been associated with inclusion of

pulses in the diet. Their low glycemic index (GI) has been linked to the management

of diabetes and diabetes-related diseases (Rizkalla et al., 2002; Sievenpiper et al.,

2009) while bioactive components have been investigated for their health potential –

e.g. lectins for their immunomodulatory effect, protease inhibitor for anti-

inflammatory effect, and angiotensin I-converting enzyme (ACE) inhibitory peptides

for their anti-hypertensive properties (Rochfort and Panozzo, 2007; Roy et al., 2010).

2.3.1 Pulse production and market

Despite their agronomic and nutritional benefits, pulses have not received the

same amount of attention or development as the main starchy staples. Between 1961

and 2014, pulse yield and production values increased by 42.3% and 90.4%

respectively, a small fraction compared to the increase of 187.2% and 219.4% in

cereals (FAO, 2016b). Much of this disparity can be attributed to developments made

during the Green Revolution, in which the focus on productivity and protein-calorie

malnutrition led to the shift from cultivation of traditional micronutrient-rich crops to

the more productive and profitable starchy cereals (Pinstrup‐Andersen and Hazell,

1985; Pingali, 2012). Poor policy and diversion of land to cereal cultivation has led

to a reduction in pulse supply, effectively driving prices up and decreasing


consumption per capita (Kennedy and Bouis, 1993; Kataki, 2002; Akibode and

Maredia, 2012).

As highlighted in the special feature on pulses in the 2014 Food Outlook (FAO,

2014), recent years have seen several key changes in pulse production and trade.

Asia remains the region with the highest pulse production, with India continuing as

the largest pulse producing country, contributing at least 20% towards global pulse

production (Figure 2.2, Table 2.2). Production in other regions except Europe has

also increased, fuelled by domestic and international demand. In contrast to these

countries is China, whose production has decreased due to a number of factors such

as population increase and decreasing availability of arable land. Despite the shift in

preference for animal-based products and protein that accompanies growing

affluence, India and China remain as major importers, consuming approximately

40% of the world’s pulse production as food, and 30% as feed. Much of this is

provided by major exporters like Canada and Australia. With other major producers

like Myanmar and Brazil, pulse consumption is primarily domestic.

Pulse production, consumption and trade are expected to increase alongside

population growth, particularly with increasing promotion from government

campaigns (Akibode and Maredia, 2012) and the declaration of 2016 as the

“International Year of Pulses” by the UN General Assembly. Increasing awareness

and concern over nutritional composition of food, particularly by food

manufacturers, has attracted greater interest in pulses, which will likely translate into

further support and development of pulses and the industry (FAO, 2014).


Figure 2.2. Average pulse production by region (FAO, 2016a).

Table 2.2. Growth in production of major pulse producers (FAO, 2014, 2016b).

Production (mmt)

Country 1961 1981 2001 2014 Principle pulse crops

India 12.9 10.8 12.2 19.98 Chickpeas, beans, pigeon peas

Myanmar 0.2 0.4 2.0 5.0 Beans, pigeon peas, chickpeas

Canada 0.1 0.2 3.4 5.8 Peas, lentils

China 8.5 6.4 5.1 4.5 Beans, broad beans, peas

Brazil 1.8 2.4 2.5 3.3 Beans

Nigeria 5 0.6 2.3 2.2 Cowpeas

Ethiopia 0.6 0.9 1.2 2.6 Broad beans, beans, chickpeas,

peas

Australia 0 0.3 2.7 3.0 Lupines, lentils, chickpeas

USA 1.1 1.7 1.3 2.4 Beans, peas

Tanzania, U.

Rep. 0.1 0.3 0.8 1.8 Beans

Rest of the world 15.0 17.5 22.6 27.02

Total 40.8 41.6 55.9 77.6

2.3.2 Challenges

Pulses are a diverse group featuring a wide variety of species and cultivars, and

this genetic richness is a treasure trove that lends itself to crop improvement.

However this diversity has also contributed to the lack of a concerted global effort,

with production and development being dispersed across various localities (FAO,

2014). Such development thus far have been on yield, disease tolerance and

macronutrient quality, though in the last decade there has been a growing interest in

micronutrient content, with increasing numbers of genotypes and cultivars being


assayed for their iron and zinc composition (Blair et al., 2013; Thavarajah et al.,

2014). In spite of this there has been little published work on pulse biofortification.

Currently the only known example of a biofortified pulse is the high iron common

bean generated from the HarvestPlus breeding program; to date, several varieties

have been produced with improvements in iron content ranging from 47 – 94%

(Katsvairo, 2015). Despite such success, bioavailability of this iron remains a

problem.

Bioavailability, as defined by Carpenter and Mahoney (1992), is the

“proportion of a nutrient present in food that the body is able to absorb and utilise by

incorporation into physiologically functional pools”. As with other plant-based

foods, iron found in pulses is non-heme iron which has lower bioavailability

compared to its heme counterpart (Björn-Rasmussen et al., 1974). This

bioavailability is further subject to other factors such as those listed in Table 2.3.

Amongst these, higher inherent levels of antinutritional factors like polyphenols and

phytate have been identified as major contributors to the poor iron bioavailability in

pulses.

This trait has presented a particular challenge to pulse biofortification efforts.

Despite the success of increasing overall iron content, feeding trials conducted in

Rwanda have indicated iron bioavailability of biofortified beans were to be similar, if

not lower, compared to the unfortified beans (Petry et al., 2012, 2014). This has been

ascribed to the influence of phytic acid, and whose reduction in concentration was

recommended by the authors as a means to improve the effectiveness of

biofortification (Petry et al., 2012, 2014).

This recommendation has been applied in several cereal crops (Larson et al.,

1998; Larson et al., 2000; Raboy et al., 2000; Guttieri et al., 2004) and more recently

in bean (Campion et al., 2009). The effectiveness of the low-phytic acid bean lines is

currently inconclusive however, as bioavailability assessments have yielded

conflicting results due to differences in experimental design (Petry et al., 2013; Petry

et al., 2016). Poor cooking quality was also observed in the low-phytic acid seeds,

which may have contributed the adverse gastrointestinal side-effects in the

participants in one of the studies (Petry et al., 2016). The relationship between phytic

acid and cooking quality have been alluded to in other studies on lentil and bean

(Kon and Sanchuck, 1981; Bhatty and Slinkard, 1989). Interestingly, no such effect


was reported in low-phytic acid maize lines (Mendoza et al., 1998); whether this this

is a legume-specific issue remains to be confirmed. Aside from influencing cooking

quality, phytic acid is also known to have antioxidant properties and protective

effects against heart disease and cancer (Sharma, 1986; Nelson et al., 1988; Vucenik

and Shamsuddin, 2003). It is unknown if reduction in phytic acid content would

affect such properties. Similarly, the subsequent long-term effect on human health is

unknown.

Currently the bulk of existing knowledge is limited to the work done on the

common bean as it is currently the forefront in pulse iron biofortification efforts.

Despite the aforementioned challenges, the iron biofortified bean has proven to be a

successful means of alleviating iron deficiency, promising much for work in other

pulses.

Table 2.3. Factors affecting bioavailability of some trace elements (House, 1999).

Host factors Dietary factors

Dietary composition Food preparation

Age

Sex

Ethnic background

o Types of food

selected

o Geographic

living area

Economic status

o Type, quality

and quantity of

selected food

Physiological status

o Pregnancy

o Lactation

o Physical

activity

Nutritional status

o Moderate or

frank

deficiency

o Lean body

mass

Disease (including

parasitism)

Protein quality

o Protein source

o Animal vs plant protein

o Amino acid balance

Protein quantity

Trace element quantity

Physiochemical form of trace

element

Nutrient interactions

o Element–element

o Element–organic compounds

Promoters

o Meat

o Ascorbate

o Citrate

o Vitamin D

o Some amino acids

o Some sugars

Inhibitors

o Phytate

o Oxalate

o Polyphenols

o Fiber

o Goitrogens

o Excess ascorbate or folate

Micronutrient deficiencies

o Ascorbate

o Riboflavin

o Vitamin E

Raw

Cooking (various

methods)

Fermentation

Malting

Milling

Extraction

Soaking


2.3.3 Chickpea

Chickpea (Cicer arietinum) is an important pulse crop that has been cultivated

by humans since the Stone Age. As of 2009, it is the second most important pulse

crop in the world after the common bean, having overtaken peas as the pulse crop

with the second highest global production values. Global production has climbed

steadily since 2008 to exceed 14.2 million tonnes in 2014, of which approximately

96 % is grown in developing countries (FAO, 2016b). India in particular, has

historically been the largest producer and consumer of chickpea; in 2013 alone it

contributed approximately 65% and 33% to total chickpea production and import

respectively (FAO, 2016b). In terms of consumption, it is difficult to obtain precise

statistics due to the lack of available data. However based on calculations using

production and trade values, the global average for chickpea consumption was

estimated to be around 1.3kg/year per person between 2006 and 2008, with South

Asia and the Middle East-North Africa regions being the biggest consumers at 4.25

kg/person and 2.11 kg/person per year respectively (Akibode and Maredia, 2012).

The demand is predicted to grow, particularly in Africa and Asia, due to population

increase and increasing support from the governments in encouraging pulse

consumption (Rao et al., 2010; Akibode and Maredia, 2012). This increase in

demand is not limited to those regions; in the USA for instance, net domestic use of

chickpea nearly doubled from 199.6g in 2010 to 322.1g in 2014 (Wells, 2016).

Much like other pulses, the nutritional qualities of chickpea have long been

recognised and documented. In addition to high protein content (20-22%), chickpeas

are also rich in micronutrients like folate, magnesium, zinc and iron (Table 2.4)

(USDA, 2013). Studies conducted by different authors have found iron content to

range from 2.4 to 11 mg/100g (e.g. USDA; Meiners et al., 1976; Wood and Grusak,

2007). Likewise, various studies have reported differing values for phytic acid and

other antinutrients (e.g. Chitra et al., 1995; Ghavidel and Prakash, 2007; Hemalatha

et al., 2007b), indicating a possible effect of genotype and environmental factors on

overall iron bioavailability. When measured as dialyzable iron generated from a

simulated gastrointestinal digest, bioavailability has been found to vary widely across

different studies, ranging from about 6% to 25% (Chitra et al., 1997; Ghavidel and

Prakash, 2007; Hemalatha et al., 2007b). The reason behind this disparity is as yet

unclear, though analytical procedures and variations in samples have been suggested


Table 2.4. Nutritional values per 100g of chickpea and important staple crops (USDA, 2013).

Nutrient Unit Chickpea,

dried

Chickpea, boiled

without salt

Corn,

yellow

Wheat,

durum

Rice, white, medium-

grain, cooked Potato, raw

Cassava,

raw

White

sorghum,

raw

Proximates

Water G 11.53 60.21 10.37 10.94 68.61 83.29 59.68 9.2

Energy kcal 364 164 365 339 130 58 160 339

Protein G 19.3 8.86 9.42 13.68 2.38 2.57 1.36 11.3

Total lipid (fat) G 6.04 2.59 4.74 2.47 0.21 0.1 0.28 3.3

Carbohydrate, by

difference G 60.65 27.42 74.26 71.13 28.59 12.44 38.06 74.63

Fiber, total dietary G 17.4 7.6 7.3 - 0.3 2.5 1.8 6.3

Sugars, total G 10.7 4.8 0.64 - -

1.7 3.39

Minerals

Calcium, Ca mg 105 49 7 34 3 30 16 28

Iron, Fe mg 6.24 2.89 2.71 3.52 1.49 3.24 0.27 4.4

Magnesium, Mg mg 115 48 127 144 13 23 21 190

Phosphorus, P mg 366 168 210 508 37 38 27 287

Potassium, K mg 875 291 287 431 29 413 271 350

Sodium, Na mg 24 7 35 2 0 10 14 6

Zinc, Zn mg 3.43 1.53 2.21 4.16 0.42 0.35 0.34 1.54

Vitamins

Vitamin C, total

ascorbic acid mg 4 1.3 0 0 0 11.4 20.6 0

Thiamin mg 0.477 0.116 0.385 0.419 0.167 0.021 0.087 0.237

Riboflavin mg 0.212 0.063 0.201 0.121 0.016 0.038 0.048 0.142

Niacin mg 1.541 0.526 3.627 6.738 1.835 1.033 0.854 2.927

Vitamin B-6 mg 0.535 0.139 0.622 0.419 0.05 0.239 0.088 0.59

Folate, DFE µg 557 172 19 43 97 17 27 20


as a possible cause (Platel and Srinivasan, 2016). Given the multifaceted nature

of nutrient bioavailability, the values obtained are at best relative.

In light of this, it would be prudent for biofortification efforts to first target

total seed iron content before progressing to bioavailability. Considerable progress

has been made to that end, particularly with the growing interest in chickpea as a

target for iron biofortification. While a concerted global effort has yet to materialise,

pockets of development have emerged with India and Canada at the forefront. To

date, the chickpea genome has been sequenced (Varshney et al., 2013). Chickpea

populations in those countries have also been screened for genetic diversity and iron

accumulation traits, allowing for identification of the associated QTLs (quantitative

trait loci) (Diapari et al., 2014; Upadhyaya et al., 2016). In terms of biofortification

via genetic modification, no work done has been yet. It is however, a viable option –

while chickpea can be considered a recalcitrant species, successful transformation

protocols have been established (Sarmah et al., 2004; Indurker et al., 2010).

Given the relative youth of this endeavour to biofortify chickpea for iron, no

biofortification targets have yet been set. As stated by Bouis and Welch (2010),

several factors need be considered in the setting of such targets. Unlike the common

bean, which is a staple, chickpea is a secondary staple and depending on the type and

cultivar, may be processed into various forms for consumption. This would in turn

affect iron content and bioavailability. Consequently, the consumption profile for

chickpea is lower and potentially more varied compared to the common bean,

particularly across different age and cultural demographics.

As with other crops, the challenge in setting biofortification targets lies

primarily in the lack of information concerning the different variables (Bouis and

Welch, 2010). Until more detailed and specific information is obtained, only gross

assumptions may be made.

Table 2.5 illustrates a crude estimate of the iron biofortification target for

chickpea, calculated using a formula modified from Bouis and Welch (2010). The

targeted demographic in this case was adult, non-pregnant, non-lactating females

from the South Asian region.


Table 2.5. Information and assumptions used to set target levels for iron

biofortification in chickpea.

*EAR (µg/day) 1460

10% EAR 146

Per capita consumption (g/day) 10

Baseline Fe content (µg/g) 5

Bioavailability (%) 6

Fe retention after processing (%) 85

Additional Fe required (µg/100g) 121

Final target as dry weight (µg/100g) 286

* – Estimated Average Requirement

2.4 IRON METABOLISM IN PLANTS

Prior to attempting any biofortification strategies, the significance of iron in the

plants, as well as the underlying mechanisms governing its metabolism, must first be

understood. Iron is the fourth most common element in the Earth’s crust and can

exist in a wide range of oxidation states, of which the most common are the ferrous

(Fe2+) and ferric (Fe3+) forms. By virtue of its high redox potential, it forms a key

component of biological processes involving electron exchange such as DNA

synthesis, oxygen transport, cellular respiration and photosynthesis, where it

participates in the form of a cofactor in iron complexes. Examples of such complexes

include haemoglobin, chlorophyll, DNA helicases, and catalase.

For all its biological significance however, iron metabolic pathways can be

summarised with the imagery of a precarious transfer of a nuclear material between

containment facilities. Biologically, free iron may result from iron overload and/or

insufficient sequestration capacity of the organism (Pietrangelo, 2003). Left alone,

free Fe2+ catalyses the formation of hydroxyl (OH) radicals through the Fenton

reaction, and the process repeats when Fe2+ is regenerated from the resultant Fe3+

through reduction by the superoxide radical (O2-) (Haber and Weiss, 1934). The

summation of this self-perpetuating reaction is known as the Haber-Weiss reaction:

𝑂2− + 𝐹𝑒3+ → 𝑂2 + 𝐹𝑒2+

𝐹𝑒2+ + 𝐻2𝑂2 → 𝐹𝑒3+ + 𝑂𝐻− + ∙ 𝑂𝐻


𝑂2− + 𝐻2𝑂2 → 𝑂2 + 𝑂𝐻− + ∙ 𝑂𝐻

Reactive oxygen species (ROS) generated as a consequence of this reaction can

react with cellular components to cause oxidative damage (Kehrer, 2000; Aisen et

al., 2001; Papanikolaou and Pantopoulos, 2005; Jeong and Guerinot, 2009;

Kobayashi and Nishizawa, 2012); however on the other hand, they also serve as

important signalling molecules and are an integral part of the stress response (Apel

and Hirt, 2004). The fine line between cytotoxicity and biological function, and the

intimate association between iron and ROS production, highlights the significance of

proper regulation of iron metabolic pathways.

Iron metabolic pathways can be divided into three main processes: uptake,

translocation and storage. Despite its abundance iron has poor solubility under

aerobic conditions, particularly in high pH and calcareous soils, necessitating its

solubilisation before uptake can occur. This process is mostly accomplished via root

exudates, the composition of which varies in response to the plant’s physiological

state and needs. In response to iron deficiency, the plant triggers the production of

factors that directly or indirectly aid iron solubilisation. Enhanced concentrations of

glutamate, ribitol and glucose were observed in the root exudates of iron-deficient

maize, which were suggested to attract and support siderophore-producing bacterial

communities to aid iron solubilisation (Carvalhais et al., 2011). Notable increases

were also observed in the production of organic acids like malate and citrate, which

increase the availability of iron through dissolution of insoluble iron compounds

(Jones et al., 1996; Sánchez-Rodríguez et al., 2014).

In addition to the aforementioned means, different plant species have adopted

specific approaches toward solubilise and acquire iron. These have been categorised

as Strategy I and Strategy II (Römheld and Marschner, 1986) (see Figure 2.3). It

should be noted that while differences between both strategies primarily affect the

uptake process, the involvement of molecular components in the translocation

process has further implications on the overall physiology; this will be discussed in a

later section.

In summary, Strategy I is a reduction-based strategy in which insoluble iron is

reduced via acidification of the rhizosphere. Phenolics are also secreted to chelate the

iron. Chelated iron is reduced at the root surface and the resulting Fe (II) ions are


absorbed across the plasma membrane by specialised transporter proteins. Strategy I

is used by non-graminaceous plants, which includes all plants except for grasses.

Strategy II on the other hand, is used by graminaceous plants (grasses) and revolves

around the chelation of insoluble iron with secreted phytosiderophores, the

production and uptake of which is specific to Strategy II plants. The resulting Fe

(III)-phytosiderophore complex is subsequently taken up via specialised transporters.

Unlike the reduction-based approach used in Strategy I, phytosiderophore uptake is

not limited by high pH, thereby conferring an advantage where such conditions are

present (Römheld and Marschner, 1986).

The use of both strategies may be present in a single species, of which the only

known example is rice (Ishimaru et al., 2006). This combination may represent an

adaptation to the submerged conditions in which rice and its wild relatives grow,

where iron is more readily available in ferrous than ferric form; whether a similar

occurrence may be found in other species remains to be seen.

Figure 2.3. Fe acquisition strategies in higher plants: Strategy I in

nongraminaceous plants (left) and Strategy II in graminaceous plants (right).

Ovals represent the transporters and enzymes that play central roles in these

strategies, all of which are induced in response to Fe deficiency. Abbreviations:

DMAS, deoxymugineic acid synthase; FRO, ferric chelate reductase oxidase; HA,

H+ -ATPase; IRT, iron-regulated transporter; MAs, mugineic acid family

phytosiderophores; NA, nicotianamine; NAAT, nicotianamine aminotransferase;

NAS, nicotianamine synthase; PEZ, PHENOLICS EFFLUX ZERO; SAM, S-

adenosyl –L-methionine; TOM1 transporter of mugineic acid phytosiderophores 1;

YS1/YSL1, YELLOW STRIPE1/YELLOW STRIPE 1-like (Kobayashi and

Nishizawa, 2012).


2.4.1 Strategy I – The reduction-based strategy

2.4.1.1 Proton extrusion

One of the components of Strategy I uptake is the acidification of the

rhizosphere via proton extrusion. While this directly encourages the protonation and

solubilisation of insoluble hydroxides (Schwertmann, 1991), a decrease in pH

facilitates uptake by enhancing Fe(III) reduction at the root surface (Wei et al., 1997)

and may also serve to promote release of other reducing (Römheld and Marschner,

1983) while stabilising organic acid-iron compounds (Jones et al., 1996).

Proton extrusion occurs through the action of H+-ATPases (HA), whose

expression has been found to increase upon detection of iron deficiency (Rabotti and

Zocchi, 1994; Dell'Orto et al., 2000; Santi et al., 2005; Santi and Schmidt, 2009). A

higher capacity for H+ release and rhizosphere acidification has been associated with

increased tolerance to iron deficiency (Wei et al., 1997; Mahmoudi et al., 2007).

However, studies in Arabidopsis and cucumber have found that not all isoforms are

involved with the iron deficiency response; some are serve as housekeeping genes or

are tissue-specific (Santi et al., 2005; Santi and Schmidt, 2009).

2.4.1.2 Phenolic production and exudation

Another component of the Strategy I uptake is the production, accumulation

and exudation of phenolics, particularly under iron deficiency (Römheld and

Marschner, 1981). Phenolics contribute directly to uptake by dissolving and

chelating insoluble iron in the soil (Dakora and Phillips, 2002). In addition, they also

facilitate remobilisation of the otherwise inaccessible apoplastic iron by stripping it

from cell walls in a process independent of proton extrusion and ferric-chelate

reductase oxidase (FRO) activity (Jin et al., 2007).

A more indirect effect on iron uptake is through management of the

rhizospheric microbial community, where their properties as both an attractant and a

form of plant defence allow for the selection for beneficial plant-microbial

interactions (Peters and Verma, 1990; Bhattacharya et al., 2010). Phenolics secreted

by iron-deficient red clover, for instance, select for siderophore and auxin producing

microbes, thereby enhancing soil iron bioavailability and root growth (Jin et al.,

2006).


2.4.1.3 Reduction and absorption

Prior to uptake into the root cell, Fe3+ chelates must first be reduced to Fe2+ at

the root surface (Chaney et al., 1972). This is done through the activity of ferric-

chelate reductase oxidase (FRO) at the root surface, which reduces Fe3+ chelates to

Fe2+ by transferring electrons across the plasma membrane (Robinson et al., 1999;

Waters et al., 2002).

FRO is a family of membrane-bound metalloreductase that transfers electrons

from cytosolic NADPH across membranes to electron-accepting substrates on the

other side. In addition to facilitating acquisition of iron from the soil, this capability

is also utilised in localisations where iron reduction is required for transport and/or

assimilation, such as in the mesophyll (Brüggemann et al., 1993), reproductive

tissues (Waters et al., 2002; Li et al., 2004a) and chloroplast membranes (Jeong and

Connolly, 2009). Several members of this family has been identified and

characterised in various species, namely Arabidopsis (Robinson et al., 1997;

Mukherjee et al., 2006), pea (Waters et al., 2002), and tomato (Li et al., 2004a). Not

all isoforms are involved in iron acquisition from the soil; this is exemplified in the

study conducted by Wu et al. (2005), in which AtFRO5, AtFRO6, AtFRO7 and

AtFRO8 were found to be shoot specific, while only AtFRO2 and AtFRO3 were

expressed in the roots.

Following reduction at the root surface, the resulting Fe(II) ions are absorbed

across the plasma membrane via the iron-regulated transporter (IRT) (Eide et al.,

1996; Vert et al., 2002). IRT is a member of the zinc-regulated transporter, iron-

regulated transporter-like protein (ZIP) family that functions as membrane-bound

uptake transporter for Zn and Fe (Lin et al., 2009). In Arabidopsis and tomato, IRT1

has been identified as responsible for uptake from the soil (Bereczky et al., 2003;

Vert et al., 2009), with loss of function producing a severely stunted and chlorotic

phenotype (Varotto et al., 2002; Vert et al., 2002). Co-regulated with IRT1 is the

AtIRT2 homologue, which facilitates subcellular transport of iron and localises to

vesicle membranes instead of plasma membranes (Vert et al., 2009).

Both ferric chelate reduction and IRT activity is regulated in response to iron

concentrations and increases in response to iron deficiency (Robinson et al., 1999;

Connolly et al., 2003; Vert et al., 2003). Enhanced ferric reduction in particular, has

been considered a hallmark indicator of iron deficiency (Römheld and Marschner,


1981; Higuchi et al., 1995) and increased capacity for FRO activity confers increased

tolerance to low iron (Connolly et al., 2003; Peng et al., 2015).

2.4.2 Strategy II – The phytosiderophore chelation strategy

Unlike Strategy I, Strategy II revolves around the use of the mugineic acid

(MA) family phytosiderophores in iron acquisition. The MA biosynthetic pathway

starts with the conversion of three units of S-adenosylmethionine (SAM) into

nicotianamine (NA) by nicotianamine synthase (NAS) (Higuchi et al., 1994; Higuchi

et al., 1999a). NA is converted to a 3” keto-intermediate by nicotianamine

aminotransferase (NAAT), before being reduced to deoxymugeneic acid (DMA) by

deoxymugeneic acid synthase (DMAS) (Kanazawa et al., 1994; Bashir et al., 2006).

DMA can subsequently be converted to other MAs through a series of

hydroxylations (Mori and Nishizawa, 1989; Ma and Nomoto, 1993), increasing

levels of which improves the affinity for Fe3+ and chelate stability under acidic

conditions (von Wirén et al., 2000). An overview of the MAs synthetic pathway is as

illustrated in Figure 2.4.

Figure 2.4. Biosynthetic pathway of mugeneic acid (MA) family of

phytosiderophores (Sharma and Dietz, 2006).


Synthesized MAs are secreted into the soil through the phytosiderophore efflux

transporter TOM1 (Nozoye et al., 2011) and the resulting ferric complexes are then

taken up by the roots through specialized transporters like YELLOW STRIPE 1

(YSL1) and YELLOW STRIPE 1-like (YSL) (Curie et al., 2001; Murata et al., 2006;

Inoue et al., 2009; Kobayashi and Nishizawa, 2012).

Under iron deficiency, the phytosiderophore biosynthetic pathway is

upregulated (Kanazawa et al., 1994; Takahashi et al., 1999; Inoue et al., 2003;

Bashir et al., 2006), leading to an increase in the MAs secretion (Higuchi et al.,

1996; Fan et al., 1997). The pattern of secretion also switches from a constant one to

a diurnal rhythm (Mori et al., 1987; Walter et al., 1995; Nozoye et al., 2011).

Expression of YS1 and YSL is also upregulated (Koike et al., 2004; Murata et al.,

2006).

2.4.2.1 Nicotianamine synthase (NAS) and nicotianamine (N A)

NA is a non-proteogenic amino acid synthesised from three units of

adenosylmethionine in a reaction catalysed by NAS. In addition to serving as a

precursor for the mugeneic acid family of phytosiderophores (Higuchi et al., 1999b),

it is also a common component in the translocation process in both Strategy I and II,

where it functions primarily as a chelator of divalent transition metals (Scholz et al.,

1992).

NA has mainly been associated with iron, zinc and, to a lesser extent,

cadmium, nickel, manganese and copper (Stephan et al., 1996; Inoue et al., 2003;

Takahashi et al., 2003a; Weber et al., 2004; Mari et al., 2006), forming stable

complexes at pH >6 (Stephan et al., 1996). Like citrate, it is a key chelator of metals

in plants (von Wirén et al., 1999; Takahashi et al., 2003a; Klatte et al., 2009) and

contributes to the translocation of metals in the vascular tissue (Stephan et al., 1994a;

Mari et al., 2006). However, unlike citrate-metal complexes which deliver to older

leaf tissues, NA-metal complexes are able to transport metals out of the vascular

bundle into the interveinal regions of leaves and reproductive organs (Takahashi et

al., 2003a; Schuler et al., 2012a).

As a result of this role, NA exerts considerable influence on the regulation of

the iron deficiency response mechanism in both graminaceous and non-graminaceous

plants. Loss of NAS function or depletion of NA results in the accumulation of iron


in the roots and stem, but as it is unable to enter the sites of requirement (i.e. young

leaves and reproductive organs) there is a perpetual induction of iron deficiency

response even in the face of excess iron. A prime example of this is the tomato

mutant chloronerva, in which a single base change in the only NAS gene has resulted

in a loss of function (Ling et al., 1999), making it the only known NA-auxotroph

amongst higher plants. It is characterised by retarded growth and sterility, and

exhibits iron deficiency symptoms despite high accumulation of iron and other heavy

metals and exposure to high concentrations of Fe-EDTA (>10µM) (Stephan and

Grün, 1989; Scholz et al., 1992). Such symptoms include intercostal chlorosis of

young leaves, and enhancement of citrate accumulation in the roots and the Strategy

I uptake process i.e. proton extrusion, Fe3+ reduction, and exudation of phenolics and

other iron chelating compounds (Scholz et al., 1992). The activity of iron-containing

enzymes was also deregulated (Pich and Scholz, 1993).

Similar effects were observed in Arabidopsis NAS-knockout mutants (Klatte et

al., 2009; Schuler et al., 2012b) and transgenic tobacco overexpressing HvNAAT-A

(Takahashi et al., 2003b). In all studies, normal growth, development, and

physiology were restored upon NA resupply, be it by application of exogenous NA

or grafting onto a wild-type or NAS-overexpressing rootstock.

2.4.3 Translocation

Following acquisition into the root symplast, iron is transported across the root

to the vascular tissue and subsequently to the rest of the plant. The translocation

process itself is a multi-step process, involving symplastic movement across the

Casparian strip and to the desired site; the loading, unloading and transport through

the vascular tissue; as well as remobilisation from source tissue (Kim and Guerinot,

2007).

During transport, iron is maintained as a chelated complex with ascorbate,

citrate or NA (Brown and Chaney, 1971; Stephan and Scholz, 1993; Pich et al.,

1994; Grillet et al., 2014). With graminaceous plants, iron may also be complexed to

DMA or MAs for transport (Koike et al., 2004; Ishimaru et al., 2010). As

demonstrated by von Wirén et al. (1999), such complexes are pH-dependent, as are

their interactions with other iron chelators. NA for instance, chelates both Fe(II) and

Fe(III) at a higher pH but will preferentially bind for the former at pH 7.0. When

bound to Fe(III) at equilibrium, the NA complex dominates at pH 7.0 to 9.0 while


the structurally similar DMA complex dominates at pH 3.0 to 6.0. Citrate removes

iron from NA at pH 5.5; and it must be converted to Fe(III)-citrate even if Fe(II) is

the major form in which Fe is loaded into xylem.

As with the uptake process, non-graminaceous plants seem to rely on

reduction-based strategy while graminaceous plants utilise a chelation-based one in

which Fe(III) undergoes little to no change in redox state. A marked difference in

stable iron isotope fractionation was observed between several graminaceous and

non-graminaceous species, with the former exhibiting a near consistent isotope

composition across all tissues, while the latter showed increasing depletion of heavy

isotopes in the younger parts corresponding with growth (Guelke and Von

Blanckenburg, 2007; Guelke et al., 2010).

2.4.4 Storage

Upon reaching the sink tissue, iron is reallocated as a cofactor in various

complexes, or bound to the iron storage molecule ferritin and stored in the apoplastic

space and vacuoles (Briat and Lobréaux, 1997). Subsequent translocation and

remobilisation may occur in response to developmental and physiological needs,

such as during iron deficiency (Waters and Troupe, 2012), seed filling (Hocking and

Pate, 1977; Burton et al., 1998; Garnett and Graham, 2005), senescence (Shi et al.,

2012; Maillard et al., 2015), and nodulation (Strozycki et al., 2007).

2.4.4.1 Ferritin

Ferritins are a superfamily of iron storage proteins found in all living things

except yeast. They consist of 24 subunits which form a spherical protein shell with a

central cavity capable of holding between 2000 and 4500 ferric iron atoms. Despite

significant structural conservation, the localisation of ferritin and much of their

structural and functional properties is organism specific. Plant ferritins are distinct in

that they straddle the middle ground between animal and bacteria ferritins – they

share conserved regions with a 39 to 49% amino acid sequence similarity with

animal ferritins though, like bacterial ferritins, plant ferritins are amorphous and have

mineral cores with high phosphate content (Briat et al., 1999). Also, unlike animal

ferritins plant ferritins are localised within the plastids and the apoplasmic space

rather than in the cytosol (Briat et al., 2010).


This localisation into apoplasmic space and plastids has been attributed to the

optimal conditions (low pH, high organic acid concentrations) which facilitate iron

deposition (Briat et al., 1999). This is at odds with the results of Laulhere and Briat

(1993) which found iron uptake into ferritin to be faster at pH 8.5 compared to pH 5

and 6. On the other hand, it was also mentioned that iron release is facilitated at low

pH, which may have hindered the sensitivity of specific radioactive detection of

exogenous iron. This is likely to be the case if one were to consider the necessity of

the presence of a reductant in the loading/unloading of iron into ferritin (Laulhere

and Briat, 1993), as it is at below pH 6 that NA-iron complexes dissociate, promoting

for iron transfer to ferritin (Stephan et al., 1996).

The exact means of uptake remains ambiguous, though it has been

hypothesised that the channels in apoferritin shells allow for some degree of access

to the core for small reducing and chelating agents/complexes (Harrison et al., 1974).

In the core, iron-binding sites allow for the initial formation of stable hydrous ferric

oxide nuclei, unto which more iron is subsequently loaded. Whether iron is

converted to an intermediate form during the transfer from chelated complex to the

core is unknown (Harrison et al., 1974).

When stored in ferritin, iron is maintained in a soluble and bioavailable form.

In plants, accumulation of ferritin mostly occurs in non-green plastids present in seed

and meristematic tissues (Seckback, 1982). Its notable absence in mature

photosynthetic tissue is indicative of its function in providing developing tissue with

the necessary stores for synthesis of iron-containing proteins, such as those required

in photosynthesis (Briat et al., 1999). A similar deduction can be made in nodule

formation in leguminous plants. Iron is required for nodule initiation and function in

a quantity exceeding that of the host roots (Tang et al., 1990); the initiation stage in

particular, is sensitive to iron deficiency (O'Hara et al., 1988). Ferritin is present in

the initial stages to supply the necessary iron stores, after which is it succeeded by

heme as the nodule matures (Bergersen, 1963; Ko et al., 1987).

Senescing tissues also accumulate ferritin, possibly as a means to safely

contain iron released from the breakdown of the photosynthetic machinery

(Seckback, 1982; Briat et al., 1999). While bound to ferritin, iron is unable to react

with oxygen and generation of reactive oxygen species (ROS) through the Fenton

reaction is avoided (Ravet et al., 2009). Consequently, it is imperative that the


release of iron from ferritin be tightly regulated and the iron quickly chelated, as

reductive release of free iron from ferritin contributes to oxidative stress and toxicity

(Thomas and Aust, 1986). Due to this ability to sequester iron and nullify its toxicity

in vivo, ferritins serve not only as a form of iron storage but also as homeostatic

rheostats against oxidative stress. Overexpression of ferritin has been reported to

confer tolerance to oxidative stress and, consequently, necrotrophic pathogens which

utilise that mechanism for infection (Deák et al., 1999; Van Wuytswinkel et al.,

1999). Loss of function results in a decreased ability to deal with excess iron, leading

to overaccumulation and altered development, with mutants having reduced

vegetative growth, defective flower development, and reduced fertility (Ravet et al.,

2009).

For the purpose of iron biofortification, ferritin has traditionally been used to

increase iron content and bioavailability. Plant ferritin-bound iron has a

bioavailability comparable to that of ferrous sulfate, a ferrous salt used in iron

supplements (Davila-Hicks et al., 2004; Lönnerdal et al., 2006), and has been used

successfully to treat anemia (Beard et al., 1996; Murray-Kolb et al., 2003). However,

the mechanism through which it is absorbed by the body has been subject to some

contention. It has been proposed that ferritin is resistant to gastrointestinal digestion

and remains largely intact when absorbed (Lönnerdal et al., 2006), with uptake

occurring through endocytic pathways (San Martin et al., 2008; Antileo et al., 2013).

Others have disagreed, asserting that ferritin is degraded during digestion and

absorbed as non-heme iron (Bejjani et al., 2007; Hoppler et al., 2008). With the

exception of Hoppler et al. (2008), most studies from both camps utilized pure

ferritin in their experiment. This makes it difficult to confirm the structural and

chemical influence of surrounding tissue on the digestion of plant ferritin (e.g.

protection by hulls, cell walls, plastid membrane). In addition, the effect of various

food processing and cooking techniques on ferritin breakdown is currently unknown;

it is possible that a mixture of degraded and undegraded ferritin may result from that,

requiring both absorption mechanisms.

Use of ferritin in monogenic approaches for iron biofortification has provided

increases in seed iron content ranging from 1.2-3 times (Goto et al., 1999;

Vasconcelos et al., 2003). On the other hand, deleterious effects have also been

observed by various authors. Iron-deficiency symptoms were found to manifest in


ferritin-transformed rice when grown under both iron-sufficient and high iron

conditions (Qu et al., 2005; Masuda et al., 2013b). Similar symptoms were also

manifested in tobacco when ferritin was expressed under a strong constitutive

promoter; additionally, ferritin over accumulated in the leaves, leading to illegitimate

iron sequestration and increased root ferric reductase activity (Van Wuytswinkel et

al., 1999). It is generally agreed that in ferritin transformants, iron storage capacity

exceeds that of uptake and that iron uptake becomes the limiting factor to iron

accumulation (Van Wuytswinkel et al., 1999; Qu et al., 2005; Masuda et al., 2013b).

This limitation can be compensated for by using iron uptake genes in conjunction

with ferritin, as proven by Masuda et al. (2013).

2.4.5 Regulation

Regulation of iron metabolism in response to internal and external iron

concentrations is achieved at both the transcriptional and post-transcriptional levels.

In non-graminaceous plants, the family of basic helix-loop-helix (bHLH)

transcriptional regulators serve as key regulators by moderating the expression of

IRT and FRO (Colangelo and Guerinot, 2004; Brumbarova and Bauer, 2005; Bauer

et al., 2007; Yuan et al., 2008). A similar mechanism present in rice affects

phytosiderophore biosynthesis and transport pathways in addition to IRT and FRO

(Ogo et al., 2006; Ogo et al., 2007; Ogo et al., 2011; Itai et al., 2013). In

graminaceous plants, the transcription factor IDEF (IDE-binding factor) binds to IDE

(cis-acting iron deficiency-responsive element) and triggers expression of genes

involved in uptake and translocation (Kobayashi et al., 2003; Kobayashi et al.,

2007). Another transcription factor is NAC (NO APICAL MERISTEM Arabidopsis

transcription factor, and CUP-SHAPED COTYLEDON), which regulates

remobilisation of iron from vegetative tissues (Waters et al., 2009).

Regulation also occurs on a hormonal level. Ferritin expression has been linked

to abscisic acid (ABA) concentrations (Lobréaux et al., 1993; Briat et al., 1999),

while ethylene have been associated with upregulation of iron uptake genes (Lucena

et al., 2006; Garcia et al., 2010) Nitric oxide (NO), a bioactive compound that serves

as a signalling molecule, has also been implicated in the transcriptional and post-

transcriptional regulation of the iron uptake, translocation and storage genes (Murgia

et al., 2002; Graziano and Lamattina, 2005; Garcia et al., 2010). Recently

epigenetics has emerged as an additional layer of iron metabolic regulation; however,


further work is required to elucidate the molecular mechanisms driving regulation

(Xing et al., 2015).

2.5 ENGINEERING FOR ENHANCED IRON CONTENT

In the interest of iron biofortification, five rate-limiting steps to grain iron

accumulation have been identified by Sperotto et al. (2012) (see Figure 2.5). These

can be classified into three main processes: uptake, translocation, and storage.

Figure 2.5. Possible rate-limiting steps for grain iron accumulation (Sperotto et

al., 2012).

Genes associated with these processes have been identified as promising

candidates for iron biofortification. Over the past decade several of them have been

used in both monogenic and multigenic approaches in different model plants, most

notably rice (Masuda et al., 2013a). These approaches and their varying levels of

success have been covered in the review by Masuda et al. (2013a), and of the

combinations investigated thus far, those containing NAS and ferritin have yielded

the most promising results. Iron accumulation was increased by approximately three

times in rice expressing with the rice YSL2, barley NAS1, and soybean ferritin genes

(Masuda et al., 2012). A four-fold increase in seed iron content was observed in


transgenic rice expressing soybean ferritin in conjunction with barley NAS1, two

nicotianamine aminotransferase genes and a mugineic acid synthase gene (Masuda et

al., 2013b). Constitutive expression of Arabidopsis NAS1 together with barley

ferritin and a fungal phytase, both under the regulation of a rice seed storage globulin

promoter, enhanced iron accumulation in rice endosperm by up to six times (Wirth et

al., 2009).

Aside from the choice of genes themselves, a key feature in these studies is the

promoters used to drive the genes. While having too weak a promoter may not

produce the desired level of iron accumulation, one too strong may lead to adverse

effects. Ferritin in particular, is liable to such side effects when overexpressed under

the strong constitutive promoter like the 35S promoter (Qu et al., 2005; Masuda et

al., 2013b). For such cases the use of tissue-specific promoters, like the seed-specific

globulin promoter, appears to negate the issue. With NAS, high expression levels

appear to be more well tolerated physiologically, giving no apparent side effects

(Johnson et al., 2011).

It should be noted that that these studies were conducted in rice, which utilises

both Strategy I and II mechanisms. Whether using similar genes and promoters will

have the same effect on iron accumulation in non-graminaceous grain crop like

chickpea is currently unknown. The effect on bioavailability is likewise unknown;

unlike rice, chickpea contains higher levels of bioavailability inhibitors (Hemalatha

et al., 2007b). Hypothetically, improving the translocation and storage capabilities

using NAS and ferritin would drive the flux in favour of iron accumulation. As both

NAS and ferritin also promote bioavailability (Davila-Hicks et al., 2004; Zheng et

al., 2010), bioavailability may also be expected to increase.

2.6 SUMMARY AND IMPLICATIONS

In summary, iron deficiency is global problem which may be alleviated

through the use of biofortified crops. Transgenic biofortification efforts, as well as

most studies on iron metabolism, thus far have largely been directed at cereal crops

like rice. As members of the Gramineae family, their molecular biology and

physiology are differ significantly from their non-graminaceous counterparts.

Consequently, biofortification strategies successfully applied in a graminaceous

species like rice may behave differently in a non-graminaceous species.


In this study, the species of interest is chickpea, the second most important

pulse crop globally. As pulse crop, chickpea is rich in protein, making it an ideal

complement to the traditional starchy staples in biofortification efforts. To date, there

have been no recorded attempts to biofortify pulse crops via a transgenic approach.

The effectiveness of the different transgenic strategies therefore remains to be

explored.

Chapter 3: General Materials and Methods 31

Chapter 3: General Materials and Methods

3.1 GENERAL MATERIALS

3.1.1 Sources of specialised reagents

All reagents used in this project were sourced from scientific companies

including Bio-Rad (USA), Invitrogen (USA), Promega (USA), Qiagen

(Netherlands), Roche Diagnostics (Switzerland), and Sigma Aldrich (USA).

Oligonucleotides were synthesized and purchased from GeneWorks (Australia)

at a concentration of 100µM. All primers were diluted to form a 10µM working

stock.

Powdered MS media (with vitamins) was obtained from PhytoTechnology

Laboratories (USA).

Antibiotics used in this project were purchased from the following sources:

Antibiotic name Source

Ampicillin Roche

Kanamycin monosulfate Austratec (Australia)

Rifampicin

Timentin (also known as ticarcillin) Pure Science (New Zealand)

Merrum (also known as meropenem) Clifford Hallam (Australia)

3.1.2 Iron metabolism genes

The soybean Ferritin H2 gene was kindly provided by Dr Alexander Johnson

from the University of Melbourne, NSW, Australia.

3.1.3 Bacterial strains

Escherichia coli strain XL1-Blue was used for general plasmid cloning. For

plant transformation, Agrobacterium tumefaciens strains AGL1 and LBA4404 were

used for chickpea and tobacco transformation respectively.

32 Chapter 3: General Materials and Methods

3.1.4 Plant material

Seeds of Cicer arietinum (chickpea cv. HatTrick) were obtained from the

Australian seed company Grainland, Moree, NSW. Wild-type Nicotiana tabacum

plants were kindly provided by Ms Maiko Kato from the CTCB.

3.1.5 General solutions: Abbreviations and composition

3.1.5.1 Media recipes and related additives

Bacterial culture

IPTG: isopropyl-β-D-thiogalactopyranoside, 20% (w/v) prepared in dimethyl

sulphoxide

LB liquid growth media: 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract,

170mM sodium chloride

LB agar: LB media solidified with 1.5% bacto agar

X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 2% (w/v) prepared in

dimethyl sulphoxide

X-gluc: 5-bromo-4-chloro-3-indolyl-β-D-glucoronide-cyclohexylamine salt

Plant tissue culture

All media and solutions in this section are prepared in a total volume of 1L unless

otherwise stated.

Chickpea tissue culture

B5 co-cultivation media: 100mL B5 Macro Stock (10x), 1mL B5 Micro Stock

(1000x), 10mL Fe-EDTA Stock (100x), 10mL B5 Vitamins Stock (100x), 3%

sucrose, 1.95g MES monohydrate, 1mL BAP (0.5mg/L), 1mL NAA (0.5mg/L), pH

5.8, 8g Difco agar, 1mL acetosyringone (100mM)

B5 Macro stock (10x): 25g KNO3, 1.5g CaCl2.2H2O, 2.5g MgSO4.7H2O, 1.4g

NaH2PO4.2H2O, 1.34g (NH4)2SO4 per 100mL

B5 Micro stock (1000x): 0.3g H3BO3, 1.36g MnSO4, 0.2g ZnSO4, 75mg KI, 25mL

NaMoO4.H2O (1mg/mL), 2.5mL CuSO4 (1mg/mL), 2.5mL CoCl2 (1mg/mL)

Fe-EDTA stock (100x): 37.25g Na2EDTA.2H2O, 27.85g FeSO4.7H2O


B5 Vitamins stock: 100mg thiamine HCl, 10mg nicotinic acid, 10mg pyridoxine

HCl per 100mL

RS0 media: 4.43g MS powder, 30g sucrose, 1.95g MES monohydrate, 8g Difco

agar, pH 5.8

RS1 media: 500µL BAP (1mg/mL), 500µL kinetin (1mg/mL), 50µL NAA

(1mg/mL) per L of RS0 media

RS2 media: 500µL BAP (1mg/mL), 500µL kinetin (1mg/mL) per L of RS0 media

RS3 media: 100µL BAP (1mg/mL), 100µL kinetin (1mg/mL) per L of RS0 media

Water agar: 0.8g Difco agar

Tobacco tissue culture

MS0 media: 4.43g MS powder, 30g sucrose, 8g Difco agar, pH 5.7

MS104 media: 1mL BAP (1mg/mL), 100µL NAA (1mg/mL) per L of MS0 media

Plant hydroponics

All media and solutions in this section are prepared in a total volume of 1L unless

otherwise stated.

Hoagland nutrient solution

Hoagland nutrient solution: 3mL Hoagland Macro stock 1, 2mL Hoagland Macro

stock 2, 0.5mL Hoagland Micro stock, 2.5mL Hoagland Fe-EDTA stock, 2.5mL

Hoagland buffer

Hoagland Macro stock 1: 101.1g KNO3, 18.99g NH4H2PO4, 81.31g MgSO4.7H2O

Hoagland Macro stock 2: 236.2g Ca(NO3)2.4H2O

Hoagland Micro stock: 2.86g H3BO3, 1.81g MnCl2.4H2O, 0.22g ZnSO4, 0.08g

CuSO4.5H2O, 0.03g NaMoO4.H2O

Hoagland Fe-EDTA stock: 3.72g Na2EDTA.2H2O, 2.78g FeSO4.7H2O

Hoagland Buffer: 39.06g MES monohydrate, pH 6.0


3.1.5.2 Solutions for molecular work

Solutions for nucleic acid extraction

CTAB buffer: 2% CTAB (cetyltrimethylammonium bromide), 2M NaCl, 25mM

EDTA pH 8, 100mM Tris-HCl, 2% polyvinylpyrrolidone (PVP

40)

CHCl3:IAA: chloroform: isoamyl alcohol (24:1)

TPS buffer (adapted from Thomson and Henry (1995)): 100mM Tris-HCl (pH

9.5), 1M KCl, 10mM NaEDTA

Solutions for gel electrophoresis

Agarose gel loading dye (6X): 0.25% (w/v) bromophenol blue, 50% TE, 50%

glycerol

TAE buffer: 10mM Tris-acetate, 0.5mM EDTA, pH7.8

TE: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA

Solutions for Southern analysis

Denaturation solution: 0.5M NaOH, 1.5M NaCl

Depurination solution: 0.25M HCl

Detection buffer: 0.1M Tris-HCl, 0.1M NaCl, pH 9.5

High stringency buffer: 0.1x SSC, 0.1% SDS

Low stringency buffer: 2x SSC, 0.1% SDS

Maleic acid buffer: 0.1M maleic acid, 0.15M NaCl, pH 7.5

Neutralisation buffer: 0.5M Tris-HCl (pH 7.0), 1.5 NaCl, 1mM EDTA

SSC (10x): 30mM sodium citrate, 0.3M NaCl, pH 7.5

Washing buffer: 0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% (v/v) Tween 20

3.2 GENERAL METHODS

3.2.1 General molecular techniques

3.2.1.1 Polymerase chain reaction (PCR)

Each reaction consisted of 5µL of 2X GoTaq green (Promega), 0.25µL of each

of 10µM forward and reverse primers, and up to 1µL of the template DNA. MilliQ


water added to reach a final volume of 10µL. Where required, 0.6µL of DMSO was

added to reach a final volume of 10.6µL.

The PCR program used was as such: initial denaturation at 95⁰C for three

minutes, followed by 30 cycles of denaturation at 95⁰C for 30 seconds, annealing at

48–60⁰C (depending on primers) for 30 seconds, and extension at 72⁰C. Extension

time was set at one minute per 1 kbp of the final product size. A final extension was

done at 72⁰C for five minutes.

3.2.1.2 Gel electrophoresis

Agarose gels with concentrations ranging from 0.8–2% (w/v) were prepared by

dissolving agarose (Roche Diagnostics – USA) in 1X TAE buffer, and adding 0.5X

SYBR Safe DNA gel stain (Invitrogen – USA). Gels were cast and run in an

EasyCast Mini Gel System. A 2-Log molecular weight marker (New England

Biolabs, USA) was loaded in at least one lane in each run to allow for determination

of size and concentration of nucleic acids. Electrophoresis was carried out in TAE

buffer at 100V for 40 minutes. Visualisation and photography of the electrophoresis

results were done using a Syngene Geldoc system (G-box and GenSnap Version

6.07) (Syngene – UK).

Purifying of electrophoresis products, if desired, was done by excising the

appropriate band and extracted using Freeze ‘N Squeeze DNA Gel Extraction Spin

Columns (Biorad) as per the manufacturer’s instructions.

3.2.1.3 DNA sequencing

All DNA sequencing was performed using the BigDye 3.1 standard methods at

the genomics facility located at the Queensland University of Technology (QUT),

Gardens Point Campus.

3.2.2 Bacterial transformation

3.2.2.1 Escherichia coli transformation

Transformation was done on chemically-competent XL1 Blue E.coli using a

heat-shock method. A 200µL aliquot of competent cells was first thawed on ice, and

1 to 5µL of the plasmid of interest mixed in. After approximately 30 minutes of

incubation on ice, the cells were heat-shocked by placing the tube in 42⁰C water bath

for 30 seconds, after which the tubes were immediately placed on ice. When cooled,


the cells were transferred to a 2mL microfuge tube containing 250µL of pre-warmed

LB broth (with no selection) and incubated at 37⁰C, 200 rpm for one hour. A 50µL

aliquot of the outgrowth culture was then spread onto an LB plate containing the

relevant selection agent, and the plates incubated overnight at 37⁰C or for two nights

at room temperature.

3.2.2.2 Agrobacterium transformation

A 100µL aliquot of electro-competent competent cells was first thawed on ice

and 1µL of the plasmid of interest added. The mixture was then mixed and

transferred to a cooled electrocuvette and electroporated using an EC100

electroporator (Thermo EC). Following this, 400µL of pre-warmed LB broth (with

no selection) was added to the cuvette. The cuvette contents were then transferred to

a 2mL microfuge tube and incubated at 28⁰C, at 200 rpm for one to two hours. A

50µL aliquot of this culture was then spread onto an LB plate containing 25µg/L of

rifampicin and 100µg/L of kanamycin, and the plates incubated for two days at 28⁰C.

3.2.3 Plant transformation

3.2.3.1 Tobacco transformation

3.2.3.1.1 Preparation of Agrobacterium culture

A starter culture was prepared by inoculating approximately 5mL of LB media

containing 25mg/L rifampicin and 100mg/L kanamycin with 100µL of LBA4404

Agrobacterium glycerol stock. The culture was incubated overnight at 28⁰C at

200rpm. To prepare the final working culture, 10µL of the starter culture was used to

inoculate 5mL of LB media containing 100mg/L kanamycin; this final culture was

then incubated overnight at 28⁰C at 200rpm.

3.2.3.1.2 Transformation, selection and regeneration

Nicotiana tabacum (cv Samsun) leaves were cut into 1 – 1.5cm2 pieces and

immersed in an overnight culture of Agrobacterium (strain: LBA4404) diluted 1:10

with liquid MS0 medium (pH 5.7). The mixture was shaken vigorously. The leaf

discs were then blotted dry on filter paper and placed adaxial side down on MS104

media. The plates were incubated at 25⁰C with moderate light for three days.

Following that, the leaf discs were transferred to MS104 media containing 200mg/L

kanamycin and incubated at 25⁰C with a 16 hour photoperiod. Leaf discs were

transferred to fresh media every two weeks. When well-defined stems have formed,


the plantlets were excised and placed onto fresh MSO selection media to allow for

further growth and rooting

3.2.3.1.3 Acclimatisation of transgenic plants

Upon sufficient rooting, plantlets were washed to remove all traces of media

from the roots, then planted in 150 mm pots filled with Plugger 222 potting mix

(Australian Growing Solutions). The soil was gently compressed around the roots

and the plantlets watered well. The plantlet was kept in a tub sealed with a clear

plastic film and placed in a growth room set at 25 ± 1⁰C, with 16 hour light/ 8 hour

dark cycle. After three days, the plastic film was loosened, then completely removed.

Hardened plants were maintained in the same growth conditions until seeds were

obtained.

3.2.3.2 Chickpea transformation

3.2.3.2.1 Seed sterilisation

Approximately 50 – 60g of dry seed was weighed out into a plastic canister and

rinsed with water before vigorous shaking in 70% (v/v) ethanol for two minutes. The

ethanol was then replaced with a freshly prepared 1.5% (v/v) sodium hypochlorite

solution (diluted with sterile MilliQ water) and the canister agitated for 7 – 8

minutes. Following this, the sodium hypochlorite was decanted and the seeds washed

5 – 7 times with sterile MilliQ water. Damaged and discoloured seeds were removed

with a pair of sterile forceps. The remaining seeds were imbibed overnight at room

temperature in sterile MilliQ water.

3.2.3.2.2 Preparation of Agrobacterium culture

A starter culture was prepared by inoculating approximately 5mL of LB media

containing 25mg/L rifampicin and 100mg/L kanamycin with 100µL of AGL1

Agrobacterium glycerol stock. This culture was incubated overnight at 28⁰C at

200rpm. To prepare the final working culture, 100µL of the starter culture was used

to inoculate 100mL of LB media containing 100mg/L kanamycin. This final culture

was then incubated overnight at 28⁰C at 200 rpm, until an OD600nm of 0.6 – 1.2 was

reached.

3.2.3.2.3 Co-cultivation of explants with Agrobacterium

An aliquot of 100µL of freshly prepared 100mM acetosyringone solution was

added to 100mL of overnight Agrobacterium culture. The culture was then incubated


at room temperature for approximately three hours, during which the explants are

prepared for infection. Approximately 1 mm was trimmed off the ‘beak’ of the

overnight imbibed seeds, and the seeds bisected along the longitudinal axis. The seed

coat was removed and the radicle of each half embryonic explant was pricked

between six to eight times with a sterile 26 gauge needle dipped in Agrobacterium

culture. The injured explants were then immersed in Agrobacterium culture for one

to two hours, during which they were incubated in a growth cabinet set at 24⁰C with

fluorescent light. Following this, the culture was drained and the explants placed cut

side-down on B5 media. The plates were then sealed with Micropore tape. The

explants were co-cultivated with Agrobacterium culture for 72 hours at 24 ± 1⁰C,

under fluorescent lights with a 16 hour light/ 8 hour dark cycle.

3.2.3.2.4 Regeneration and selection of transformed plants

After co-cultivation, the explants were transferred on RS1 media, with up to 16

explants per plate. After 14 days, the explants were subcultured to RS2 media, during

which the roots, and any dead or dying tissue were trimmed off. Two-thirds of the

cotyledon was also removed, and a small incision made at the cotyledonary node, to

an approximate depth approximately 1mm, with a size 10 scalpel blade. The explants

were incubated for 14 to 21 days before transferral to fresh RS2 media for the third

round of selection. Bleached, dead and dying tissues were trimmed off, as was the

remaining cotyledon. The explants were arranged with 9 explants per plate. The base

of another deep Petri dish (90 x 25mm) was used as a lid and the plates sealed with

Micropore tape. The explants were subcultured for up to eight rounds of selection,

with each round lasting up to 21 days. Clumps of secondary shoots that emerged

from the base of the explants during the selection process were isolated and

transferred to separate RS2 plates for further multiplication. Each clump was

numbered and considered a single putative transgenic event. Upon reaching an

appropriate size, shoots from such clumps were then grafted onto non-transgenic

rootstocks. Throughout the selection process, culture conditions were maintained at

24 ± 1⁰C, with full light and a 16 hour light/ 8 hour dark cycle.

3.2.3.2.5 In vitro grafting

Non-transgenic chickpea seeds were sterilised and germinated for four days on

deep petri dishes containing ½ MS media with no sucrose. After four days, the most

of the hypocotyl was removed with a transverse cut above the first node. A silicon


ring was placed over the decapitated hypocotyl and an incision made along the

longitudinal axis to prepare for insertion of the scion. Healthy looking shoots were

selected for use as scions; they were removed from shoot clumps and the base cut

into a V-shape. The scion was then inserted into the incision in the rootstock and the

silicon ring pulled up to secure the graft. The base of another deep Petri dish was

inverted to serve as a lid for the plate, and the setup was sealed with Micropore tape.

The grafted plants were incubated in a growth chamber at 24 ± 1⁰C, with 16 hour

light/8 hour dark cycle, for eight to ten days to allow the graft to seal. Any side

shoots emerging from the rootstock were removed to promote growth of the grafted

scion.

3.2.3.2.6 Acclimatisation of grafted plants

Grafted plantlets were carefully removed from the medium and the roots

washed to remove all agar. The seed coats were removed to reduce fungal growth.

The grafted plantlets were then transferred to 150mm diameter pots half filled with

sterilised University of California (UC) mix and the roots covered with additional

UC mix. The soil was then gently compressed around the roots and the plantlets

watered. During the transfer, care was taken to keep the plantlet upright and the graft

above the soil. The plantlet was then covered with a transparent plastic jar and placed

in a growth cabinet set at 22 ± 1⁰C, with 16 hour light/8 hour dark cycle. Checks

were performed every day, during which the transparent plastic jars were wiped free

of condensation. After 15 – 20 days, or when the plants had grown big enough to

nearly touch the walls of the jar, the jars were left partially open for two to three days

to reduce humidity, and then completely removed. The plants were transplanted to

400mm x 250mm pots and transferred to a glasshouse upon successful establishment

in the soil.

3.2.3.2.7 Generation of transgenic seed material

Seeds were harvested from T0 plants and sown into potting mix in 100mm tube

stocks. The soil was kept damp, but not saturated. After emergence, samples were

collected from fully expanded leaves for PCR screening. PCR-positive progeny were

transplanted to 400mm x 250mm pots with slow release fertiliser for further growth

and seed production. The process of harvesting, screening and transplanting was

repeated with subsequent generations. Generation time was approximately 90 days

from sowing to harvest. In all harvests, seeds were collected only from dried pods


and the quantity obtained from each plant was recorded. Collected seeds stored in

small labelled envelopes and allowed to dry further at room temperature for at least

three days, after which they were stored at 4⁰C until ready for planting. At least five

PCR-negative siblings from the same generation were also maintained to serve as

controls.

3.2.4 Plant growth conditions

Conditions for the growth chamber and glasshouse were set at 23⁰C and 28⁰C

respectively, with a 16 hour photoperiod.

3.2.5 Verification and molecular characterisation of transgenic plants

3.2.5.1 Nucleic acid extraction

3.2.5.1.1 Quick release method (for rapid screening)

A piece of tissue at least 2 mm2 was homogenised in 100µL of TPS buffer and

incubated at 65⁰C for ten minutes, then cooled on ice. When cool, 100µL of

choloroform/IAA (24:1) was added and mixed by vortex or inversion. The mixture

was then centrifuged at 18, 000 rcf for ten minutes at room temperature. A 10µL

aliquot of the upper phase was removed and diluted in 40µL of MilliQ water for use

as template in PCR.

3.2.5.1.2 Small scale DNA extraction

Fresh leaf samples were homogenised with a TissueLyser or with mortar and

pestle. The homogenised tissue was mixed with 800µL of pre-warmed CTAB

solution and incubated at 65⁰C for thirty minutes. An 800µL aliquot of

choroform:IAA (24:1) was added and the mixture mixed by inversion. Samples were

then centrifuged at 18, 000 rcf for five minutes. The resulting upper phase was

transferred to a fresh tube and 2µL of RNAse A added. The mixture was then

incubated at 37⁰C for 15 minutes, following which 800µL of choroform:IAA (24:1)

was mixed in and the mixture centrifuged at 18, 000 rcf for five minutes. The upper

phase was transferred to a fresh tube and an equal volume of choroform:IAA (24:1)

mixed in. The mixture was centrifuged at 18, 000 rcf for five minutes and the

resulting upper phase was transferred to a fresh tube, to which 2.5 times the volume

of 100% ethanol was added. DNA allowed to precipitate at 4 ⁰C for at least 30

minutes. The samples were centrifuged at 18, 000 rcf for ten minutes and the

supernatant discarded. A 1mL aliquot of 70% ethanol was added to the remaining


DNA pellet and the samples centrifuged again at 18, 000 rcf for five minutes. The

supernatant was discarded and the DNA pellet allowed to dry on the benchtop at

room temperature, after which it was resuspended in 30µL of MilliQ water.

3.2.5.1.3 Large scale DNA extraction

Fresh leaf samples were homogenised with a TissueLyser or with mortar and

pestle. The homogenised tissue was mixed with 2 mL of pre-warmed CTAB solution

and incubated at 65⁰C for ten minutes. A 2 mL aliquot of choroform:IAA (24:1) was

added and the mixture mixed by inversion. Samples were then centrifuged at 3000

rcf for ten minutes. The resulting upper phase was transferred to a fresh tube and 3µL

of RNAse A added. The mixture was then incubated at 37⁰C for 15 minutes,

following which 2mL of choroform:IAA (24:1) was mixed in and the mixture

centrifuged at 3000 rcf for ten minutes. The upper phase was transferred to a fresh

tube with 2.5 times the volume of 100% ethanol, and the DNA allowed to precipitate

at 4⁰C for at least 30 minutes. Precipitated DNA threads were harvested via spooling

with a glass hook, on which it was dried on the bench at room temperature. Upon

drying, the spooled DNA was resuspended in a 400µL of 1M NaCl solution, then re-

precipitated with 2 times the volume of 100% ethanol. The precipitated samples were

centrifuged at 18, 000 rcf for ten minutes and the supernatant discarded. A 1mL

aliquot of 70% ethanol was added to the remaining DNA pellet and the samples

centrifuged again at 18, 000 rcf for five minutes. The supernatant was discarded and

the DNA pellet allowed to dry on the benchtop at room temperature, after which it

was resuspended in 100µL of MilliQ water.

The samples were centrifuged at 18, 000 rcf for ten minutes at 4⁰C and the

supernatant discarded. The resulting DNA pellet was allowed to dry on the benchtop

at room temperature, then resuspended in 30µL of MilliQ water.

3.2.5.1.4 RNA extraction

RNA extraction was performed using RNeasy Mini kits (QIAGEN). All

reagents were prepared as per manufacturer’s instructions. Briefly, up to 100mg of

frozen fresh tissue or 30mg of freeze-dried tissue was used as starting material. All

tissues were homogenised using a TissueLyser until a fine powder was obtained.

Fresh tissues were first snap-frozen in liquid nitrogen prior to homogenisation, and

cooled between 20 second milling rounds to prevent thawing of samples. An aliquot

of 450µL of Buffer RLT with 1% β-mercaptoethanol was added to each sample and


the tubes vortexed vigorously to mix. The lysate was then transferred to a

QIAshredder spin column and centrifuged at 18, 000 rcf for two minutes. The

supernatant was transferred to a fresh 2mL microfuge tube and 0.5X volume of

100% ethanol mixed in by pipetting. The sample was then transferred to an RNeasy

spin column and centrifuged for 15 seconds at 18, 000 rcf. The flowthrough was

discarded and 700µL of Buffer RW1 added to wash the column. The samples were

again centrifuged and the flowthrough discarded. This was followed by two washes

with 500µL of Buffer RPE; in the final wash, the samples were centrifuged for two

minutes to dry the membrane. The flowthrough was again discarded and the samples

centrifuged for an additional one minute to remove all traces of Buffer RPE. The

column was then transferred to a 1.5mL microfuge tube. Elution was done by adding

30µL of RNAse-free water to the column and centrifuging at 18, 000 rcf.

3.2.5.1.5 Quantification of nucleic acids

Estimation of nucleic acid quantity was performed using a NanoDrop2000

spectrophotometer (Thermo Scientific – USA). Absorbance was measured at 230,

260, and 280nm. The purity of gDNA and RNA was evaluated based on the ratios of

absorbance at 260 and 280nm, and 260 and 230nm respectively. The quantity and

quality was confirmed through electrophoresis of 500 ng of nucleic acids on a 1% gel

for gDNA, and a 1.5 – 2% gel for RNA.

3.2.5.2 Gene expression analysis

3.2.5.2.1 DNAse treatment

Prior to cDNA synthesis, contaminating DNA was removed from RNA

samples using RQ1 DNAse (Promega). One unit of RQ1 RNAse-free DNAse was

used per 1µg of RNA. Up to 8µL of RNA was mixed with 1µL of RQ1 RNAse-free

DNAse 10X reaction buffer and 1µL of RNAse-free DNAse, and nuclease-free water

added to a final volume of 10µL. The reaction was incubated at 37⁰C for at least 30

minutes, then transferred to -20⁰C for temporary storage. A 0.5µL aliquot of the

treated RNA was used as a template for PCR to check for the presence of DNA.

Upon confirming the absence of DNA, the reaction was incubated at 65⁰C for 10

minutes to deactivate the DNAse. The treated RNA was stored at -80⁰C.

3.2.5.2.2 cDNA synthesis

cDNA synthesis was performed using SuperScript™ IV Reverse Transcriptase

(ThermoFisher Scientific) as per the manufacturer’s instructions. Briefly, 1µL of


50µM oligod(T), 1µL of 10mM dNTP mix was added to 500ng of RNA, and

nuclease-free waster was added to a final volume to 13µL. The reaction was mixed,

heated at 65⁰C for five minutes, then incubated on ice for one minute.

Following this, 7µL of RT reaction mix was added to the annealed RNA. The

RT reaction mix consisted of 4µL of SSIV buffer, 1µL of 100mM DTT, 1µL of

RNseOUT™ Recombinant RNase Inhibitor, and 1µL of SuperScript® IV Reverse

Transcriptase (200U/µL). The combined reaction mixture was incubated at 55⁰C for

10 minutes, then inactivated at 80⁰C for 10 minutes. The resulting cDNA was stored

at -20⁰C.

3.2.5.2.3 Qualitative analysis of gene expression

Qualitative analysis of gene expression was done via PCR (see Chapter

3.2.1.1), using 1µL of cDNA as the template.

3.2.5.2.4 Quantitative real-time PCR (qPCR)

Measurement of transgene expression was performed on a CFX384 Touch™

Real-Time PCR Detection System (BIO-RAD) using the SYBR Green PCR Master

Mix kit (Applied Biosystems). A 1:30 dilution of cDNA was used for gene

expression analysis. Primers for three housekeeping genes were included in each run

to serve as internal controls.

3.2.5.2.5 Primer design.

Primers targeting the transgenes were designed the using the Primer3 software

(Koressaar and Remm, 2007; Untergasser et al., 2012). The length of all primers

ranged from 60 to 200bp, with a GC content of 40-60% and a melting temperature of

58 - 63⁰C. All primers were tested against gDNA and cDNA. The products generated

were sequenced as per section 3.2.1.3 to ensure amplification of the right product.

Primer sequences for reference genes were selected based on the publication by

Garg et al. (2010). The housekeeping genes actin 1, elongation factor 1-α (EF1α) and

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected for use as

internal references. The stability of these genes was confirmed in all tissue types

following the guidelines recommended by Hellemans et al. (2007). The

recommended values are as listed below, where CV refers to the coefficients of

variation, and M to the gene stability value.


Sample type CV M

Homogenous <0.25 <0.5

Heterogeneous <0.5 <1.0

Gene expression was calculated using the following formula:

𝐹𝑜𝑙𝑑 𝑐ℎ𝑎𝑛𝑔𝑒 = 𝑙𝑜𝑔2[𝐺𝑂𝐼 − (𝐻𝐾1 × 𝐻𝐾2 × 𝐻𝐾3 … )1𝑛]

GOI represents the gene of interest, HK represents the housekeeping gene, and

n represents the number of housekeeping genes.

3.2.5.3 Southern analysis

To determine transgene copy number, 15µg of gDNA was initially digested

overnight with an appropriate restriction enzyme which cut once between the T-DNA

borders of the respective plasmids used for the generation of the transgenic events.

Digested DNA was electrophoresed through a 0.8% agarose gels in TAE buffer

containing 0.5X SYBR Safe (Invitrogen) at 45V for 5 hours. Gels were prepared for

transfer by incubation for 10 minutes in depurination solution, followed by 30

minutes in denaturation solution and finally twice for 30 minutes in neutralisation

solution; gels were rinsed in double distilled water between each treatment. Gels

were then equilibrated in 10X SSC for 5 minutes before transfer to a positively

charged nylon membrane (Roche) overnight in 10X SSC according to the capillary

method of Southern (1975). Following transfer, the membrane was washed twice in

2X SSC for two minutes to remove salt residues and the DNA fixed to the membrane

by baking at 80˚C for 2 hours. Pre-hybridisation of the nylon membrane was done for

1 hour at 42ºC in DIG-easy Hyb Solution (Roche) with agitation. The DIG-labelled

PCR probe was then added and allowed to hybridise to the membranes overnight

with rotation at 42ºC. The following day, the membrane was washed twice for 5

minutes with low stringency buffer at room temperature. This was followed by two

rounds of washing with pre-warmed high stringency buffer, each done for 15 minutes

at 68 ºC. The membrane was rinsed briefly in maleic acid buffer and blocked in

maleic acid buffer containing 3% skim milk powder. After blocking, the membrane

was incubated in a solution containing 1:20,000 diluted mouse-derived anti-DIG

antibody (Roche) in maleic acid buffer with 3% skim milk powder for 30 minutes at

room temperature. Unbound antibody was removed by two 15 minutes rounds of


washing in washing buffer. Prior to detection, all membranes were equilibrated in

detection buffer. Detection of DIG-labelled DNA was achieved using CDP-star

(Roche), as per manufacturer’s instructions.

3.2.5.3.1 Preparation of DIG-labelled probe

DIG-labelled probes used in for Southern analysis were generated via PCR.

The components for the labelling reaction are as listed below. An unlabelled

reaction, in which the DIG labelling mix was replaced with unlabelled dNTPs, was

run done simultaneously as a control.

10X buffer 3µL

25mM MgCl 1µL

Forward primer (10µM) 1µL

Reverse primer (10µM) 1µL

DIG labelling mix (10X) 3µL

Plasmid DNA (1:10 dilution of miniprep) 1µL

HybriPol DNA polymerase 1µL

dH2O added to final volume of 30µL

The PCR conditions for the reactions are as follows: initial denaturation at

94⁰C for two minutes, 35 cycles of denaturation at 94⁰C for ten seconds, annealing at

55⁰C for 55 seconds, extension 72⁰C for one minute, and a final extension at 72⁰C

for five minutes. The resulting PCR product was separated via electrophoresis,

purified, then stored at -20⁰C.

3.2.6 Trace element analysis

To avoid trace element contamination, washed nitrile gloves were worn and

only non-metal implements (e.g. plastic, wood) were used to handle samples.

Likewise, only PP tubes with HDPE screw caps from Greiner Bio-One were used for

extraction or storage purposes. Following appropriate preparation, leaf samples were

analysed via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-

MS). Seed samples were analysed via inductively coupled plasma optical emmision

spectrometry (ICP-OES).


3.2.6.1 Sample preparation

To avoid contamination from external sources of iron, all large foreign

particulate matter (e.g. soil, agar) was removed. Seeds were briefly brushed down

while leaf samples were rinsed twice in MilliQ water. All samples were freeze-dried

for at least 48 hours using a BenchTop Pro with Omnitronics freeze-dryer (SP

Scientific).

3.2.6.1.1 Preparation of samples for trace element analysis by laser-ablation

inductively coupled plasma mass spectroscopy (LA-ICP-MS)

Leaf samples were transferred to 1.5mL microfuge tubes containing a 3mm

tungsten carbide bead (QIAGEN) and milled using a TissueLyser II for ten minutes

at 30/s. To ensure complete and even milling, tubes were filled to a maximum of a

third of the total volume. Milled samples were then pressed into 5mm diameter

pellets, which were then stuck onto a hard plastic sheet using double-sided tape.

3.2.6.1.2 Preparation of samples for trace element analysis by inductively coupled

plasma optical emission spectroscopy (ICP-OES)

For analysis of seed material by ICP-OES, a minimum of five freeze-dried

whole seeds were ground with an IKA Tube Mill (IKA®, Germany) at 25, 000 rpm

for five rounds of 30 seconds. Milling was repeated as needed until a fine powder

was obtained. All processed material was stored in labelled 50mL PP tubes.

3.2.6.1.3 Extraction of trace elements for ICP-OES

When ready for analysis, no more than 350mg of processed freeze-dried tissue

was transferred to a labelled 50mL tube. Three technical replicates were prepared for

each sample. Digestion was initiated by the addition of 2mL HNO3 and 0.5mL H2O2.

The tubes were vortexed to ensure complete wetting of samples and allowed to stand

overnight at room temperature. The following day, the tubes were shaken at 200 rpm

for 20 minutes and heated first to 80⁰C for 30 minutes, then to 125⁰C for two hours.

Prior to shaking and increasing the temperature, the caps were briefly loosened to

release the accumulated pressure. The sample was allowed to cool to room

temperature before addition of MilliQ water to a total volume of 25mL. The caps

were then sealed and the samples agitated at 300 rpm for five minutes. Undissolved

material (e.g. silicates) was allowed to settle for 60 minutes. Filtering was performed

as required, such as with leaf tissue. The settled extract was then decanted into 15mL


falcon tube, capped and sealed with Parafilm, and stored at room temperature until

analysis.

3.2.6.2 Trace element quantification

All quantification of trace elements was performed in the analytical

laboratories located at the Queensland University of Technology (QUT), Gardens

Point Campus unless otherwise specified.

LA-ICP-MS was done using an Agilent 8800 Inductively Coupled Plasma

Mass Spectrometer attached with an ESI 193nm Excimer Laser. The laser ablation

settings used were as follows:

Laser ablation

Pulse width = 4 ns

Laser energy = 2.00 J/cm2

Repetition rate = 10 Hz

Laser helium flow = 515 mL/min

Speed of each line scan = 10 microns/sec

Spot size = 85 microns

ICP-OES was done on a Perkin ElmerOptima 8300 DV Inductively Coupled

Plasma Optical Emission Spectrometer.

3.3 DATA ANALYSIS

All graphs and standard errors were prepared using Microsoft Excel. Statistical

analysis was performed using one-way ANOVA, and comparison of means done

using Tukey’s HSD test or Dunnett’s test. All statistical analysis was done using

Minitab statistical software (Arend, 2010).

Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 49

Chapter 4: Assessment of Iron Content in

Chickpea cv Hattrick

4.1 INTRODUCTION

Cicer arietinum, or more commonly known as chickpea, is an annual self-

pollinating diploid (n=16) pulse crop belonging to the Fabaceae family that serves as

an important secondary staple. It is cool season legume that can be cultivated in a

wide range of soils and environments across both tropical and sub-tropical region,

and can been utilised in a variety of cropping systems (Saxena, 1987).

As a food crop, chickpea can be utilised in a variety of ways. Green pods,

immature seeds and young leaves can be consumed as a vegetable while the stover

and pod husks can be used as animal feed (Ibrikci et al., 2003; Yadav et al., 2007).

The primary commodity however, is the dried mature seed which can be used as

animal feed or for human consumption. With the latter, the long history of

consumption in various regions such as India, the Middle East and Europe has given

rise to a diversity of dishes in which chickpea can be utilised. Chickpeas are

consumed on their own or with other foods; seeds may be eaten whole, hulled, or

ground into flour from which other products may be derived. Preparation for

consumption can be done via various processing methods such as soaking, sprouting,

fermenting, boiling, steaming, roasting, extrusion and puffing (Yadav et al., 2007),

all of which exert different effects on the overall nutritional quality (Poltronieri et al.,

2000; Sebastiá et al., 2001; Ghavidel and Prakash, 2007; Hemalatha et al., 2007a).

Most of the chickpea in the global market can be classified into two main types

which are primarily distinguishable by their seed morphology, specific aspects of

which influence their end-use. The first type is the kabuli, also known as garbanzos.

Kabuli seeds are large and round, weighing approximately 400 mg per seed (Pulse

Australia, 2016). The seed coat is thin and light-coloured, ranging from shades of

white to cream and the seeds are typically consumed whole or made into hummus

(Gaur et al., 2015; Pulse Australia, 2016). Kabuli cultivation areas are mostly located

in Southern Europe, Northern Africa, Afghanistan, Pakistan, Chile and India (Gaur et

al., 2015).

50 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick

The second type is the desi, which forms the bulk of the international export

market (Rao et al., 2010). Desi seeds are small, wrinkled and angular, with an

approximate weight of 120 mg per seed (Pulse Australia, 2016). The seed coat is also

1.2 to 3 times thicker than the kabuli (Umaid et al., 1984; Wood et al., 2011) and can

be found in a greater variety of colours ranging from brown to yellow, as well as

orange, black and green. Desi seeds are commonly dehulled and split to obtain the

cotyledons, which are then known as chana dhal and can in turn be milled to flour,

known as besan or gram flour. Australian-grown desi chickpea, in particular, are

known to be superior quality for dhal production, with up to 90% of total export used

for that purpose (Agbola et al., 2000).

Desi and kabuli types are generally similar in terms of nutritional profiles with

comparable starch and amino acid contents. However there are differences in a few

key aspects which have been linked to seed coat morphology – kabuli seeds for

instance, tend to have a higher fat content while desi seeds are higher in fibre and

tannin contents (Petterson and Mackintosh, 1994; Khan et al., 1995; Rincón et al.,

1998). However considerable variations can be found across different cultivars and

sites, particularly when it comes to the mineral composition and distribution within

the seed (Wood and Grusak, 2007). Iron content for instance, have been reported to

range from 2.4 to 11 mg/100g (e.g. Jambunathan and Singh, 1981; Petterson and

Mackintosh, 1994; Bueckert et al., 2011; Thavarajah and Thavarajah, 2012; Nobile

et al., 2013). Even where overall iron content of the seeds may be similar,

accumulation patterns may vary. As demonstrated in the study by Jambunathan and

Singh (1981), the seed coat of kabuli cultivars was found to be significantly richer in

iron compared to the desi. A significant difference was also found in the phosphorus

content from seeds grown in two different locations; this is of especial interest as that

it may reflect phytate localisation and may thus be an indicator of iron bioavailability

of the end product. For that reason, both total iron content and distribution within the

seed require due consideration in biofortification approaches, particularly with

downstream processing methods in mind.

The significance of the interactions between genotype and environment (and by

extension, management practices) on the grain micronutrient profile has been

documented in several crop species including wheat (Ficco et al., 2009), rice (Norton

et al., 2014), sorghum, bean, pea, lentil, and chickpea (Ray et al., 2014). However up


until recently, crop development has primarily focused on productivity with little

consideration to the nutritional aspect. Current efforts to improve micronutrient

content of chickpeas are few (e.g. Diapari et al., 2014; Ray et al., 2014; Upadhyaya

et al., 2016), with no existing program within Australia. Consequently information

on the micronutrient-accumulating behaviour of recently developed Australian

cultivars is relatively sparse, if not non-existent, making it difficult to ascertain the

degree to which various factors may influence the micronutrient profile.

The aim of this chapter therefore, is to determine the range of iron

accumulation in the seeds of the Australian commercial desi cultivar PBA HatTrick

and identify potential factors that influence it. As part of this study, other commercial

cultivars from a range of field locations were assessed for comparison. The

approximate distribution of trace elements within PBA HatTrick seed was also

examined to establish a baseline for comparison in later chapters.

4.2 MATERIALS AND METHODS

4.2.1 Seed material and locations

The three kabuli and three desi cultivars were used in this study (Figure 4.1).

The details of each cultivar are as described in

Table 4.1, and the sites from which samples were obtained are listed in

Table 4.2. Seeds from field trials at Billa Billa, Warra, Roma, and Kingaroy

were provided by Dr Yash Chauhan and the Queensland Department of Agriculture

and Fisheries (DAF). Seeds from New South Wales (NSW) were obtained from the

seed company Grainland, Moree.

All seeds used in this study were grown in the 2014 growing season. Sowing

was done during winter between June and July, 2014. Harvesting was done during

summer between December, 2014, and January, 2015. Information on the cultivation

sites and conditions during the growing period, where available, are listed in Table

4.3. The soil types of the field locations were provided by Dr Yash Chauhan from the

Agricultural Production Systems sIMulator (APSIM) database. Whole undamaged

seeds were selected and the samples were sent to Waite Analytica Laboratories

(University of Adelaide) for trace element analysis.


Figure 4.1. Chickpea cultivars used in this study. Top row, from left: Kabuli

culitvars Genesis090™, Kalkee™ and PBA Monarch. Bottom row, from left:

Desi cultivars PBA Boundray, CICA0912 and PBA HatTrick.

Table 4.1. Description of chickpea cultivars used in this study.*

Genotypes Description Seed weight

(g/100 seeds)

Genesis090™ Small seeded kabuli. Cream coloured seed coat. 31.3

Kalkee™ Medium to large seeded kabuli. Cream coloured

seed coat. 45.0

PBA

Monarch

Medium sized kabuli. Cream coloured seed

coat. 40.5

PBA

Boundary

Medium sized desi. Dark brown coloured seed

coat. Suitable for splitting and direct

consumption.

19.5

CICA0912 Medium sized desi. Dark brown coloured seed

coat. 22.6

PBA

HatTrick

Medium sized desi. Dark brown coloured seed

coat. Suitable for splitting and direct

consumption.

20.1

*Details as published by Pulse Australia (2016)


Table 4.2. Locations from which seed samples were obtained.

Genotypes Billa

Billa Roma Warra Kingaroy NSW

Kabuli

Genesis090™ Yes Yes Yes n/a n/a

Kalkee™ Yes Yes Yes n/a n/a

PBA Monarch Yes Yes Yes n/a n/a

Desi

PBA Boundary Yes Yes Yes Yes n/a

CICA0912 Yes n/a Yes Yes n/a

PBA HatTrick Yes Yes Yes Yes Yes

Table 4.3. Cultivation conditions for each location.

Site Australian soil

classification Total fertiliser used

Approximate rainfall

during growing period

(mm)

Billa Billa Gray vertosol 25kg/ha Zinz star 25 64.5

Roma Black vertosol 25kg/ha Zinz star 25 85

Warra Gray vertosol 25kg/ha Zinz star 25 80.2

Kingaroy Ferrosol n/a 594

NSW n/a n/a n/a

4.2.2 Measurement of trace element distribution within the chickpea seed

A total of 100 undamaged seeds were rinsed and imbibed for 20 hours in

MilliqQ water at 4 ⁰C. Following imbibition, seeds were rinsed again in MilliqQ

water and carefully dissected to separate into three parts: seed coat, cotyledons, and

radicle. To determine the approximate distribution of mass, the weights of individual

components of ten randomly selected seeds were measured and calculated as a

percentage of the total seed weight. To determine trace element profile of each

component, tissues (from all 100 seeds) of the same type were then pooled and

prepared as described in Chapter 3.2.6.1. Following milling, three subsamples of up

to 250 mg per tissue type were analysed by ICP-OES following the protocol in

Chapter 3.2.6.1.3. The exception to this was the radicle tissue – due to the low

quantity of the material only a single sample was analysed. The relative distribution

of trace elements proportional to the mass of each tissue was calculated using the

following formula:


𝑇𝑜𝑡𝑎𝑙 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑡𝑖𝑠𝑠𝑢𝑒

= 𝐸𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔

100𝑔) × 𝑀𝑎𝑠𝑠 (𝑔)

𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑑𝑖𝑠𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛

=𝐸𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑡𝑖𝑠𝑠𝑢𝑒

𝑆𝑢𝑚 𝑜𝑓 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 𝑎𝑙𝑙 𝑡𝑖𝑠𝑠𝑢𝑒𝑠 × 100%

4.2.3 Statistical analysis

Data was analysed using one-way ANOVA and Tukey’s HSD test (MINITAB

17 Statistical Software, 2010). The elements of interest expressed as mean ± standard

deviation (SD). Pearson correlation analysis and Principal component analysis (PCA)

were performed on seed trace element concentrations using the XLSTAT statistical

package (Addinsoft, 2016).

4.3 RESULTS

4.3.1 Trace element composition of Australian-grown chickpea

The macro and micro-elemental profile of the desi cultivar PBA HatTrick was

assessed to determine the approximate range within which different elements

accumulated in the dry seeds. Seeds were sourced from five different locations

within Eastern Australia to determine the extent of variation within the cultivar. The

profiles of five other commercial cultivars were also assessed for comparison.

In general, the seeds used in this study were found to be rich in calcium (105–

257 mg/100g), magnesium (118–180 mg/100g), potassium (836–1117 mg/100g),

phosphorus 186–420 mg/100g), iron (3.36–5.20 mg/100g), zinc (2.60–4.35mg/100g)

and copper (0.44–1.10 mg/100g) (Table 4.8 and Table 4.9).

In terms of average iron content, higher values were measured in kabuli

samples compared to the desi ones from the same site though the difference was not

significant (Table 4.4). Iron concentrations also corresponded with location, with the

highest concentrations being found the samples from Roma, followed by Warra and

Billa Billa (Table 4.4 and Appendix A, Table 8.3). This pattern was not observed

amongst the desi cultivars sourced from those sites. However a location-specific

effect was apparent upon inclusion of the Kingaroy desi samples in the assessment,

which had lower average iron concentrations compared to the samples from sites

with vertosol-type soils (Table 4.4 and Appendix A, Table 8.3). Also unique to the


Kingaroy samples was the higher manganese content to iron ratio, which was likely

due to the soil type. Within the cultivars themselves, no significant differences were

found between samples from different sites. The exception to this was the Kingaroy-

grown Boundary and CICA0912, which had significantly lower iron compared to

those grown on vertosol-type soils. Little significant difference was also seen

between the iron contents of different cultivars from the same location. The

exception to this was Genesis090™, which was significantly higher compared to

PBA HatTrick at Warra and Roma. Genesis090™ was in general the richest in iron

even amongst the kabuli cultivars, while PBA HatTrick was similar to the other desi

types in terms of low iron content (Appendix A, Table 8.3).

As with iron, Genesis090™ generally had the highest zinc content while PBA

HatTrick ranked amongst the lowest of the cultivars. However comparison of all

samples found zinc accumulation to be primarily influenced by location rather than

genotype. The distinction between the kabuli and desi types was not as distinct, and

significant differences could be observed within the same cultivar across different

locations (Appendix A, Table 8.3). The trend resulting from this was similar to that

of iron, with highest zinc accumulation in the seed from Roma, followed by Warra,

Billa Billa and Kingaroy (Table 4.4and Appendix A, Table 8.3).

Similar to the trend observed in zinc content, grain phosphorus content

appeared to be primarily influenced by location, albeit in a different pattern. Despite

have having higher iron and zinc concentrations compared to the other locations,

samples from Roma were distinctly lower in phosphorus concentration (Table 4.4

and Appendix A, Table 8.2). Even lower still were the phosphorus contents of

Kingaroy-grown samples. No significant differences were found between the desi

and kabuli types except at Warra.

An examination of the relationship between iron and other trace elements in

PBA HatTrick revealed a moderate negative correlation only with manganese, while

positive correlations were observed with zinc, phosphorus, sulphur, potassium and

magnesium (Table 4.5). Zinc and phosphorus in particular, shared the strongest

correlation with seed iron content.


Table 4.4. Summary of Fe, Zn and P concentrations in kabuli and desi cultivars

grown at different locations. Data are expressed as mg/100g and presented as a

mean of all cultivars collected for that site. For each cultivar from each site, n>3.

Significance was calculated using Tukey’s HSD test, and values sharing the same

superscript letters indicate no significant difference (p-value>0.05).

Kabuli Desi PBA HatTrick

Location Range Mean Range Mean Range Mean

Fe

Billa Billa 4.12 - 4.67 4.42 bc 3.97 - 4.53 4.20 c 3.97 - 4.33 4.10

Roma 4.43 - 5.22 4.87 a 4.10 - 4.91 4.45 abc 4.10 - 4.30 4.23

Warra 4.13 - 5.54 4.63 ab 4.23 - 4.65 4.46 bc 4.23 - 4.36 4.31

Kingaroy n/a n/a 3.31 - 4.24 3.63 d 3.57 - 4.24 4.31

NSW n/a n/a n/a n/a 4.08 - 4.37 4.23

Zn

Billa Billa 2.99 - 3.37 3.23 c 2.78 - 3.19 3.02 c 3.01 - 3.10 3.06

Roma 3.86 - 4.42 4.19 a 3.85 - 4.33 4.02 a 3.85 - 3.93 3.06

Warra 3.18 - 4.12 3.61 b 3.82 - 3.96 3.88 ab 3.84 - 3.88 3.86

Kingaroy n/a n/a 2.53 - 3.74 2.96 c 2.53 - 2.68 2.60

NSW n/a n/a n/a n/a 3.39 - 3.61 3.49

P

Billa Billa 370 – 410 389 ab 340 - 410 382 abc 340 - 390 363

Roma 270 – 350 322 d 300 - 400 343 cd 300 - 310 303

Warra 310 – 440 367 bc 400 - 430 412 a 410 - 430 420

Kingaroy n/a n/a 183 - 248 204 e 194 - 202 199

NSW n/a n/a n/a n/a 400 - 420 408


Table 4.5. Pearson’s correlation coefficient between the different trace elements in PBA HatTrick. Analysis was performed on PBA

HatTrick samples collected from all locations. Values marked with an * indicate a significant correlation between two elements (p-value <0.05).

Ca Mg Na K P S Fe Mn Zn B Cu

Ca 1

Mg 0.618* 1

Na -0.352 -0.026 1

K 0.544* 0.844* 0.120 1

P 0.388 0.831* 0.453 0.735* 1

S 0.061 0.644* 0.523* 0.469 0.905* 1

Fe 0.439 0.498* 0.385 0.542* 0.673* 0.554* 1

Mn -0.542* -0.839* 0.171 -0.745* -0.691* -0.498* -0.474 1

Zn 0.638* 0.439 0.192 0.329 0.626* 0.471 0.692* -0.529* 1

B 0.654* 0.945* -0.295 0.816 0.669* 0.467 0.361 -0.886* 0.342 1

Cu 0.095 -0.255 0.602* -0.178 0.155 0.139 0.362 0.312 0.574* -0.443 1


4.3.2 Relationships between location and cultivar on seed trace elemental

composition

Identification of factors contributing to trace element composition was done

using principal component analysis (PCA) and agglomerative hierarchical clustering

(AHC). Both methods serve to determine relationships between samples within the

data set with no reference to prior knowledge. PCA is a bilinear modelling method

which reduces multivariate data to a few principal components in which maximum

data variation is found, allowing for the visualisation of data structure. Cluster

analysis serves to group a set of objects based on similarity.

Amongst the elements, iron, manganese, magnesium, and calcium were

identified as the ones in which the greatest variations were found (see Table 4.6).

The results of both PCA and AHC on seed trace elemental composition showed a

segregation of samples into three main groups based on cultivation location (Figure

4.2 and Figure 4.3). The first group consisted of the samples from Kingaroy which

were grown in ferrosol soil, while the second group comprised of the sole sample

from NSW whose cultivation conditions are unknown. These outgroups had a strong

correlation to iron and manganese contents and magnesium and calcium contents

respectively (Figure 4.2, Table 4.6).

The third and largest group consisted of samples grown in Billa Billa, Warra

and Roma. These locations had vertosol-type soils and similar environmental

conditions (see Table 4.3). This group was further segregated into the desi and kabuli

types along the Y-axis, to which magnesium and calcium had the strongest

correlation (Table 4.6); this corresponds to the higher average calcium and

magnesium concentrations in the desi types compared to the kabuli (see Appendix A,

Table 8.2). No such distinction was observed in the distribution along the X-axis

where the range of both types overlapped. Within both the desi and kabuli groups,

samples appeared to cluster loosely based on source location (Figure 4.3).


Figure 4.2. PCA of trace element composition of chickpea grown in QLD and

NSW based on overall mineral composition.

Table 4.6. Representation quality of a variable for each axis. The greater the

value, the greater the association with the axis.

Fe Mn P S Zn Cu B K Mg Ca Na

PC1 0.84 0.61 0.57 0.56 0.53 0.51 0.44 0.35 0.04 0.02 0.10

PC2 0.01 0.01 0.12 0.00 0.01 0.09 0.23 0.02 0.73 0.68 0.23


Figure 4.3. Clustering analysis of chickpea grown in QLD and NSW based on

mineral composition of samples. The dotted line indicates the level at which

truncation had been carried out to generate homogenous groups.

4.3.3 Cotyledons the primary storage for iron in PBA HatTrick seeds

To determine the storage site of the various trace elements, PBA HatTrick

seeds from NSW from the same stock as those used in later plant transformation

work were imbibed and split into the seed coat, cotyledon and radicle. The weight of

each individual part was measured. Each component was pooled and three

subsamples taken to determine trace element concentration via ICP-OES. The

exception to this was the radicle tissue, where the low volume allowed for only one

sample to be taken.

The weights of whole, imbibed seeds ranged from 361.3 – 511.2 mg. When

split into the three parts, a large variation was observed in the mass across the ten

replicates, ranging from 60.1 – 81.4 mg for the seed coat, 293 – 422 mg for the

cotyledons, and 5.3 – 9.2 mg for the radicle (see Table 4.7). When converted to a

percentage of the total seed mass, the cotyledons was found to constitute 82.28 ±

1.58%, followed by the seed coat and radicle at16.07 ± 1.54% and 1.65 ± 0.21%

respectively.


In terms of mineral concentrations, the results obtained for PBA HatTrick were

found to be consistent with range reported by Jambunathan and Singh (1981). The

seed coat was found to have the highest values for calcium, magnesium, and

manganese while being low in all other elements assayed (see Table 4.8 and Table

4.9). The opposite was seen in the elemental profile of the radicle, which was the

richest in sodium, potassium, phosphorus, sulphur, iron, zinc, boron and copper. The

cotyledons were not exceptionally high in any particular element, but were

moderately rich in potassium, phosphorus, sulphur, iron and zinc while being low in

calcium, magnesium, sodium, boron, copper and manganese. However due to their

large mass, they contained the bulk of the total content of all elements except

calcium and manganese (see Figure 4.4). Calcium was predominantly held in the

seed coat (75%), while and manganese was split between the cotyledons and seed

coat at 55% and 43% respectively. Despite having high concentrations of several

elements, the radicle contributed less than 5% of the total content of any of the

elements studied due to its small mass.

Table 4.7. Relative mass distribution within chickpea seeds of PBA HatTrick.

n=10. Data are presented as a mean ± SD.

Component Weight (mg) Average (mg) Weight (%) Average (%)

Seed coat 60.1 – 81.4 68.67 ± 6.24 13.60 – 18.95 16.07 ± 1.54

Cotyledon 293.0 – 422.0 354.15 ± 44.15 79.29 – 84.89 82.28 ± 1.58

Radicle 5.3 – 9.2 7.08 ± 1.08 1.33 – 1.99 1.65 ± 0.21

Total 361.3 – 511.2 429.89 ± 48.25

Figure 4.4. Relative distribution of trace elements within PBA HatTrick seeds.


Table 4.8. Concentrations of macro-elements in the different chickpea parts. Data are presented as a mean ± SD. All samples had n>3 except

the radicle, where n=1 due to small volume.

Ca

(mg/100g)

Mg

(mg/100g)

Na

(mg/100g)

K

(mg/100g)

P

(mg/100g)

S

(mg/100g)

Whole seed (dry) 242 (± 11) 180 (± 4.6) 6.34 (± 0.63) 1016 (± 23) 408 (± 8.4) 182 (± 2.8)

Seed coat 934 (± 50) 265 (± 14) 9.74 (± 0.62) 665 (± 48) 26.13 (± 1.2) 26.33 (± 1.1)

Cotyledon 58.4 (± 0.87) 145 (± 2.2) 9.25 (± 0.11) 913 (± 25) 520 (± 11) 246 (± 6.8)

Radicle 88.7

204

11.24

1081

926

374

Table 4.9. Concentrations of micro-elements in the different chickpea parts. Data are presented as a mean ± SD. All samples had n>3 except

the radicle, where n=1 due to small volume.

Fe

(mg/100g)

Zn

(mg/100g)

Mn

(mg/100g)

B

(mg/100g)

Cu

(mg/100g)

Whole seed (dry) 4.23 (± 0.1) 3.49 (± 0.09) 2.54 (± 0.07) 1.41 (± 0.02) 0.44 (± 0.03)

Seed coat 1.60 (± 0.7) 0.46 (± 0.03) 5.56 (± 0.3) 1.38 (± 0.09) 0.05 (± 0.01)

Cotyledon 4.96 (± 0.3) 4.44 (± 0.1) 1.40 (± 0.02) 1.11 (± 0.04) 0.54 (± 0.02)

Radicle 9.52

7.40

1.84

2.07

1.28


4.4 DISCUSSION

HatTrick is one of the highest yielding desi varieties across all chickpea

growing areas in NSW and southern Australia, and is widely grown due to its

resistance to ascochyta blight and phytopthera root rot (Queensland Department of

Agriculture and Fisheries, 2015). Despite its widespread use, there is no information

concerning its nutritional value. In order to determine the baseline from which the

iron content of the commercial chickpea cultivar PBA HatTrick may be improved,

the elemental profiles of samples from five different locations were assessed. Other

commercial cultivars were also included in this study determine the extent of the

influence exerted by genotype and environment and/or management practices.

Mineral compositions of the seeds used in this study fell within the ranges

previously reported in the literature (Wood and Grusak, 2007). Iron was found to be

positively correlated to phosphorus as well as another important micronutrient, zinc.

Comparison of the overall mineral composition showed segregation of samples into

distinct groups based firstly by location and cultivar types. Specifically, that

distribution was found to be particularly influenced by variations in iron, manganese,

calcium and magnesium contents. Comparison between the cultivars studied

generally found little significant difference between them, though the cultivar of

interest, PBA HatTrick, ranked among the lowest in terms of iron and zinc contents.

An examination of its seed confirmed the localisation of almost all iron and zinc to

the cotyledons, which was also the primary site of phosphorus storage.

Effect of location and soil type on grain micronutrient content

In this study, seeds obtained from various sites exhibited distinct mineral

profiles which corresponded to their source location. The locational effect was

particularly pronounced in the zinc contents of the grain. This is in agreement with

the findings of Ray et al. (2014) and Diapari et al. (2014), who have also reported

significant year to year variation even within the same site, further highlighting the

impact on environmental and management factors on zinc accumulation in the seed.

Where the management is concerned however, the effects of zinc fertilisation on

grain zinc content are unpredictable and may differ between seasons (Akay, 2011).

This is worth noting given that majority of the soil types within the Australian Soil


Classification (ASC) have been identified to be at risk of zinc deficiency and

administration of zinc fertilisers is a recommended practice in chickpea cultivation

(Norton, 2013; Pulse Australia, 2016).

Iron was identified to be partially subject to environmental influence, which

corresponds with the results obtained by Diapari et al. (2014) and Jambunathan and

Singh (1981). Accounting for genotype effect, grain iron content was not markedly

different amongst seeds grown on similar soil types. However while one might

expect enhanced iron accumulation in plant tissue where soil conditions are

favourable for uptake, this was not necessarily the case, as demonstrated by the

samples grown in Kingaroy. The high levels of iron oxides and low pH characteristic

of ferrosol soils, both of which favour plant uptake, did not translate to enhanced

seed iron content. Ironically the opposite was observed, with those samples having

the lowest iron contents amongst the locations. Considering the reversal of

manganese to iron ratios within those samples and the high manganese content in

Kingaroy soils (Appendix A, Table 8.1), it is likely that iron accumulation was

inhibited by the manganese concentrations. Similar cases of iron-manganese

antagonism have also been documented in other crops like rice, lettuce, tomato and

oat (Twyman, 1951; Tanaka and Navasero, 1966; Alvarez-Tinaut et al., 1980). As

both elements share components of the uptake and translocation processes (e.g.

Stephan et al., 1996; Vert et al., 2002), competition may occur where the metabolic

pathways overlap. How high iron-genotypes would respond to such conditions

cannot be concluded in this study due to lack of available material, but further

investigation should be conducted to guide grower decisions in this aspect.

Effect of genotype on trace element profile of seeds

When compared to the maximum levels reported in chickpea, the iron and zinc

levels of all cultivars in this study can at best be considered mid-range (Petterson and

Mackintosh, 1994). The highest average values were found in the kabuli cultivar

Genesis090™, while desi cultivars tended to be at the bottom of the spectrum for

iron. Whether this is biotype specific currently cannot be confirmed due to the

insufficient information on these traits. Studies in wheat and more recently in

Canadian chickpea have reported a negative correlation between seed micronutrient

content and yield, though the extent is subject to environmental effects (Garvin et al.,


2006; Ficco et al., 2009; Diapari et al., 2014). In light of this, it is possible that

cultivars developed with productivity at the main objective would suffer from a

trade-off in terms of micronutrient content. Further work is required to verify this in

the Australian context.

In terms of other elements, a clear distinction in calcium content was observed

between the kabuli and desi cultivars used in this study. The higher concentrations in

the desi cultivars were consistent with the findings of Jambunathan and Singh (1981)

and is likely to be attributed to the differing morphologies of the seed coat in which

most of the total calcium is stored. Unless the seeds are consumed whole however,

this calcium is likely to be lost as seed coats are discarded in the dhal-making

process. Most of the nutrition and trace elements are otherwise retained due to their

localisation to the cotyledons (Lal et al., 1963), a trait which can be exploited for

biofortification efforts.

Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 67

Chapter 5: Characterisation of Chickpea

Nicotianamine Synthase Genes

5.1 INTRODUCTION

Nicotianamine synthase (NAS) is the enzyme responsible for the biosynthesis

of the nicotianamine (NA). NA is a common component of Strategy I and II plants

that serves as a chelator for the systemic translocation of divalent cations, and is also

a precursor of mugineic acids (MAs) in Strategy II plants.

As a consequence of its role in iron transport, NAS holds significant influence

over iron content in plant tissue. For that reason, it has been extensively investigated

for use in iron biofortification. When expressed constitutively at elevated levels,

NAS has consistently increased the accumulation of iron and other transition metals

like zinc in plant tissues (Douchkov et al., 2005; Johnson et al., 2011; Lee et al.,

2011). The enhancement of seed iron content in transgenic rice overexpressing

endogenous NAS genes has been reported to be as high as 2.9 to 4 times (Lee et al.,

2009; Johnson et al., 2011) with a concomitant enhancement in bioavailability

(Zheng et al., 2010; Trijatmiko et al., 2016). In addition, NAS transformants have

also been demonstrated across various studies to have greater tolerance to heavy

metals (Douchkov et al., 2005; Kim et al., 2005; Pianelli et al., 2005) and iron

deficiency (Douchkov et al., 2005) compared to untransformed plants. No penalty on

general plant growth has been reported in these studies. Sensitivity to iron starvation

however, may be affected depending on the amount of accumulated NA – severe

overaccumulation (up to 100 times) may enhance sensitivity to iron deficiency

(Cassin et al., 2009). No such characteristic was noted in when NA accumulation

was increased by 4.8 fold (Wirth et al., 2009). It should be noted that the species

used by Cassin et al. (2009) and Wirth et al. (2009) differ in iron uptake strategies;

as such, the threshold for NAS accumulation remains inconclusive.

Despite extensive investigation into its function, there is a general lack of

information on the behaviour of the NAS family. Consequently, the potential of

different members for biofortification purposes remains largely unexplored save for a

select few. The number of NAS genes may range from one, such as in tomato (Ling

68 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes

et al., 1999), to twenty-one such as in wheat (Bonneau et al., 2016). To date, the

most well-characterised homologues are those from Arabidopsis (e.g. Schuler et al.,

2012b; Koen et al., 2013), tomato (Ling et al., 1999), rice (e.g. Inoue et al., 2003;

Nozoye et al., 2014b), and barley (e.g. Herbik et al., 1999; Higuchi et al., 1999b).

While not as extensively studied, NAS homologues from other species have also

been characterised to some degree (see Table 5.1).

Table 5.1. List of characterisation studies done on NAS from selected species.

Species Gene ID Source

Arabidopsis halleri AhNAS

Weber et al. (2004)

Deinlein et al. (2012)

Cornu et al. (2014)

Lotus japonicas LjNAS Hakoyama et al. (2009)

Thlaspi caerulescens TcNAS Mari et al. (2006)

Wheat (Triticum aestivum) TaNAS Bonneau et al. (2016)

For the purposes of biofortification, the native expression patterns of a gene

requires due consideration. Given that the physiological role of NAS in Strategy I

and II is fundamentally different, the behaviour of the different homologues is

expected to vary. In species utilising the Strategy II uptake mechanism, NAS

expression and activity are generally upregulated in the roots during iron deficiency

(e.g. Higuchi et al., 1996; Inoue et al., 2003; Mizuno et al., 2003). The reverse is true

for Strategy I plants however, where downregulation is observed instead (Stephan

and Scholz, 1990; Higuchi et al., 1995).

Tissue-specific expression also occurs under various iron regimes. In the study

conducted on rice by Inoue et al. (2003), constitutive expression of OsNAS1 and

OsNAS2 was confined to the root cells involved in long-distance transport (e.g. stele,

pericycle and xylem parenchyma cells). Under iron deficient conditions however,

expression of both was induced in all tissues including the leaf sheaths and vascular

bundle of leaves. In contrast, OsNAS3 expression in the same tissues was lowered in

response to iron deficiency. On top of external iron concentrations, NAS expression

is also subject to specific developmental needs. In the legume Lotus japonicas,

LjNAS2 expression was nodule-specific while LjNAS1 served a more systemic

housekeeping role (Hakoyama et al., 2009).


The degree to which these specific expression patterns can be extrapolated to

homologues in other species is currently unclear, given the varying number of NAS

genes as well as differing iron requirements. Leguminous plants in particular, may

have specific iron requirements due to nodulation, which may in turn influence

overall NAS behaviour.

The aim of this chapter therefore, was to characterise chickpea NAS2 for use in

iron biofortification. As part of this study, the native expression of CaNAS2 was

investigated. Expression plasmids were also generated for overexpression studies in

tobacco and chickpea, the former of which is covered in this chapter.


5.2.1 Designation of chickpea NAS2

The sequences of annotated chickpea NAS genes were obtained from NCBI

and the coding sequences translated to a hypothetical protein. A BLAST check of the

putative protein sequence was performed on the resulting protein to ensure correct

annotation and open reading frames (ORFs). Upon confirmation, a progressive

pairwise alignment was done to determine the degree of similarity between the rice

and chickpea NAS sequences. The chickpea NAS protein with the highest similarity

to OsNAS2 was designated as CaNAS2.

5.2.2 Assessment of NAS amino acid sequence and protein properties

Following the designation of CaNAS2, a range of bioinformatics tools were

used to predict the biochemical properties and localisation of the enzyme. The

theoretical isoelectric point (pI) and molecular weight were calculated using the

Compute pI/Mw tool on ExPASY (Bjellqvist et al., 1993; Bjellqvist et al., 1994;

Gasteiger et al., 2005b). The hydrophobicity profile of the protein was assessed using

ProtScale (Gasteiger et al., 2005a) and potential transmembrane sections identified

using TMpred (http://www.ch.embnet.org/software /TMPRED_form.html). A check

for motif sequences was conducted using ScanProsite (De Castro et al., 2006) and

MOTIF Search (GenomeNet). Phobius (Käll et al., 2004) and iPSORT (Bannai et al.,

2001, 2002) was used to identify potential signalling peptides. Hypothetical 3D

structures were generated using Phyre2 (Kelley et al., 2015).

http://www.ch.embnet.org/software%20/TMPRED_form.html


5.2.3 Phylogenetic analysis of NAS proteins

A progressive pairwise alignment was performed on full length protein

sequences of chickpea and other species to determine the degree of homology at the

amino acid level. Of the latter, only NAS genes and/or proteins whose activity or

expression patterns have been experimentally verified were used. The alignment was

done using the default settings of Geneious alignment (global alignment with free

end gaps, Blosum62, gap open penalty 12, gap extension penalty 3). An unrooted

neighbour-joining tree was constructed with Juke-Cantor as the genetic distance

model. No outgroups were selected.

All sequences were obtained from NCBI and the accession numbers are as

listed in Appendix B, Table 8.4. Amino acid sequences used were checked against a

translated cDNA sequence to confirm the correct open reading frames (ORFs). All

alignments and phylogenetic analysis were done using Geneious 7.1.9.

5.2.4 Assessment of CaNAS2 expression

5.2.4.1 Preliminary study

A preliminary study was conducted to determine approximate spatial and level

of expression of CaNAS2 and other CaNAS genes. Samples were taken from a

healthy, non-transgenic chickpea plant. The plant was approximately one month old

at the time of sampling, and had been grown on potting mix under iron-sufficient

conditions. The following tissues were sampled: mature green leaf, senescing leaf,

stem, cotyledon, and root. All samples were snap-frozen in liquid nitrogen

immediately after collection and stored in -80⁰C. RNA extraction and cDNA

synthesis was done following the method described in Chapter 3.2.5.1 and 3.2.5.2.

Gene expression was assessed qualitatively using end-point PCR with cDNA as the

template. Primers used for CaNAS2 and other CaNAS family members are as

described in Table 5.4 and 5.2 respectively.

5.2.4.2 Iron deficiency experiment

Chickpea seeds cv Hattrick were sterilised and germinated on half MS media

as described in Chapter 3.2.3.2.1. One week post-germination, the seed coats were

removed and the seedlings were acclimatised for four days in tap water in a loosely

covered beaker. Healthy seedlings of approximately the same size and developmental

stage were selected and transferred to the mini hydroponics set-up illustrated below


(Figure 5.1). A total of twenty seedlings were chosen, with ten per set-up. Both set-

ups were placed in a growth cabinet set at 23⁰C, with a 16 hour photoperiod.

All plants were grown on tap water for four weeks from the time of

acclimatisation. Full-strength Hoagland solution with or without Fe-EDTA (Chapter

3.1.5.1) was then provided in the subsequent weeks until sampling. All solutions

used were topped up every two to three days as required. During the treatment,

MilliQ water was used every third top-up instead of Hoagland solution to dilute

accumulated salts. Sampling was done two weeks following the onset of chlorosis in

the iron-deprived plants. Three plants of similar conditions and growth stage were

selected from each treatment for analysis of gene expression. The selected plants

were rinsed in tap water, photographed, and the following tissue types were

collected: mature leaf, stem, cotyledon, and root. Senescent leaf and chlorotic leaf

were also collected from the iron-sufficient and iron-deficient plants respectively. All

samples were snap-frozen in liquid nitrogen immediately after collection and stored

in -80⁰C. RNA extraction and gene expression analysis was done following the

method described in Chapter 3.2.5. Primers used in qPCR are as listed in Table 5.2.

Figure 5.1. Schematic diagram of the mini-hydroponics system.

5.2.4.3 Measurement of CaNAS2 expression level

Measurement of CaNAS2 expression was done as described in Chapter 3.2.5.

RNA was extracted from the sampled tissues and the quality confirmed via gel

electrophoresis. DNase treatment was then performed on 1 µg of RNA, from which

cDNA was generated. The cDNA was checked via PCR using a housekeeping gene


to confirm successful synthesis, following which it was used for qPCR. Primers used

in qPCR are as listed in Table 5.2.

Table 5.2. List of primers used in qPCR.

Gene Sequences (5’-3’)

Expected

amplicon

size (bp)

Actin 1 Fw GCCTGATGGA CAGGTGATCA C

62 Rv GGAACAGGAC CTCTGGACAT C

EF1α Fw TCCACCACTT GGTCGTTTTG

64 Rv CTTAATGACA CCGACAGCAA CAG

GAPDH Fw CCAAGGTCAA GATCGGAATC A

65 Rv CAAAGCCACT CTAGCAACCA AA

CaNAS2 Fw AGTAGTGCCT TTCTAAATGG CC

116 Rv CATGTCACCA ATCCCCAACA T

CaNAS

(XP_004487761.1)

Fw GTCACTCAAG TCTGATTCGA CC 172

Rv TGAGGTGGTG CATGTTGTTA C

CaNAS

(XP_004488704.1)

Fw TAGCAAGATC GTGGCATCGG 135

Rv CCTCTACTCA TACCAACAAG TGC

CaNAS

(XP_004494544.1)

Fw AGTGCTTTGT ATCTCATGGA GC 133

Rv TGCATGCCCT TATATACGGC T

5.2.5 Isolation and cloning of chickpea NAS2 and other genes of interest

The OsNAS2 gene was kindly provided by Dr Alex Johnson from the

University of Melbourne. The GmFER gene was synthesized. CaNAS2 was isolated

from chickpea genomic DNA using primers designed from the predicted sequence

from Genbank database, accession number XM_004495601.

The genes of interest were amplified and restriction sites added to the ends via

site-directed mutagenesis using high fidelity PCR (Phusion®, NEB) with the primers

listed in Table 5.3. For OsNAS2 and CaNAS2, AscI and PacI restriction sites were

added to the 5’ and 3’ ends respectively. For GmFER, SalI and BstEII restriction

sites was added to the 5’ and 3’ end respectively. Following this, the mix was

incubated at 72⁰C for one hour with 5µL of GoTaq (Promega) to restore the A

overhang in the ends blunted by high fidelity PCR.

The resulting PCR products were separated by gel electrophoresis, purified

using Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Biorad) and ligated

overnight into pGEM®-T Easy vectors (Promega). A 2µL aliquot of ligation reaction

was transformed into E.coli using an in-house heat shock method, and the culture


spread onto Luria-Bertani (LB) plates containing 14% IPTG, 2% X-gal and 100mg/L

of ampicillin. White colonies were selected and their DNA sequenced to verify gene

integrity.

Table 5.3. List of cloning primers. Restriction sites have been highlighted in gray.

Gene Restriction

site Sequences (5’-3’)

Expected

amplicon

size (bp)

OsNAS2

Fw AscIF GGCGCGCCAT GGAGGCTCAG

AACCAAGAG 997

Rv Pac1R TTAATTAATC AGACGGATAG

CCTCTTGG

CaNAS2

Fw AscIF GGCGCGCCAT GGTTTGCAAG

GAAGATATAT TAATC 939

Rv Pac1R TTAATTAATC ATTCTTCAAT

GACCAATTCC TC

GmFER

Fw SalIF GTCGACCCTA GGATGGCCCT

TTCTTGCTCC 793

Rv BstEIIR GGTGACCTTA TACATGATCT

TCATCGTGAA GAA

5.2.6 Generation of expression plasmids

The pOpt-EBX expression vector was obtained from the Gates research group

from the Centre for Tropical Crops and Biocommodities (CTCB). The pOpt-EBX-

35s-UidA expression vector and pGEM-T cloning vector with a cassette containing a

Nos promoter and CaMV 3’ UTR was obtained from Hao Long from the Abiotic

Stress research group in the CTCB.

Upon verification of sequence, the genes of interest were excised from

sequence verified pGEM-T® Easy vectors. OsNAS2 and CaNAS2 were digested

using AscI and PacI. GmFER was digested using SalI and BstEII. The digested

products were separated by gel electrophoresis and the genes ligated into their

respective backbones/sub-backbones in a 5’ to 3’ direction. GmFER was ligated into

the pOpt-EBX backbone, in between the CaMV 35s promoter and Nos terminator.

To add the promoter and terminator sequences to OsNAS and CaNAS, those genes

were ligated with a pGEM-T cloning vector with a cassette containing a Nos

promoter and CaMV 3’ UTR. The constructs were sequenced verified to determine

gene integrity and orientation, following which the NosP-CaNAS/OsNAS-CaMV 3’

UTR cassette was digested and ligated with the pOpt-EBX-GmFER backbone to


form the complete vector. The final constructs were sequence verified and

transformed into Agrobacterium tumefaciens strains Agl1 and LBA4404.

To generate the NAS-only constructs, the verified pOpt-EBX-OsNas2/CaNas2-

GmFER constructs were digested with BamHI and StuI to remove GmFER and its

CaMV 35s promoter and Nos terminator. The digested constructs (without ferritin

and its promoter and terminator) were then separated by gel electrophoresis. As

BamHI produces a sticky end incompatible to the blunt end from StuI, blunting of

the sticky end was done using a high-fidelity polymerase (Phusion®, NEB).

Following this, self-ligation was performed to join the now compatible ends of the

construct to each other. The newly generated NAS2-only constructs were cloned into

E.coli for amplification, then sequenced to confirm the integrity of the remaining

genes and complete removal of GmFER and its promoter and terminator. The

constructs (Figure 5.2) were subsequently transformed into Agrobacterium

tumefaciens strains Agl1 and LBA4404.

To confirm the presence of the gene/s of interest in Agrobacterium cultures

used for transformation, genomic DNA was isolated from the respective AGL1 and

LBA4404 cultures for PCR verification. PCR was performed using GoTaq

(Promega) and gene-specific primers (Table 5.4). The resulting products were

separated via electrophoresis in a 1% agarose gel and made visible by staining with

SYBR® Safe (Life Technologies).


Figure 5.2. Expression plasmids generated for plant transformation. The

combinations of genes of interest and the backbone are as illustrated in (A) and (B)

respectively. NosT represents the nopaline synthase terminator, NPTII represents the

neomycin phosphotransferase II gene, S1 Pro represents the S1 promoter, GUS

represents the β-glucuronidase gene, CaNAS2 represents the Cicer arietinum

nicotianamine synthase 2 gene, OsNAS2 represents the Oryza sativa nicotianamine

synthase 2 gene, GmFER represents the Glycine max ferritin H1 gene, CaMV 35sP

represents the cauliflower mosaic virus 35s promoter, CaMV 3’UTR represents the

cauliflower mosaic virus 3’UTR terminator, and NosP represents the nopaline

synthase promoter.

A)

B)


Table 5.4. List of primers used for screening.


Expected

amplicon

size (bp)

uidA (GUS) Fw TGAACATGGC ATCGTGGTGA

507 Rv GCTAACGTAT CCACGCCGTA

OsNAS2 Fw CTGAGCAAGC TGGAGTACGA

660 Rv TCAGACGGAT AGCCTCTTGG

CaNAS2 Fw GCATGTCACC AATCCCCAAC

568 Rv CGCAGCATCA AAAGTGCTCC

GmFER Fw ATGGCCCTTT CTTGCTCCAA

604 Rv GTTCTGCCAC ACTGTGAACG

Neomycin

phosphotransferase II

Fw ATTCGGCTAT GACTGGGCAC 675

Rv TAAAGCACGA GGAAGCGGTC

5.2.7 Generation and molecular characterisation of transgenic tobacco

Transgenic tobacco was generated following the procedure outlined in Chapter

3.2.3.1. The resulting lines were screened for the genes of interest and RT-PCR was

performed on PCR-positive plants to check for gene expression. All lines were

acclimatised and grown as per Chapter 3.2.3.1.3, and seeds collected upon maturity.

5.2.8 Assessment of iron accumulation in transgenic tobacco leaf

T1 tobacco seeds were sterilised and germinated on half-strength MS with

100mg/L of kanamycin for two weeks. Germinated seedlings were transferred to

half-strength MS with no selection for two weeks.

Selected plants were subcultured to fresh MS media containing 150µM Fe-

EDTA and after eight days, the second, third and fourth youngest fully expanded

leaves were harvested for trace element analysis. Samples were rinsed twice in DI

water and prepared for LA-ICP-MS following the protocol outlines in Chapter 3.2.6.

5.3 RESULTS

5.3.1 Designation and sequence analysis of CaNAS2

Depending on the species, the number of NAS genes may range from one to

twenty-one. In chickpea, four hypothetical NAS proteins are annotated on NCBI. A

progressive pairwise alignment was done to compare the sequences to OsNAS2,

which had been successfully applied in other biofortification efforts. Hypothetically,

similarity in amino acid sequence would translate into a comparable activity and


effectiveness for biofortification. Of the four CaNAS sequences, XP_004495658.1

had the highest sequence similarity to OsNAS2, as well as the other OsNAS proteins

(see Table 5.5). Visual inspection of the predicted 3D structure however, showed

XP_004487761.1 to be more similar to OsNAS2 – this observation was arrived at by

several independent parties (see Figure 5.3). Given potential inaccuracies in

predictive 3D modelling however, sequence similarity was set as the criteria for

designation of CaNAS2. XP_004495658.1 was therefore selected and designated as

CaNAS2.

Table 5.5. Similarity between the CaNAS and OsNAS amino acid sequences.

Comparison was done using a progressive pairwise alignment. Similarity values are

expressed as a percentage.

OsNAS1 OsNAS2 OsNAS3

CaNAS2 XP_004495658.1

44.089 44.66 46.326

XP_004487761.1 41.009 40.317 41.195

XP_004488704.1 42.908 42.199 41.176

XP_004494544.1 42.547 41.304 45.652

Figure 5.3. Predicted 3D structures of OsNAS and CaNAS2 proteins. Structures

are coloured based on progression from the N (red) to C terminus (dark blue).


Upon designation of CaNAS2, predictive modelling was done to identify the

potential biochemical properties of the enzyme, from which the subcellular

localisation and behaviour may be alluded. CaNAS2 was predicted to have an

approximate molecular weight of 34.36kDA and a pI of 5.52. Assessment of the

hydrophobicity profile and transmembrane topology showed the enzyme to be

primarily hydrophilic (Figure 5.4.A) with a potential transmembrane domain at

position 126 to 151 (Figure 5.4.B). The lack of significant hydrophobic regions

suggests that membrane association, if any, occurs only at a peripheral level. A

subsequent check for motifs revealed a single NAS motif from position 5 to 275

which occupied approximately 88% of the entire protein (see Figure 5.5). A

methyltransferase domain was detected within the NAS motif from positions 151 to

250, just outside the potential transmembrane domain.

Figure 5.4. Predicted biochemical properties of CaNAS2. A) Hydrophobicity plot,

and B) predicted transmembrane topology. The potential transmembrane site has

been highlighted in blue.


1 MVCKEDILIE QVCDLYNQIS NLDTLKPCKI VNTLFTKLVL TCMSPIPNID VTKLATNVQE

61 IRSKLIILCG EAEGHLESHY STILASHNNP LNHLNIFPYY TNYLKLSLLE FNILNQHITN

121 NVPKNVAFIG SGPLPLTSIV LATNYLPSTI FHNYDIDPLA NSKASCLISS NPDLSNRMLF

181 HTNDILNVTN DLKEFEVVYL AALVGMNNEE KNKIIDHLGK YMAHGALLML RSAHGARAFL

241 YPVVDTSDLR GFEVLSIFHP TDEVINSVLI ARKYNPIVLL PNKCCGDEIQ VFKPLNNMIE

301 ELVIEE

Figure 5.5. Amino acid sequence of CaNAS2. Red text marks out the predicted

NAS motif, underlined text marks out the methyltransferase domain, and highlighted

section is the YXXΦ and LL motifs.

Scans using Phobius and iPSORT predicted a non-cytoplasmic localisation and

detected no signal peptides, mitochondrial targeting or chloroplast transit signals.

However, a cross-check with existing studies on other NAS proteins (Nozoye et al.,

2014a; Bonneau et al., 2016) pointed to the presence of two motifs at the N-terminus

region. The YXXΦ (Y refers to tyrosine, X to any amino acid residue, and Φ to

bulky hydrophobic residues) and di-leucine (LL; leucines may be substituted with

isoleucines) motifs were located at the position 103 and 118 respectively. Both are

conserved in the NAS superfamily, with the former implicated in vesicular

localisation and movement, and the latter with maintenance of enzyme structure

(Nozoye et al., 2014a).

However, whether this culminates in actual vesicular localisation is uncertain.

Despite the conservation of YXXΦ and LL motifs, studies with GFP-tagged NASes

from Arabidopsis, rice and maize have reported localisation to both vesicles and

cytoplasm; this has been suggested to reflect physiological roles (Mizuno et al.,

2003; Zhou et al., 2013a; Nozoye et al., 2014b). To verify the results obtained with

CaNAS2, the OsNAS, AtNAS and ZmNAS sequences (accession numbers as listed

in Appendix B, Table 8.4) were also assessed. The results obtained were similar to

that of CaNAS2 despite the reported cytoplasmic localisation of all the inputs except

OsNAS2, ZmNAS1 and ZmNAS2.

5.3.2 Phylogenetic analysis of CaNAS2

To determine the relationship between CaNAS2 and other NAS proteins, a

pairwise multiple alignment was done on the amino acid sequences and a

phylogenetic tree generated. Consistent with the findings of other authors (Filipe de


Carvalho et al., 2012; Zhou et al., 2013b), the result show a clear segregation

between the graminaceous and non-graminaceous sequences (Figure 5.6). Amongst

the non-graminaceous plants, two groups could be observed. The first is a legume-

specific outgroup containing a NAS from chickpea and Medicago truncatula (Group

1). The second group comprises of all other non-graminaceous NAS, within which is

another legume-specific cluster containing the remaining legume NAS sequences.

This cluster contained two distinct groups (Figure 5.6, Group 2 and 3). In

Group 3 an additional duplication event appears to have taken place in the Medicago

and chickpea lineages giving rise to CaNAS2 and CaNAS (XP_04494544.1), and

MtNAS XP_003591220.1 and XP_13450461.1 respectively. Based on existing

studies, it is unclear if each group fulfils specialised functions. Differing patterns of

expression have been exhibited in Group 2 members LjNAS2 and MtNAS

(XP_003594753.1), with the former being nodule-specific (Hakoyama et al., 2009),

and the latter having low expression in all tissues (Medicago truncatula Gene

Expression Atlas).

On the other hand, the tissues in which Group 3 are expressed or upregulated

appear to be more uniform. LjNAS1 was reported to be expressed in leaves, stem and

cotyledons (Hakoyama et al., 2009). Similar patterns were also observed with

MtNAS XP_003591220.1 and XP_013450461.1, which showed general upregulation

in the various aerial tissues as well as the roots (Medicago truncatula Gene

Expression Atlas). Amongst these three genes, the stem appears to be a common site

of expression. It is likely that the CaNAS homologues would behave in a comparable

manner as the genomes of both M. truncatula and chickpea share approximately 95%

synteny, and MtNAS (XP_003591220.1) was the most similar to CaNAS2 with

74.8% sequence similarity.


Figure 5.6. Phylogenetic relationship between CaNAS2 and NAS proteins from

other plants. The scale bar and branch labels represent the number of substitutions

per site. The legume-specific clades are as indicated by the green boxes and the red

box shows the locations of CaNAS2 in the unrooted phylogenetic tree. Species

included in this tree are Arabidopsis thaliana (AtNAS), Arabidopsis halleri

(AhNAS), barley (HvNAS), Lotus japonicus (LjNAS), Medicago truncatula

(MtNAS), rice (OsNAS), Thlaspi caerulescens (TcNAS), tomato (SlNAS) and maize

(ZmNAS).


5.3.3 CaNAS2 expression is downregulated in response to iron deficiency

NAS expression has been reported to vary between species, particularly

between the graminaceous and non-graminaceous ones (Higuchi et al., 1995). This is

further subject to the number of homologues, which may be differentially expressed

in various tissues in response to external and internal iron supply. A preliminary

study was done on a one month-old non-transgenic chickpea (cv HatTrick) to

determine the approximate pattern of expression for CaNAS2. Five different tissue

types were surveyed: mature green leaf, senescing leaf, stem, cotyledons, and root.

Qualitative assessment of gene expression showed CaNAS2 to be expressed in

almost all tissues except senescing leaf (see Figure 5.7). The strongest expression

occurred in the stem and cotyledons, while a fainter signal was detected in the green

leaf and root samples. The same assessment was carried out for the other CaNAS

family members showed differing patterns of expression (see Figure 5.8).

XP_004487761.1 was not expressed in any of the tissues tested, while

XP_004488704.1 was expressed in the leaf, cotyledons, and roots. XP_00449454.1

was expressed in all tissues.

Figure 5.7. Qualitative assessment of CaNAS2 expression in different chickpea

tissues via PCR.

M = 2-log ladder (NEB)

“-” = No template control

“+” = gDNA positive control

Leaf (GR) = Mature green leaf

Leaf (SC) = Senescing leaf

Stem = Stem

Coty = Cotyledon

Root = Root


Figure 5.8. Qualitative assessment of the expression of other CaNAS family

members in different chickpea tissues via PCR.






Stem = Stem

Coty = Cotyledon

Root = Root


To confirm these results and to determine the effect of iron status on CaNAS2

expression, plants were grown in a hydroponics set-up under iron-sufficient and iron-

deficient hydroponic conditions. Chlorotic symptoms were allowed to develop for

two weeks after onset before tissues were sampled for expression analysis. Initial

germination and establishment was done in a nutrient-free solution did not produce

visible symptoms of iron deficiency. Subsequent treatment with nutrient solution

allowed for further vegetative growth, however it still took approximately four weeks

before visible symptoms of iron deficiency, chlorosis, were observed. Including an

additional two weeks to allow for further development of symptoms, the treated

plants were deprived of iron for a total of 9½ weeks. By the time of harvest, young

leaves of the treated plants were severely chlorotic (see Figure 5.9). The whole plant

in general was also more yellowish compared to their iron-sufficient counterparts,

though no apparent senescing leaves were present. Consequently, the chlorotic leaves

were harvested in place of senescing leaves. In addition to the leaves, the roots of the

treated plants were also paler compared to the iron-sufficient controls. No nodules

were observed in either treated or untreated plants.

Following harvest, RNA was extracted from the sampled tissues and treated

with DNAse. Upon confirmation of gDNA removal, cDNA was synthesized. Good

quality RNA was obtained from all tissues except the roots, which were excluded in

this analysis. The removal of gDNA and subsequent synthesis of cDNA was verified

by PCR (Figure 5.10).

Figure 5.9. Morphology of iron-sufficient (+Fe) and iron-deficient (-Fe)

chickpea grown under hydroponics conditions.


Figure 5.10. Representative photos of verification of RNA and cDNA for qPCR

analysis. A) Quality of extracted chickpea RNA, B) Removal of contaminating

gDNA, and C) confirmation of cDNA synthesis using GAPDH.




1, 2, 3 = Biological replicates



Stem = Stem

Coty = Cotyledon

Root = Root


Evaluation of CaNAS2 expression in the sampled tissues found it to be

generally upregulated under iron-sufficient conditions, with expression

predominantly occurring in the stem and roots (see Figure 5.11). Under iron-deficient

conditions however, an overall downregulation of CaNAS2 was observed. No

expression was observed in the cotyledons of treated plants, possibly due to the

exhaustion of iron stores and resulting lack of iron for export to the rest of the plant.

Lower levels of CaNAS2 expression was observed in the senescing and chlorotic

leaves compared to the mature green leaves (see Figure 5.11).

Figure 5.11. Expression of CaNAS2 in different tissues under iron-sufficient

(+Fe) and iron-deficient conditions (-Fe). n=3. Error bars represent standard error.

Expression was measured via qPCR and calculated as relative to that of the

housekeeping genes GAPDH and EF1α. No viable RNA could be extracted from the

iron-deficient root samples and they were therefore excluded from this analysis.



Leaf (CHL) = Chlorotic leaf


5.3.4 Generation of expression plasmids

Following the characterisation of CaNAS2 in the native host, transgenic

vectors were generated for overexpression studies in the model plant tobacco

(Nicotiana tabacum cv Samson). These vectors were also used in Chapter 6 for

overexpression studies in chickpea. OsNAS2 was used as a positive control for

comparison. All Agrobacterium cultures containing transgenic constructs were

screened prior to use in plant transformation, and the presence of their respective

genes of interest confirmed (see Figure 5.12).

Figure 5.12. PCR detection of the genes of interest in the Agrobacterium strains

AGL1 and LBA4404 used for plant transformation work.


“H2O” = No template control

“WT” = Untransformed Agrobacterium control

“+” = Positive plasmid control

GUS = Agrobacterium transformed with pEBX-UidA

FO = Agrobacterium transformed with pEBX-GmFER-OsNAS2

O = Agrobacterium transformed with pEBX-OsNAS2

FC = Agrobacterium transformed with pEBX-GmFER- CaNAS2

C = Agrobacterium transformed with pEBX- CaNAS2


5.3.5 Generation and molecular characterisation of transgenic tobacco

Following determination of CaNAS2 expression profile in chickpea, transgenic

tobacco overexpressing CaNAS2 was generated to determine its influence on iron

concentrations. To also determine its effect in a multigenic approach like the one

used by Trijatmiko et al. (2016), transgenic tobacco plants expressing both CaNAS2

and soybean ferritin (GmFER) were generated. OsNAS2, a homologue from rice that

has successfully been used in other biofortification studies, was used as a positive

control in both the monogenic and multigenic constructs.

Putative transgenic lines were screened via PCR using gene-specific primers

(see Figure 5.13). Amongst the PCR positive lines, one GUS, 12 GmFER-CaNAS2

and 11 GmFER-OsNAS2, CaNAS2, and OsNAS2 lines were randomly selected for

further work. Expression of the genes of interest was confirmed via end-point RT-

PCR (see Figure 5.14). All lines were subsequently grown to seed to obtain the T1

generation. Not all lines flowered successfully; amongst those that did, at least three

lines per construct did not produce viable seed; this occurrence was consistent across

all the clones from the same line. This failure to produce flowers or seed was not

limited to any particular construct – at least two lines per construct were affected.

The final summary of transgenic events progressing to the T1 generation is as listed

in Table 5.6.


Figure 5.13. Representative photos of T0 tobacco PCR screening with gene-

specific primers. The following genes were screened: A) GmFER, B) CaNAS2, and

C) OsNAS2. Each lane bearing FO, O, FC, or C represents an individual transgenic

event.




NT = Untransformed tobacco control

FO = pEBX-GmFER-OsNAS2

O = pEBX-OsNAS2

FC = pEBX-GmFER-CaNAS2

C = pEBX- CaNAS2


Figure 5.14. Detection of transgene expression in GM tobacco lines via PCR.

The constructs are as follows: A) pEBX-Ferr-CaNAS2, B) pEBx-Ferr-OsNAS2, C)

pEBX-CaNAS2, and D) pEBX-OsNAS2. The target genes are as listed in each

image. Each lane bearing a number represents an individual transgenic event.




NT = Untransformed tobacco control

GUS = pEBX-UidA transgenic tobacco lines

Table 5.6. Summary of transgenic tobacco lines generated and progressing to

the T1 generation.

Construct RT-PCR +

lines

Lines progressing to

T1 generation

pEBX-GUS 1 1

pEBX-GmFER-CaNAS2 12 10

pEBX- GmFER-OsNAS2 11 6

pEBX-CaNAS2 11 3

pEBX-OsNAS2 8 7


5.3.6 Transgenic tobacco exhibit to significant increase in leaf iron or zinc

contents

To assess the impact of the genes of interest on iron accumulation, the leaves

of one month old T1 plants were assessed for iron content using LA-ICP-MS. Prior

to commencement of the experiment, all plants used were screened for the presence

of the transgenes (Appendix B, Figure 8.1). Only plants testing positive for the genes

of interest were used in this study. Of the lines tested, FC6 was positive for GmFER,

but not for CaNAS2. It was included in this study as a Ferr-only control.

To determine viability of further work, an initial assessment was done on the

CaNas2 and Ferr-CaNAS2 line. Amongst those samples, leaf iron content ranged

from 132 to 177ppm (see Figure 5.15). While considerable variability was observed

between different lines within the same construct, there was no significant difference

between them. Similarly, no significant difference was found when compared to the

non-transgenic and vector controls. The same result was obtained after including

three randomly chosen OsNAS2 and Ferr-OsNAS2 lines for further comparison

(Figure 5.15). Examination of individual transgenic plants showed that, save for a

few individuals within each line, iron contents were comparable to that of the non-

transgenic controls (Appendix B, Figure 8.2).

A similar observation was made with zinc content where, despite more

variability between lines, the average values were not markedly different from the

non-transgenic control (see Figure 5.15 and Figure 8.2). However, the difference was

significant compared to the GUS vector control line, which contained the lowest iron

and zinc content.


Figure 5.15. Average A) iron and B) zinc content in non-transgenic (n=10) and

transgenic tobacco leaves (n=4 to 7). Gray bars represent the non-transgenic (NT)

and GUS control lines, dark orange bars represent the GmFER-CaNAS2 (FC)

transgenic lines, light orange bars represent the CaNAS2 (C) transgenic lines, dark

blue bars represent the GmFER-OsNAS2 (FO) transgenic lines, and light blue bars

represent the OsNAS2 (O) transgenic lines. Error bars indicate standard error.

Statistical significance was calculated using Dunnett’s test, and p-values<0.05 were

considered statistically significant.

NT = Non-transgenic tobacco control

GUS = pEBX-UidA transgenic tobacco lines

FO = pEBX-GmFER-OsNAS2 transgenic tobacco lines

O = pEBX-OsNAS2 transgenic tobacco lines

FC = pEBX-GmFER-CaNAS2 transgenic tobacco lines

C = pEBX-CaNAS2 transgenic tobacco lines

* = Statistically significant compared to GUS line

= Statistically significant compared to non-transgenic control


5.4 DISCUSSION

Compared to their counterparts in the Gramineae family, relatively little is

known about the NAS orthologues in non-graminaceous species. Chickpea has four

NAS genes, and this chapter focuses on the characterisation of the one of the

homologues. Dubbed CaNAS2, it was selected due to its sequence similarity to

OsNAS2, which has been successfully applied in biofortification work by other

authors (e.g. Johnson et al., 2011; Trijatmiko et al., 2016). Using a combination of

modelling and phylogenetic analyses, we predicted the subcellular localisation and

expression profile of CaNAS2. Transgenic tobacco overexpressing CaNAS2 were

subsequently generated to determine its effect on iron accumulation.

Predicted subcellular location of CaNAS2

To determine potential subcellular behaviour, predictive modelling was

performed on the CaNAS2 amino acid sequence. CaNAS2 was predicted to be a

hydrophilic protein with a single transmembrane domain. No other signalling

sequences were detected aside from the YXXΦ and LL vesicular translocation

motifs. Whether this is reflective of actual vesicular localisation however, is

contentious. Studies on NAS homologues from other species have showed

conflicting results – despite conservation of the YXXΦ and LL motifs in the NAS

family, only OsNAS2, ZmNAS1 and ZmNAS2 were confirmed to localise to

vesicles (Mizuno et al., 2003; Nozoye et al., 2014a). Others, like ZmNAS3 and the

AtNAS family, localised to the cytoplasm (Mizuno et al., 2003; Nozoye et al.,

2014b). For comparison, the same analysis performed on CaNAS2 was repeated on

these NAS homologues. The results obtained were inconclusive – no other

localisation signals were detected despite the proven vesicular localisation of some

homologues (data not shown). Whether this discrepancy can be attributed to

limitations on existing databases or post-translation modifications is uncertain, and

further study required.


Systemic expression of Group 3 NAS homologues and their potential housekeeping

role

Consistent with the findings of other authors (Higuchi et al., 2001; Hakoyama

et al., 2009; Filipe de Carvalho et al., 2012), phylogenetic analysis of NAS proteins

showed a clear distinction between monocots and dicots. This corresponds to the

differing physiological roles of NAS between the two lineages. Incidentally all the

monocots used in this study belonged to the Gramineae family, and it is unclear if

this clustering will change upon inclusion of non-graminaceous monocots. No

conclusive statements can be made in that regard due to a current lack of

experimentally verified NAS of non-graminaceous monocot origins. While this lack

of inputs may mask potential phylogenetic links, the results thus far indicate that

specific physiological capabilities such as MAs synthesis can exert a selective

pressure on NAS evolution.

With that in mind, it is possible that symbiosis with Rhizobium may provide

sufficient selective pressure to produce NAS specific for that function. LjNAS2 for

instance, was reported to be nodule-specific (Hakoyama et al., 2009). However,

including the closely related MtNAS (XP_003594753.1), no other genes used in this

study were known to share that trait. This can partly be attributed to the lack of

information on dicot NAS homologues as a whole, and future studies may uncover

more nodule-specific homologues. In the interim, existing information only permits

reasonable allusion to the potential function of the one of the three NAS groups

observed within the leguminous lineage.

Group 3 of leguminous NAS homologues are characterised by their widespread

expression in various tissues, ranging from roots, to leaves and cotyledons. This

expression pattern was also exhibited by CaNAS2 and XP_00449454.1, both

members of Group 3. While the range of expression sites was found to vary between

the homologues, the stem remained a common site of expression. As suggested by

Hakoyama et al. (2009), such an expression pattern points to a housekeeping role in

the systemic redistribution of iron. Given that NA is involved in both long and short-

distance translocation (Stephan et al., 1994b; Takahashi et al., 2003b; Schuler et al.,

2012b), the NAS expressed in the various sites may operate at different scales.

Expression in the stem for instance, may serve to feed NA into the vascular tissue


and symplast for systemic transport, while expression in the other locations may

provide NA for more localised translocation.

Differential expression of CaNAS2 in response to external iron conditions

In this study, CaNAS2 transcripts were detected in a wide range of tissues,

particularly in the stem and roots. Higher expression was also noted in the iron-

sufficient plants. This supports the idea of a housekeeping role in systemic

translocation of iron, particularly from the roots to the leaves. That a general

downregulation was observed in the iron-deficient plants suggests that CaNAS2 is

not heavily involved in the iron deficiency response, and this housekeeping role

occurs mostly under iron-sufficient conditions.

This downregulation was also observed by Higuchi et al. (1995), who reported

decreased NAS activity in tomato, soy, and several tobacco species in response to

iron deficiency. Douchkov et al. (2005) on the other hand, reported enhanced NA

accumulation in Arabidopsis under iron-deficient but not iron sufficient conditions.

These accounts hint at potential species-specific responses even amongst the non-

graminaceous species, though the regulatory mechanisms underlying this disparity or

the levels at which they occur is unknown. Given that the role of NA in the iron

metabolism of chickpea is also not fully understood, it is plausible that other CaNAS

homologues may be involved in the iron deficiency response even is CNAS2 is not.

With NA being a key ligand for iron translocation, it was expected that NAS

expression would be upregulated in the cotyledons to facilitate iron export during

iron deficiency. The absence of CaNAS2 transcripts in that tissue under iron-

deficiency was therefore quite unusual. Tiffin et al. (1973) had previously

demonstrated the significance of the cotyledons as a nutritional support and buffer

during both germination and later vegetative growth. They had found iron to be

exported from the cotyledons during iron deficiency, while the reverse was observed

under iron sufficient conditions. Assuming a similar phenomenon had occurred in

this study, there are two probable reasons behind the expression pattern of CaNAS2

in the cotyledons. First, is the exhaustion of iron reserves in the cotyledons and the

resulting lack of iron to transport. Second, is that CaNAS2 is not involved in the


export of iron from the cotyledons during iron-deficiency. Further work is required

for confirmation.

Overexpression of CaNAS2 in N.tabacum leads to enhancement of zinc, but not

iron, content in the leaf

Overexpression of NAS have been demonstrated to enhance iron content in

both monocot and dicot species (Douchkov et al., 2005; Masuda et al., 2009;

Johnson et al., 2011). To determine if overexpression of CaNAS2 would produce a

similar result, several transgenic tobacco lines overexpressing CaNAS2 on its own or

in combination with GmFER were generated. The same expression constructs, but

with OsNAS2 instead of CaNAS2, were used as positive controls.

Between the OsNAS2 and CaNAS2 overexpressing lines, no notable

differences were observed in the leaf iron and zinc contents. Compared to the non-

transgenic and GUS vector control, no significant differences were also observed in

the average leaf iron contents of the NAS and NAS-GmFER lines. However,

considerable variability was also observed amongst the replicates in each line. In

contrast, consistent enhancements were seen in the zinc contents, which were

significantly higher in most transgenic lines compared to the GUS vector control.

This increased zinc accumulation was unsurprising. NA has previously been

implicated in the zinc homeostasis, particularly in the zinc hyperaccumulating

species Arabidopsis halleri (Pich and Scholz, 1996; Deinlein et al., 2012; Tsednee et

al., 2014). NAS has also been successfully used in zinc biofortification efforts in rice

(Johnson et al., 2011; Trijatmiko et al., 2016). However increases in zinc and iron

levels have typically been observed to occur in tandem (Douchkov et al., 2005;

Johnson et al., 2011), and the absence of such a phenomenon in this study was

unforeseen. This was particularly so for the NAS-GmFER transgenic lines in which

GmFER was driven by the 35s promoter. Unlike NA, which interacts with a range of

divalent cations, GmFER is known to be specific to iron. Also, the positive effect of

FER overexpression on iron accumulation has been consistently demonstrated by

several authors (Van Wuytswinkel et al., 1999; Drakakaki et al., 2000; Goto et al.,

2000).


Considering that NAS was a common factor in all the expression cassettes, a

possible reason behind this discrepancy might be external iron concentrations. When

grown under iron-limiting conditions, elevated iron contents have been observed in

transgenic tobacco and Arabidopsis overexpressing AtNAS2 and TcNAS1

respectively (Douchkov et al., 2005; Cassin et al., 2009). With the latter, shoot iron

content was also noted to be lower than the wild-type under iron-sufficient

conditions (Cassin et al., 2009). In this chapter, all plants were grown under iron

sufficient conditions and the response to iron deficiency was not tested. Whether a

similar response to that reported by Cassin et al. (2009) and Douchkov et al. (2005)

will be evoked bears further investigation.

Chapter 6: Generation and Characterisation of Transgenic Chickpea 99

Chapter 6: Generation and

Characterisation of Transgenic

Chickpea

6.1 INTRODUCTION

Iron deficiency is one of the most common micronutrient deficiencies in the

world. More than 60% of global anaemia cases are attributed to iron deficiency, and

it afflicts both developing and developed nations (Alvarez-Uria et al., 2014). Several

strategies have been developed to combat this problem, amongst which the

generation of ‘self-fortifying’ crops (also known as biofortification) demonstrates

significant promise. Recently, pulses have gained recognition as suitable targets for

biofortification. Despite this pique in interest, there remains a paucity of information

in this aspect compared to the more well-studied crops like cereals.

Considerable progress has been made in the past five years, particularly with

chickpea. Latest advancements include the sequencing of the chickpea genome and

mapping of quantitative trait loci (QTLs) associated with iron and zinc accumulation

(Varshney et al., 2013; Diapari et al., 2014; Upadhyaya et al., 2016). Several genes

involved in iron metabolism have also been identified amongst the QTLs

(Upadhyaya et al., 2016). Although these genes remain uncharacterised, this

information has shed light on key mechanisms, thereby allowing for more targeted

approaches.

While such information accelerates the development of elite micronutrient-rich

cultivars, the breeding process itself is laborious and time-consuming. This selection

of traits may be expedited through the use of transgenic technologies. As an added

benefit, closer investigation of the underlying molecular mechanisms may be done

due to the specificity of this approach. The generation of transgenic chickpea,

however, remains a challenging undertaking. Chickpea transformation has been

attempted since 1993, and several protocols have been reported in the literature.

Most involve Agrobacterium-mediated transformation of explants like axillary

meristems (Bhatnagar-Mathur et al., 2009), cotyledonary nodes (Sanyal et al., 2005;

Indurker et al., 2010), embryos and its derivative tissues (Polowick et al., 2004;

100 Chapter 6: Generation and Characterisation of Transgenic Chickpea

Sarmah et al., 2004; Tewari-Singh et al., 2004). As observed by Sarmah et al.

(2004), early protocols (Fontana et al., 1993; Kar et al., 1996; Krishnamurthy et al.,

2000) were fraught by three major hurdles, namely 1) lack of reproducibility, 2) low

transformation efficiencies, and 3) poor transmission of transgenes to subsequent

generations. Such issues appear to have been somewhat addressed in later

publications (Polowick et al., 2004; Sarmah et al., 2004). The protocol used by

Sarmah et al. (2004) and Acharjee et al. (2010) for instance, reported transformation

efficiencies of 0.3 – 0.72% and transmission of transgenes up to the T3 generation.

Transgenic work in chickpea thus far has revolved around development of

drought tolerance (Bhatnagar-Mathur et al., 2009) and insect resistance (Acharjee et

al., 2010; Ganguly et al., 2014). To date, no known attempts at biofortification of

chickpea have been made. In addition, while several iron biofortification strategies

have been developed (e.g. Masuda et al., 2013a), the focus has been on cereals.

General information on the effectiveness of these strategies in dicots is lacking.

Consequently, it is difficult to reliably estimate the influence of these strategies on

seed iron accumulation.

This chapter aims to fill that gap. In this study, chickpea (cv HatTrick) was

transformed with a combination of rice nicotianamine synthase 2 (OsNAS2) and

soybean ferritin H1 (GmFER). This combination was recently proven to be highly

effective in rice when overexpressed under a constitutive and seed-specific promoter

respectively (Trijatmiko et al., 2016). However due to the lack of prior study in

chickpea, both genes were constitutively overexpressed to determine the

effectiveness of these genes in chickpea. To also evaluate the effectiveness of a

cisgenic approach, a chickpea nicotianamine synthase (CaNAS2) and GmFER

combination was assessed. The progeny of transformed plants were assessed for iron

accumulation in the leaf and seed tissues.


6.2.1 Generation of transgenic chickpea

Plants were transformed with the pEBX-GmFER-CaNAS and pEBX-GmFER-

OsNAS2 constructs generated in Chapter 5.2.6. The chickpea transformation process

was initially done following the protocol developed by Sarmah et al. (2004).

However, modifications were later made to adapt and optimise it for laboratories in


the Queensland University of Technology (QUT). The modified protocol is as

outlined in Section 3.2.3.2 and an overview of the transformation process is as

illustrated in Figure 6.1. The modifications made are listed in Table 6.1.

Figure 6.1. Overview of the chickpea transformation process. A) Half-embryonic

axis explant, B) micro-injury with needle in Agrobacterium culture, C) explants after

3 days of co-cultivation, D) explants after first selection, E) shoot multiplication, and

F) putative transgenic shoot grafted onto non-transgenic rootstock in vitro.


Table 6.1. Summary of modifications made to the original protocol.

Step Original protocol

(Sarmah et al., 2004) Modified protocol

Explant

preparation Bisection along embryonic

axis only

Bisection along embryonic

axis and additional injury to

radicle using needle

Elimination of explant

washing step after co-

cultivation.

Co-

cultivation

Explants placed directly onto

B3 media after incubation

with Agrobacterium

Explants washed after 3 days

co-cultivation, before

transfer to selection media

Explants placed on a layer of

sterile filter paper on B3

media

Direct transfer of explants to

selection media (elimination

of washing step)

Selection

media

Single round on

multiplication media,

followed by successive

rounds on B3 (elongation)

media

Timentin used as selection

against Agrobacterium

Successive rounds on

multiplication media and

eliminated use of R3

(elongation) media

Injury to the cotyledonary

node after first selection

round

Replacement of timentin with

merrem as selection against

against Agrobacterium

Culture

vessels Tissue culture pots

Replacement of 250mL pots

with deep Petri dishes (90 x

25mm)

6.2.2 Molecular characterisation of transgenic chickpea

Shoots emerging from the same explant were considered as a single

independent transgenic event. Following successful acclimatisation, each T0 plant

was screened for the genes of interest following the protocol described in Chapter

3.2.3.2. Seeds from the PCR-positive individuals were collected, sown, and

screening was performed on all germinated seedlings. This process was repeated for

every generation produced.

Expression of the genes of interest was assessed via the method outlined in

Chapter 3.2.5.2. A qualitative analysis was performed on the T1 generation to

confirm gene expression, while a quantitative analysis performed on seven week old

plants used in the final glasshouse trial. RNA was extracted from the leaf tissue of


three biological replicates per line and the quality confirmed by gel electrophoresis.

DNAse treatment was performed on 1 µg of RNA, from which cDNA was generated.

The cDNA was checked via PCR using a housekeeping gene to confirm successful

synthesis, following which it was used for qPCR. Gene expression was normalised to

the same housekeeping genes used in Chapter 5.2.4.3. Primers used in qPCR are as

listed in Table 6.2.

Table 6.2. List of primers used for qPCR.


Expected

amplicon

size

Actin 1 Fw GCCTGATGGA CAGGTGATCA C

62 Rv GGAACAGGAC CTCTGGACAT C

EF1α Fw TCCACCACTT GGTCGTTTTG

64 Rv CTTAATGACA CCGACAGCAA CAG

GAPDH Fw CCAAGGTCAA GATCGGAATC A

65 Rv CAAAGCCACT CTAGCAACCA AA

CaNAS2 Fw AGTAGTGCCT TTCTAAATGG CC

116 Rv CATGTCACCA ATCCCCAACA T

OsNAS2 Fw TGATCAACTC CGTCATCGTC

175 Rv TCAGACGGAT AGCCTCTTGG

GmFER Fw GTGCAATCGG AACAGCAAGA

138 Rv TTGGGTCTTT CTAAGGGTGT TG

To determine transgene copy number, DNA was extracted from leaf tissue and

Southern analysis carried out following the protocols described in Chapter 3.2.5.3.

The DNA was digested with KpnI, and a DIG-labelled probe against the NPTII

selectable marker was used to detect for the transgene. The probe was generated

using the NPTII-specific primers listed in Chapter 5, Table 5.4.

6.2.3 Glasshouse trial for T3, T4 plants

All plant material was germinated and screened following the protocols

described in Chapter 3.2.5.1.1. At 4 weeks of age, verified plants were moved to the

Queensland Crop Development Facility (Redlands, Queensland, Australia and

transplanted to 10 inch pots. The potting mix used was a 1:1 mixture of University of

California (UC) mix and regular potting mix. Plants were watered twice a day with

100 mL of water. To ensure sufficient quantities of iron in the soil, 16 mL of 50 µM

Fe-EDTA was added to the base of each plant at the 7 and 10 week stage. When the

plants began to senesce, watering was reduced by half to 50mL per dose for three


days, then cut completely in preparation of harvest. The plants were dried for two

weeks before harvesting and measurement of agronomic parameters.

6.2.4 Assessment of agronomic parameters

After drying, the aerial portion of the plants was harvested to measure the

following parameters: biomass, yield, 100 seed weight, harvest index and pod

abortion rate. Biomass was measured as the dry weight of the entire aerial portion of

the plants including the pods. Yield was calculated as the total seed count, in which

only filled seeds were considered. The 100 seed weight, harvest index, and pod

abortion rates were calculated using the following formulas:

100 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡

𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑐𝑜𝑢𝑛𝑡× 100

𝐻𝑎𝑟𝑣𝑒𝑠𝑡 𝑖𝑛𝑑𝑒𝑥 (𝐻𝐼) = 𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡

𝑇𝑜𝑡𝑎𝑙 𝑏𝑖𝑜𝑚𝑎𝑠𝑠× 100

𝑃𝑜𝑑 𝑎𝑏𝑜𝑟𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 = 𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑑 𝑐𝑜𝑢𝑛𝑡 − 𝑛𝑜. 𝑜𝑓 𝑓𝑖𝑙𝑙𝑒𝑑 𝑝𝑜𝑑𝑠

𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑑 𝑐𝑜𝑢𝑛𝑡× 100%

6.2.5 Assessment of iron content in transgenic chickpea plants

A preliminary study was conducted on the leaves of T1 plants to determine

iron accumulation. The third and fourth youngest leaves were collected from a single

branch of seven week old plants grown in the P9 glasshouse at QUT Gardens Point

campus. These plants were grown on Plugger 222 potting mix (Australian Growing

Solutions) and fertilised with full-strength Hoagland solution every two weeks.

Samples were prepared for analysis via LA-ICP-MS following the procedure

described in Chapter 3.2.6.

For the final glasshouse trial, iron accumulation in both leaf and seed were

assessed. For leaf iron content, the third, fourth and fifth youngest leaves of 7 week

old plants were pooled and prepared for LA-ICP-MS analysis as described in

Chapter 3.2.6. For seed iron content, three to four plants were selected to represent

each line, with each plant representing a single biological replicate. Ten seeds


harvested from each plant were used for the analysis. Sample preparation was done

as described in Chapter 3.2.6 and the trace element content measured by ICP-OES.

6.3 RESULTS

6.3.1 Optimisation of chickpea transformation procedure

Various chickpea transformation protocols have been reported since 1992

(Anwar et al., 2010). Amongst these, the protocol developed Sarmah et al. (2004)

was selected for use in this study. Initial attempts were unsuccessful, and over the

course of work several factors were identified as major contributors to this outcome,

namely humidity, Agrobacterium overgrowth, and poor induction of transgenic

shoots. A few key modifications were therefore made to address these challenges and

adapt the protocol to local conditions.

One of the major problems was excessive condensation within the plant tissue

culture vessels, which led to tissue vitrification and contributed to Agrobacterium

overgrowth. This was particularly prevalent during the selection steps, resulting in

poor quality of shoots for grafting as well as the complete loss of explants. To

overcome this challenge, pots were replaced with petri dishes as culture vessels,

which helped to promote airflow and reduce the humidity and condensation. Despite

this change, Agrobacterium overgrowth remained a recurring problem. Attempts to

salvage affected explants by removing contaminated sections and washing in sterile

water supplemented with timentin were unsuccessful. This problem was resolved

through two modifications. Firstly, a layer of sterile filter paper was placed on top of

the media during the co-cultivation step. Secondly timentin (200mg/L) was replaced

with merrem (25mg/L) as the selection agent. The combination of these two

modifications eliminated the incidence of recurring Agrobacterium contamination

and also allowed for omission of the washing and drying steps following co-

cultivation.

The next challenge was the induction of putative transgenic shoots. Explants

produced a single primary shoot that was able to withstand successive rounds of

selection. Despite this, such shoots did not harbour the genes of interest. Preliminary

studies with plants transformed with the GUS vector control showed expression to be

weak with a patchy distribution (Appendix C, Figure 8.3), and scions generated from

these primary shoots tested negative for the genes of interest (data not shown).


Figure 6.2. Morphology of emerging putative transgenic shoots. The region

highlighted with black is the primary shoot, and the region highlighted with red is the

putative transgenic shoot.

True transgenic shoots on the other hand, typically stemmed from the

secondary shoots emerging from the base of the explant near the cotyledonary node.

A common characteristic of such shoots was their emergence through the selection

media with varying degrees of shoot multiplication (see Figure 6.2). Induction and

multiplication of such shoots rarely occurred using the original protocol (less than

1% induction rate).

To increase exposure to Agrobacterium and allow deep-seated transgenic cells

access to the growth hormones in the media, additional injury was applied at two

stages. The first was to the radicle before incubation with Agrobacterium, using a

needle dipped in the Agrobacterium culture. The second injury was inflicted during

the first subculture to multiplication media, where a shallow incision was made in the

cotyledonary node using a scalpel blade. Successive rounds of selection on

multiplication media was also done to improve shoot proliferation. This was found to

greatly enhance the number of putative lines obtained per transformation experiment,

as most secondary shoots emerged after the second cycle on multiplication media.

This modification also allowed for the continuous proliferation of these shoots and

thus increased supply of material for grafting. With these modifications, induction

rate of these secondary shoots was increased to approximately 3%.


6.3.2 Generation and molecular characterisation of transgenic chickpea

Several rounds of transformation were performed to optimise the procedure

and later produce the number of lines required for later trials. Each transformation

attempt started off with approximately 500 explants. Attempts to further scale-up

operations made unfeasible by logistical constraints (i.e. lack of space).

Following grafting and acclimatisation, putative transgenic plants were

screened for the genes of interest. This screening was also performed on every plant

in successive generations to confirm the inheritance of the genes of interest (see

Figure 6.3). Testing of the acclimatised T0 plants showed the transformation

efficiency to be approximately 1%. In total, eight OsNAS2 lines and seven CaNAS2

lines were generated. Of these, four of the former and two of the latter were

successfully carried down to the T1 and subsequent generations (see Table 6.3).

Despite several attempts, no GUS transgenic plants were successfully generated; as

negative control, a line established from a PCR negative sibling from the T1

generation was used instead. Expression of the genes of interest was confirmed in all

lines at the T1 generation via end-point RT-PCR (see Figure 6.4).

Transgenic lines 7.1, 7.2, 1.1, 6.6, and 6.14 were selected for the glasshouse

trial. Due to low number of seeds, line 7.15 was not included in this trial. All seeds

used were at the T3 generation. The exception was Line 1.1, which was assessed at

the T4 generation. T3 seeds from a PCR-negative sibling were included in this trial

as the null control.


Figure 6.3. Representative photo of PCR screening of T0 plants. Each number

represents an individual transgenic line, within which each lane represents a single

plant.



“+” = Plasmid positive control

WT = Untransformed chickpea

Table 6.3. Summary of transgenic lines generated.

Genes of interest

GmFER-OsNAS2 GmFER-CaNAS2 GUS

No. of explants 5508 4840 2727

Lines generated: 8 7 0

No. of lines passing

to T1 generation 4 2 0

Line ID

7.1

7.2

7.15

1.1

6.6

6.14 -


Figure 6.4. Detection of transgene expression in transgenic chickpea lines via

PCR. A) GmFER expression, B) CaNAS2 expression, C) OsNAS2 expression in T1

plants. Each number represents an individual transgenic line. All lines were at the T1

generation; the exception was Line 1.1, which was at the T2 generation.




WT = Untransformed chickpea

Null = Non-transgenic sibling

FO = pEBX-GmFER-OsNAS2

FC = pEBX-GmFER-CaNAS2


To determine the levels of transgene expression in the material used for the

glasshouse trial, qPCR was performed on leaf tissue harvested from seven week old

plants. The results confirmed the continued expression of the genes of interest in

most lines (see Figure 6.5). Relative expression of GmFER was at least 18-fold in

Lines 1.1, 6.6, and 6.14. With the CaNAS2 overexpressing lines 6.6 and 6.14,

CaNAS2 expression was enhanced by 49 and 100 times respectively compared to the

null control. With the OsNAS2-overexpressing line 1.1, relative expression of

OsNAS2 was approximately 0.032.

In lines 7.1 and 7.2, negligible expression of OsNAS2 was detected (Figure

6.5). GmFER expression was similarly affected, with relative expressions of 0.01 and

0.11 respectively. Southern analysis using a probe for the selection marker showed

multiple integration sites for those two lines. With the other line, only a single copy

was found (see Figure 6.6).

Figure 6.5. Relative expression of transgenes in transgenic chickpea. A) GmFER,

B) CaNAS2, and C) OsNAS2. The gray bar represents the non-transgenic segregant

(Null), the orange bars represent CaNAS2-GmFER lines, while blue bars represent

OsNAS2-GmFER lines. n=3. Error bars indicate standard error. Expression was

measured using qPCR calculated as relative to that of the housekeeping genes

GAPDH and EF1α. All lines were at the T3 generation; the exception was Line 1.1,

which was at the T4 generation.


Figure 6.6. Southern analysis of transgenic chickpea lines used in the glasshouse

trial. gDNA was digested using KpnI and 15µg loaded onto the gel. A probe for the

selection marker, NPTII, was used to detect for presence of the integrated transgenes.

All lines were at the T3 generation; the exception was Line 1.1, which was at the T4

generation.

Null = Non-transgenic sibling

FO = pEBX-GmFER-OsNAS2 transgenic chickpea lines

FC = pEBX-GmFER-CaNAS2 transgenic chickpea lines

Restriction enzyme = KpnI

DNA quantity = 15 µg

Probe target = NPTII

Exposure time = 15 minutes

The lanes containing the 2-log ladder (NEB) and the plasmid control were

covered during the exposure period due to excessively strong signal strength.


6.3.3 Morphology and agronomic properties of transgenic chickpea

To establish sufficient numbers for a glasshouse trial, transgenic plants were

grown to the T3 and T4 generation. Lines 7.1 and 7.2 consistently yielded the highest

seed count (data not shown) and germination rate (appendix C, Table 8.5). Lines

7.15 and 6.14 produced low yields with poor seed viability, and the former being

dropped for the glasshouse trial due to insufficient numbers.

In the final trial, no morphological differences were observed between the

transgenic lines when compared to the null controls during the vegetative and

flowering states (see Figure 6.7). Flower and pod morphology were similarly

unaffected. In terms of agronomic performance, the average biomass of the null

controls was noticeably higher than that of line 7.2, 6.6, and 6.24 (see Figure 6.8 A).

The difference was insignificant and did not appear to affect the other parameters

measured. No significant differences were observed between the transgenic lines and

null controls in other parameters measured (see Figure 6.8).


Figure 6.7. Morphology of 9 week old transgenic chickpea at the flowering/pod-filling stage.


Figure 6.8. Agronomic properties of transgenic chickpea under glasshouse

conditions. A) Biomass, B) harvest index (HI), C) seed count, D) seed weight, and

E) pod abortion rate. n= 3 to 20. The gray bar represents the non-transgenic

segregant (Null), blue bars represent the GmFER-OsNAS2 transgenic lines, and the

orange bars represent the GmFER-CaNAS2 transgenic lines. Error bars indicate

standard error. Statistical significance was calculated using Dunnett’s test and p-

values<0.05 were considered significant. No significant differences were found

between the null control and transgenic lines. All lines were at the T3 generation; the

exception was Line 1.1, which was at the T4 generation.


6.3.4 Iron content in transgenic chickpea

A preliminary assessment was conducted at the T1 generation to determine the

effect of the transgenes on iron accumulation in the plants. Line 1.1 was not included

in this analysis due to insufficient numbers in this generation. In general, all

transgenic lines except line 7.1 were found to have higher average leaf iron content

than the null controls (see Figure 6.9). A similar observation was made with the zinc

and manganese contents; the exception was line 7.2, which had a manganese content

similar to the null control (see Figure 6.9). Amongst the lines tested in this

preliminary assessment, line 7.15 in particular, was significantly higher than the null

in terms of leaf iron, zinc, and manganese contents. Due to insufficient numbers

however, subsequent data for later generations are unavailable.

Following this preliminary assessment, a large scale glasshouse trial was

conducted using plants at the T3 and T4 generation. The trends observed were

similar to that of the preliminary study, in that the average iron content of all

transgenic lines (except 7.1) was higher than the null (see Figure 6.10). As with the

preliminary study, the difference was not significant. Zinc and manganese levels in

the leaves were also not significantly different except for line 1.1, which had the

highest manganese contents compared to all other lines and the null (see Figure

6.10).

This higher manganese content was not reflected in the subsequent analysis of

the seed. Examination of the micronutrient content in the seeds generated from this

trial showed a trend unlike that observed in the leaf. Of the lines, line 7.1 was shown

to be the highest in terms of average seed iron and zinc contents at 9.3mg/100g,

despite consistently low concentrations of the said elements in the leaf. However, as

observed with the leaves, there were no significant differences between the

transgenic lines and null controls (see Figure 6.11). The lower manganese to iron

content also indicated that there was, at least, no inhibition of iron accumulation by

manganese. Total seed phosphorus were also similar between the null and transgenic

lines (Appendix C, Figure 8.4).


Figure 6.9. Preliminary study on leaf iron, zinc and manganese contents in 7

week old transgenic chickpea at the T1 generation. n=3. The gray bar represents

the non-transgenic segregant (NULL), blue bars represent the GmFER-OsNAS2

transgenic lines, and the orange bars represent the GmFER-CaNAS2 transgenic lines.

Error bars indicate standard error. Statistical significance was calculated using

Dunnett’s test and p-values<0.05 were considered significant. “*” indicate a

significant difference compared to the null control. Line 1.1 was excluded from this

preliminary study due to insufficient replicates.

Figure 6.10. Leaf iron, zinc and manganese contents in 7 week old transgenic

chickpea. n=3 to 23. The gray bar represents the non-transgenic segregant (NULL),

blue bars represent the GmFER-OsNAS2 transgenic lines, and the orange bars

represent the GmFER-CaNAS2 transgenic lines. Error bars indicate standard error.

Statistical significance was calculated using Dunnett’s test and p-values<0.05 were

considered significant. “*” indicate a significant difference compared to the null

control. Line 7.15 was excluded from this preliminary study due to insufficient


replicates. All lines were at the T3 generation; the exception was Line 1.1, which was

at the T4 generation.

Figure 6.11. Iron, zinc and manganese contents in transgenic chickpea seeds.

n=4. The gray bar represents the non-transgenic segregant (NULL), blue bars

represent the GmFER-OsNAS2 transgenic lines, and the orange bars represent the

GmFER-CaNAS2 transgenic lines. Error bars indicate standard error. Statistical

significance was calculated using Dunnett’s test, and p-values<0.05 were considered

significant. “*” indicate a significant difference compared to the null control. Line

7.15 was excluded from this preliminary study due to insufficient replicates. All lines

were at the T3 generation; the exception was Line 1.1, which was at the T4

generation.

6.4 DISCUSSION

Crop biofortification efforts to date have primarily been focused on primary

staple crops such as rice and wheat, with pulses only garnering attention recently. As

such, knowledge on the pulse biofortification can be considered to still be in its

infancy. With transgenic work in particular, there is currently no known precedent.

In this chapter, transgenic chickpea expressing iron metabolism genes were

generated. In this strategy, soybean ferritin was used in combination with the rice

nicotianamine synthase 2 (NAS2) gene. A variation of this strategy was also used in

this study, where OsNAS2 was replaced by CaNAS2 – this was done to compare the

effects of a trans-genic versus a cis-genic approach. The effect of the transgenes was

assessed in terms of the leaf and seed iron content at the T3 and T4 generation.


Optimisation of chickpea transformation protocol

Despite the availability of several published protocols, transformation of

chickpea remains a challenge with low reported efficiencies, ranging from 0.2 to 5%

(e.g. Krishnamurthy et al., 2000; Polowick et al., 2004; Senthil et al., 2004). Most

protocols also are tend to lack reproducibility, as evident in the general lack of

reported downstream work using the transgenic chickpea.

One of the most successful protocols thus far is the one developed by Sarmah

et al. (2004), which has been successfully replicated by other authors (Acharjee et

al., 2010; Ganguly et al., 2014). As such, this was the protocol of choice used in this

study. Concurrently, attempts were also made to use calli as explants instead (data

not included). These proved fruitless – despite reported successes by other authors

(Barna and Wakhlu, 1993; Sagare et al., 1995), no plants could be regenerated from

the calli.

In the course of work, the original procedure by Sarmah et al. (2004) was

generally found to be insufficient in generating transgenic material. Several

modifications were required for effective implementation within the CTCB

laboratories.

One of the key modifications was the infliction of additional injuries to the

explant. Despite the extensive injury caused by bisection of the along the embryonic

axis, poor induction of putative transgenic shoots was observed when using the

original protocol. This was suspected to be due to either poor penetration of

Agrobacterium into the tissue, or lack of contact between transformed cells and the

growth hormones in the media, or both. To test this hypothesis, additional injury was

applied during the infection step and transfer to multiplication media. This was

observed to increase both shoot induction rate and overall transformation efficiency

at the T0 generation.

This use of micro-injury to enhance plant transformation efficiency is not a

new development, having been applied in other plants transformation systems (Trick

and Finer, 1997; Trick and Finer, 1998; Bakshi et al., 2011). In addition to increasing

the surface area for Agrobacterium colonisation and interaction, this technique

allows access to and infection of deeply-embedded germ layers. In other chickpea

transformation protocols, sonication of explants increased the number of transient


transformants by at least three-fold (Sanyal et al., 2005; Tripathi et al., 2013). Stable

transformation frequency at the T0 generation was reported to range from 0.32 –

1.12% (Sanyal et al., 2005) and 1.60 – 2.08% (Mishra et al., 2013; Tripathi et al.,

2013). In this study, transformation efficiency was found to be approximately 1%.

This was higher than the 0.72% reported in the original protocol by Sarmah et al.

(2004). Given that PCR screening was only performed on acclimatised plants and

accounting for loss of lines during the grafting and acclimatisation processes, actual

transformation efficiency is likely to be higher.

Currently, the grafting and acclimatisation processes are the main bottlenecks

in this chickpea transformation system. Despite reports of successful root induction

(Sanyal et al., 2005; Tripathi et al., 2013), attempts to induce rooting in this project

were unsuccessful. Grafting, while laborious, was found to be more efficient though

success was directly affected by the condition of the material and handler skill. As

such, the availability of good quality scions is vital. The supply of healthy grafted

plants in turn increases the chances of survival of the lines past the acclimatisation

process. This is particularly significant given the humidity of local conditions, which

is conducive for Botrytis cinerea growth (Pande et al., 2006). Deterioration of shoot

health inevitably led to infection and loss of the plants. Repeated use of the same

growth cabinet or room also increased spore load and chance of subsequent losses.

In addition to its significance in the acclimatisation process, humidity was also

a key factor in vitro. Vitrification of shoots was an ongoing problem, and similar

issues have been reported by other authors (Brandt and Hess, 1994; Sarker et al.,

2005). However this problem was not observed in the CSIRO laboratory in Canberra,

where the same transformation and regeneration protocol by Sarmah et al. (2004)

was used. Attempts to replicate their growth conditions (e.g. light, temperature,

culture vessels) were unfruitful, though a solution was eventually found by using

different culture vessels which increased airflow.

Collectively, one may suggest variations in local environmental conditions to

be a major contributor to the successful implementation of this chickpea

transformation system. Whether this can be said of other protocols is uncertain. It is

clear however, that adoption of this protocol in a different laboratory may require

further modification to adapt it to local conditions.


Iron accumulation on transgenic chickpea is not consistent between generations

This chapter documents the first example of a transgenic biofortification

attempt in chickpea. This strategy consisting of a combination of NAS and FER

genes had been applied with great success in rice (Johnson et al., 2011; Trijatmiko et

al., 2016). In this chapter, a total of three GmFER-OsNAS2 lines and two GmFER-

CaNAS2 lines were generated and iron accumulation in their leaf and seed assessed.

Initial measurements at the T1 generation showed higher average leaf iron content in

most transgenic lines compared to the null control. However such differences were

considerably reduced in the subsequent assessment of leaf and seed of the T3 and T4

generation. Zinc content, which have been demonstrated in other studies to be

correlated to iron content (e.g. Johnson et al., 2011; Trijatmiko et al., 2016), shared a

similar pattern.

This discrepancy between generations was unexpected, though it likely evolved

as an artefact of experimental design and the environment in addition to genetic

influence. This can primarily be attributed logistical limitations early in the project,

which necessitated the use of different growth facilities and potting mixes between

the trials. Also, only a small population was assessed in the preliminary trial due to

the limited number of plants in the early generations. Consequently the transgenic

lines therefore not have been sufficiently or accurately represented. The results

obtained from this preliminary trial should therefore be interpreted with caution.

As both NAS and GmFER have individually been demonstrated to enhance

leaf iron content in other non-graminaceous species (Van Wuytswinkel et al., 1999;

Douchkov et al., 2005; Cassin et al., 2009), a similar effect was expected when they

were used in conjunction. However, results from the second trial showed the

transgenic chickpea lines to be similar to the controls in iron content, contrasting

with the observations in biofortified rice transformed with the same genes. This

disparate result might have arisen due to the different physiologies of Strategy I and

II plants – rice uses a combination of both Strategy I and II (Ishimaru et al., 2006),

while chickpea uses only Strategy I. Interestingly, the results also showed that iron

contents in the leaf and seed were not necessarily correlated, as was most evident in

Lines 7.1 and 6.6. This was unusual as iron remobilisation from vegetative tissues to

seeds occurs during development; higher leaf iron content was therefore expected to

produce similarly high seed iron content. Several factors may have contributed to this


discrepancy. For instance, the extent of iron contribution to the seeds differs between

tissues (Hocking and Pate, 1977; Burton et al., 1998), and variations in iron

accumulation in said tissues may affect seed iron levels. Another possible reason

may be overaccumulation of iron in a way that exceeds the plant’s sequestration

ability. Excessive accumulation has been known to result in the formation of

insoluble precipitates in the apoplast as a means of avoiding toxicity (Becker et al.,

1995; Garnett and Graham, 2005), though the iron pool available for seed loading

would be simultaneously reduced. Given that NAS was overexpressed however, such

a scenario is improbable as the NA produced would aid in solubilising such

precipitates. Perhaps another unknown mechanism was at work, and further

investigation is required to determine what it is.

Regardless, it is clear that a measure of physiological disruption had occurred

in the transgenic lines, as evident in the reduced yield and seed viability of the early

generations. This was most prominent in the high expressing lines like 7.15, 1.1 and

6.14. Such physiological penalties may have inadvertently selected for individuals

with reduced expression and/or iron accumulation, which may somewhat explain the

recovery of the yield during the glasshouse trial. This may also explain why the

transgenic seeds did not contain that much more iron than the controls – this is

difficult to prove though, as the early generations lacked sufficient seeds to allow for

elemental analysis. Additional trials are required to determine if this is a consistent

trend across the generations.

A consideration for such future trials is the setting in which they are conducted.

As previously mentioned, the use of different glasshouses may contributed to the

difference in leaf iron contents between the generations. The effect however, is

unlikely to be limited to leaf iron content. Several authors have reported an

environmental influence on both yield and seed micronutrient content, though the

specific factor (or combination of factors) has yet to be identified (Garvin et al.,

2006; Ficco et al., 2009; Diapari et al., 2014). It is possible that aside from the

aforementioned genetic and physiological selection, the environmental conditions

have contributed to the recovery of yield in the glasshouse trial. Again, more trials

are required for confirmation. For future reference though, glasshouse trials should

ideally be conducted within the same compound to maximise comparability.

122 Chapter 7: General Discussion

Chapter 7: General Discussion

Iron deficiency is one of the major micronutrient deficiencies worldwide.

Several methods have been developed to alleviate this global problem. Amongst

them, biofortification has gained considerable attention over the past decades. Iron

biofortification via genetic modification (GM) typically target the three main

processes that make up iron metabolism: uptake, translocation, and storage. Several

strategies have been developed using one or more components from these processes,

and varying levels of success have been achieved in several species (Drakakaki et al.,

2000; Kumar et al., 2011; Ihemere et al., 2012). The effectiveness of these strategies

however, is best illustrated in rice, a species used extensively in iron biofortification

studies (Masuda et al., 2013a). Targeting of grain sink strength using soybean ferritin

(GmFER) for instance, increased seed iron content by 1.5 to 3.7-fold (Vasconcelos et

al., 2003; Qu et al., 2005), while a 2.9 to 4.5-fold increase was attained when the

uptake and translocation processes were targeted using nicotianamine synthase

(NAS) (Lee et al., 2009; Masuda et al., 2009). Recently, a strategy combining both

NAS and GmFER was reported to have achieved up to 7.5-fold increase in iron

content (Trijatmiko et al., 2016).

Whether a similar phenomenon may be observed when applied to a dicot grain

crop like chickpea remains unknown. Being a non-graminaceous species, chickpea

differs significantly from rice on both the molecular and physiological levels. This is

of interest particularly where nicotianamine synthase (NAS) is concerned – its

product, nicotianamine (NA), is a precursor for mugineic acid (MA) synthesis and

thus contributes to iron uptake in addition to translocation in graminaceous plants.

While overexpression of NAS has been demonstrated to enhance iron content in both

graminaceous and non-graminaceous species, the effect is more pronounced in the

former (Masuda et al., 2009; Johnson et al., 2011) than the latter (Douchkov et al.,

2005; Cassin et al., 2009). With the latter, the effectiveness of multigenic

biofortification approaches such as the one using NAS and FER is also currently

unknown.

The aim of this project therefore, was the biofortification of chickpea using a

combination of NAS and GmFER. To compare between the effectiveness of cisgenic

Chapter 7: General Discussion 123

and transgenic approaches, the novel chickpea NAS2 (CaNAS2) gene was also

characterised and investigated for its use in the biofortification of chickpea. The

well-characterised rice NAS2 (OsNAS2) gene was used as positive control, and

homology with its amino acid sequence was the basis on which CaNAS2 was

designated.

7.1 THE PHYSIOLOGICAL ROLE OF CANAS2 IN THE SUBCELLUALR

AND SYSTEMIC CONTEXT

Members of the Fabaceae family are unique in that most are capable of

nitrogen fixation via symbiosis with Rhizobium. This relationship imposes an

additional demand for iron (Tang et al., 1990; Strozycki et al., 2007), which may

result in different homeostatic behaviour compared to other non-graminaceous

species. The extent of this difference, if any, on NA homeostasis is unknown, and

examination of NAS subcellular localisation may reveal more about downstream

usage and regulation.

In this project, predictive modelling was performed to determine the protein

properties and localisation of CaNAS2. However, aside from its hydrophilic nature,

the results obtained did not allow for other conclusive statements. Much of this could

be attributed to discrepancies in the existing literature concerning predicted

localisation sites and signals (Mizuno et al., 2003; Nozoye et al., 2014b). Using the

AtNAS homologues as an example (Nozoye et al., 2014b), CaNAS2 may be

speculated to be a cytoplasmic protein. However actual localisation studies are

required for further verification – this was unable to be accomplished within the time

constraints of this project, and can be investigated in future works.

Based on that hypothesis however, some degree of physiological function can

be guessed. Vesicular localisation of NAS has only been reported in some

graminaceous homologues, and this has been linked to the synthesis and secretion of

DMA and MAs (Nozoye et al., 2014a; Nozoye et al., 2014b). Cytoplasmic

localisation on the other hand, has been confirmed in both graminaceous and non-

graminaceous NAS homologues (Mizuno et al., 2003; Nozoye et al., 2014b).

Interestingly, the examples of such graminaceous homologues like OsNAS3 and

ZmNAS3 are known to be downregulated during iron deficiency (Inoue et al., 2003;

Zhou et al., 2013b). As pointed out by Inoue et al. (2003), such behaviour is contrary

to expectations as MAs production, and thus iron uptake, is directly linked to NA


production. It may be inferred implied that such homologues serve other

physiological functions. Such functions may be shared with the non-graminaceous

counterparts, and may include systemic transport or regulation of subcellular iron

levels to prevent toxicity (Pich et al., 2001).

Alternatively, or in addition to the above, NA may serve in iron uptake under

certain limiting conditions. Recently, Tsednee et al. (2014) discovered the presence

of NA in the root exudates of Arabidopsis halleri and Arabidopsis thaliana. This was

linked to zinc tolerance, as the secreted NA served to prevent toxicity by regulating

zinc bioavailability in the soil. Notably, this phenomenon was not seen in any of the

graminaceous species examined by the authors. It is unclear if NA secretion is a trait

shared by all non-gramineae or is limited to the Arabidopsis species; nonetheless it

points to a secretory pathway unique from that of MAS. However, outside of aiding

iron uptake in the presence of excess zinc, there is no indication of the secreted NA

affecting iron uptake under other circumstances like iron deficiency.

Concerning the general role of NA and NAS in the non-gramineae under iron

deficiency, reports by other authors have been conflicting. As previously mentioned

in Chapter 5.4, the response appears species-specific (Higuchi et al., 1995;

Douchkov et al., 2005). Interestingly, while NAS activity in iron-deficient tobacco

was reduced (Higuchi et al., 1995), overexpression of AtNAS1 conferred tolerance

to iron deficiency (Douchkov et al., 2005). This tolerance is unlikely to be due to NA

secretion, if any, facilitating uptake – in the latter study, iron was present as a chelate

and content, rather than bioavailability, was limited. Tolerance may instead be due to

the increased remobilisation capability, as iron from senescing leaves would be more

efficiently moved to younger tissues. Whether increased remobilisation led to

enhanced iron uptake is unknown, as the authors did not report on plant iron content

under iron-sufficient conditions. The higher iron accumulation in our CaNAS2 and

OsNAS2 tobacco lines suggests that there might be a slight increase in uptake. As to

whether NAS overexpression will lead to iron deficiency tolerance, it is difficult to

say. While most studies typically simulate iron deficiency through removing or

reducing iron in the media, iron deficiency in plants is caused by limited

bioavailability in the soil rather than content. Replicating such conditions within this

project was not logistically feasible. Nonetheless this environmental interaction is

important to biofortification efforts, and should be investigated in future studies.


For the purpose of investigating gene expression within the plant however,

manipulation of external iron contents would suffice and was thus used to assess

changes in NAS expression in chickpea. Consistent with the results of studies done

in other species (Inoue et al., 2003; Schuler and Bauer, 2011; Bonneau et al., 2016),

differential expression across various tissues was observed in the CaNAS family.

Specifically, the expression profile of CaNAS2 indicated that it was not involved in

the iron deficiency response, but may have a systemic housekeeping role under iron-

replete conditions. Similar expression profiles were noted in the Medicago truncatula

and Lotus japonicas orthologues that share a common root, hinting at a conserved

function within this group of legume-specific NAS. At this point, this cannot be

confirmed until additional studies are performed due to the general lack of

characterised NAS genes. Also based on observations in Arabidopsis (Schuler et al.,

2012b), considerable functional overlap may exist between the homologues given

their small number.

7.2 NAS-FER TRANSGENE COMBINATION HAS LIMITED EFFECT ON

IRON ACCUMULATION IN TOBACCO AND CHICKPEA

To characterise the CaNAS2 gene and determine the effectiveness of the NAS-

FERR biofortification approach on chickpea, transgenic tobacco and chickpea lines

overexpressing NAS and FERR were generated. Interestingly, despite confirmation

of gene expression in both species, no notable enhancements in iron content

observed. This was unexpected, given previous reports on the positive effect of both

NAS and GmFER on leaf and seed iron content (Van Wuytswinkel et al., 1999; Goto

et al., 2000; Douchkov et al., 2005). The precise reason behind this is difficult to

ascertain; there are, however, several factors that may have contributed to this

outcome. These include, but are not limited, to the following: 1) the differing roles of

NA between graminaceous and non-graminaceous species, 2) promoter strength, 3)

external trace element concentrations, and 4) insufficient uptake capacity.

As mentioned previously, NAS and NA serve different physiological roles in

graminaceous and non-graminaceous species, which translates into different

regulatory mechanisms. However, while the general pattern of NA production

appears to be conserved in the former, conflicting results have been reported in the

latter. Differing patterns of NA accumulation in response to iron deficiency have

been observed in Arabidopsis compared to tomato, soy and tobacco (Higuchi et al.,


1995; Douchkov et al., 2005), and it is unclear if these differences arise from

regulatory mechanisms at the transcriptional, translational or feedback level.

Given that CaNAS2 is upregulated in chickpea during iron-sufficient

conditions, transcript abundance in the overexpressing plants may lead to

suppression of the iron deficiency response and thus iron uptake. This, however, is

unlikely. Firstly, the Nos promoter driving CaNAS expression produced only a small

increase in expression. Secondly, this phenomenon was also observed in tobacco, as

well as transgenic chickpea and tobacco overexpressing OsNAS2. It is possible that

the NAS expression, and thus NA production, was too low to produce a noticeable

effect. A certain threshold may need to be reached for iron content to be significantly

enhancement, and even so the effect is dependent on external iron concentrations.

Such a phenomenon was evident in the study by Cassin et al. (2009) – despite having

100-fold higher NA levels, iron content was not significantly higher in the leaves of

transgenic plants compared to the wild type under iron-sufficient conditions. With

this in mind, it is possible that a stronger promoter than the Nos promoter may be

used without incurring any detrimental effects.

An additional consideration is the concomitant overexpression of GmFER with

the NAS genes. Overexpressing NAS may elicit differing responses between

graminaceous and non-graminaceous species. However no such distinction has been

reported for FER. While ferritin accumulation is not necessarily paralleled by the

iron accumulation of the same extent (Qu et al., 2005), the positive effect of FER

overexpression on iron accumulation has been extensively documented in several

species (e.g. Goto et al., 1999; Van Wuytswinkel et al., 1999; Goto et al., 2000).

Such an effect was noticeably absent in the transgenic tobacco and chickpea lines

used in this project.

The reason behind this is unclear, though there are a few plausible scenarios.

One such scenario is that rather than promoting iron accumulation, concurrent

overexpression of NAS with GmFER may serve instead to redistribute internal

stores. Such a mechanism may temper the added sink strength afforded by GmFER,

thereby avoiding the side-effects of excessive sequestration observed by Van

Wuytswinkel et al. (1999) and Masuda et al. (2013b). For such a purpose, a low

NAS expression may suffice. An alternative scenario is that the iron uptake ability is

unable to match up to the increased translocation and storage capacities. Iron


entering the system is therefore limited, translating to a similar restriction on iron

accumulation. Like the previous scenario, the enhanced translocation capability may

sidestep the physiological problems associated with high FER expression.

It is uncertain which of these scenarios is accurate and in this regard, the results

of this project raise more questions than answers. Some degree of confirmation may

be obtained via quantification of NA and ferritin proteins. Measuring the expression

of genes associated with iron uptake such as IRT (iron-regulated transport) or FRO

(ferric chelate reductase) would also help verify the impact on the overall iron

metabolism. Such assessments could not be performed within this project due to time

and logistical limitations, but can be included in future studies.

Nonetheless, for the purposes of biofortification, the general increase in iron

content produced by this strategy is promising. Further optimisation can be done,

perhaps through the use of different promoters. As plants seem to tolerate higher

levels of NA rather than ferritin, the same strategy employed by Trijatmiko et al.

(2016) may be used instead. Their strategy, unlike the one used in this study, had

NAS constitutively expressed using the 35s promoter, while FER was driven by an

endosperm-specific promoter. By limiting ferritin overaccumulation to the seed, the

side effects on general growth appear to be bypassed. However whether revising the

promoters in chickpea will be as effective remains to be seen; again due to the lack

of MAS biosynthesis in chickpea.

Other aspects may also be examined in such future studies. One of this is iron

bioavailability. While the increase in iron content in the transgenic chickpea seeds

was not as dramatic as in rice (Trijatmiko et al., 2016), the bioavailability of that

iron is uncertain. Hypothetically, improved bioavailability is expected since NA is an

promoter of bioavailability (Zheng et al., 2010), while ferritin-bound iron has been

touted to have a bioavailability equivalent to ferrous sulfate (Davila-Hicks et al.,

2004; Lönnerdal et al., 2006). Should it prove true, then the NAS-FER strategy may

be considered to be somewhat effective, though the effects of downstream processing

still need to be considered. Experiments with cell cultures or animals are required to

verify this hypothesis. Based on existing results however, the effect of inhibitors,

specifically phytic acid (PA), on iron bioavailability may be inferred. PA is a major

inhibitor of iron bioavailability and is often present as a phytate salt of mineral

cations like potassium, magnesium, calcium, manganese and zinc (Sandberg et al.,


1989). It serves as the principal form of phosphorus storage in seeds and depending

on the species, constitutes 40–84% of total phosphorus (Lolas et al., 1976; Griffiths

and Thomas, 1981; Ravindran et al., 1994). As such, phosphorus content was used as

an indication of bioavailability in this project. To this end, the transgenic and non-

transgenic chickpea seeds were found to be similar, which suggests transgene

expression to have no particular impact on phytate accumulation. However such a

method does not account for other bioavailability inhibitors and verification in a

biological system is required.

Another aspect that may be explored in later studies is the influence of the

environmental conditions, particularly soil mineral composition. Relatively little

work has been done in this area concerning transgenic crops, though the effect of the

environmental on crop micronutrient profile has been well documented in several

species (Diapari et al., 2014; Matovu, 2016). This was effect also exemplified in this

project, where a large disparity was seen between the field-grown (Chapter 4) and

glasshouse-grown samples (Chapter 6). In the field-grown samples, levels of iron,

zinc and manganese in the seed mineral were subject to locational variations and

mineral-mineral interactions. These three elements have been associated with NA

(Stephan et al., 1996) and their accumulation in planta may be affected by NAS

overexpression. This is particularly relevant if potential for concurrent zinc

biofortification is to be explored, as zinc accumulation was dependent on

environmental conditions. Assessing the response of the transgenic lines to different

soil types would therefore serve in advising managerial decisions to maximise the

effectiveness of biofortified crops.

Such information would also applicable in the non-transgenic content,

particularly in light of the recent interest in breeding for seed micronutrient content

(Diapari et al., 2014; Upadhyaya et al., 2016). The results obtained in Chapter 4

showed most cultivars surveyed to be fairly similar in trace element composition.

While this is no representative of diversity amongst Australian chickpea, it provides

an idea on the current state of the common germplasm. It is plausible that

micronutrient-accumulating traits may have been bred out of the more current

cultivars due to focus on yields and stress tolerance, and potential yield penalties

accompanying micronutrient accumulation (Garvin et al., 2006; Ficco et al., 2009;


Diapari et al., 2014). At the moment, the extent to which seed micronutrient content

in chickpea can be improved via breeding is yet unexplored.

131

Chapter 8: Concluding remarks

The work presented in this thesis illustrates the first known attempt at the

biofortification of chickpea using a GM approach. The body of work was divided

into three main sections, the summaries of which are described below.

In Chapter Four, the iron contents of modern Australian chickpea cultivars

were assessed. This served to determine existing iron contents in common cultivars

and to identify the factors influencing it. Cultivar was found to exert a greater

influence on seed iron content, though an environmental effect was apparent where

soil iron bioavailability was limited. The environmental effect was also more

prominent in zinc and phosphorus levels, which were correlated to iron and are of

nutritional significance. For further work, HatTrick, a low iron desi cultivar, was

selected. Examination of the distribution of iron within its seed showed that iron was

mainly stored in the cotyledons; this was ideal for biofortification as iron would not

be lost during downstream processing.

Chapter Five focused on the characterisation of the novel chickpea NAS2 gene.

CaNAS2 was found to be closely related to MtNAS, and to be systemically

expressed and upregulated during iron-sufficient conditions, suggesting that it may

have a housekeeping role under iron-sufficient conditions. The gene was then

isolated and cloned into transgenic vectors for overexpression in tobacco, whether on

its own or in conjunction with GmFER. The transgenic lines exhibited slight

increases in leaf iron content compared to the GUS control, though the difference

was mostly insignificant. Similar results were obtained with the OsNAS2 and

OsNAS2-GmFER lines, which were also generated to serve as positive controls.

In Chapter Six, the NAS-GmFER vectors from the previous chapter were used

to transform chickpea. Despite challenges of chickpea transformation, various

modifications allowed for efficiency to be raised to 1% from the 0.7% reported in the

original protocol. A total of four OsNAS2-GmFER and two CaNAS2-GmFER lines

were successfully generated. Preliminary measurement of leaf iron content noted

enhanced levels in the transgenic lines compared to the non-transgenic control,

though later generations showed a less marked increase. While the difference was

132

mostly statistically insignificant, enhanced seen iron content was also observed with

up to a 1.3-fold increase attained.

Collectively, this study demonstrates that the NAS-FER approach is capable of

increasing the iron accumulation in non-graminaceous species like chickpea and

tobacco. However whether this increase of around 1.3-fold can be considered

sufficient is uncertain, as biofortification targets have yet to be set for chickpea.

Regardless, it is likely that further increase of seed iron content, along with

elimination of the physiological penalties, can be attained upon revision of the

promoters used. The use of other genes, such as those targeting the uptake process, is

also viable option – this may confer an added benefit of tolerance to suboptimal iron

concentrations (Connolly et al., 2003). All these can be explored in future studies.

Several other things may also be examined in future studies. For instance, to

determine the precise physiological impact of the transgenes, the expression of genes

and transcription factors associated with iron metabolism can be explored. The effect

of transgenes on nodulation and the response to different soils, particularly those

with limited iron bioavailability, would also be of great interest to growers. Last but

not least, the bioavailability of the seed iron should be assessed to determine the

actual effectiveness of the transgenic approach.

All in all, while exceptional improvements in iron content was not attained

within this project, this study is the first of its kind in chickpea. The information

gleaned can therefore provide a foundation on which future iron biofortification work

can be built on, not just for chickpea, but also other pulses. Ultimately, this is but a

step forward in alleviating global iron deficiency.

Appendices 133

Appendices

Appendix A

Chapter 4 supplementary figures

Table 8.1. Profile of ferrosol soil from Kingaroy (Chauhan, 2015).

Location Memerambi

Depth 0 – 10 cm

pH 6.4

C.E.C. 31 m.eq/100g

Macro elements Micro-elements

Organic C 2.9% Fe 24 ppm

N 0.33% Mn 186 ppm

P 0.073% Cu 3.8 ppm

K 0.28% Zn 4.0 ppm

S 0.053%

134 Appendices

Table 8.2. Concentration of macro-elements in dry chickpea seed. Data are presented as a mean ± SD (n= >3). Values sharing the same

superscript letters indicate groups that are not significantly different at p<0.05.

Ca

(mg/100g)

Mg

(mg/100g)

Na

(mg/100g)

K

(mg/100g)

P

(mg/100g)

S

(mg/100g)

Genesis090

Billa Billa efg 142 (± 8.33) cdefg 134 (± 1.73) cde 16.72 (± 3.10) bcde 1007 (± 11.55) abc 400 (± 10.00) abcd 193 (± 1.73)

Roma cdef 160 (± 20.50) fg 124 (± 1.00) cde 17.08 (± 2.73) bcde 1010 (± 45.83) efg 333 (± 11.55) abcd 187 (± 1.53)

Warra cdef 164 (± 23.52) defg 132 (± 5.29) cde 15.91 (± 3.88) fgh 913 (± 30.55) ab 410 (± 26.46) a 204 (± 14.00)

Kalkee

Billa Billa g 105 (± 4.93) efg 131 (± 1.00) cde 15.53 (± 1.84) ab 1080 (± 10.00) abc 393 (± 5.77) abcd 191 (± 3.61)

Roma fg 124 (± 11.15) efg 128 (± 0.00) cde 12.14 (± 1.89) a 1117 (± 5.77) def 343 (± 11.55) bcd 186 (± 4.51)

Warra fg 121 (± 10.21) fg 122 (± 12.49) cde 14.89 (± 10.56) defg 940 (± 60.83) cde 360 (± 26.46) abcd 190 (± 8.72)

Monarch

Billa Billa fg 127 (± 11.59) bcdef 138 (± 2.08) a 63.33 (± 14.57) ab 1077 (± 25.17) bcde 373 (± 5.77) cdef 180 (± 1.53)

Roma cdef 160 (± 20.03) fg 122 (± 7.00) b 33.00 (± 6.56) bcde 1020 (± 10.00) g 290 (± 20.00) defg 176 (± 6.66)

Warra def 157 (± 11.72) fg 124 (± 3.46) bc 24.67 (± 3.06) gh 863 (± 37.86) efg 330 (± 26.46) ab 198 (± 10.58)

Boundary

Billa Billa defg 148 (± 26.21) bcde 144 (± 10.5) cde 12.41 (± 3.63) abc 1047 (± 25.17) abcd 387 (± 11.55) abcd 189 (± 4.36)

Roma ab 217 (± 5.77) bcde 143 (± 5.51) cde 14.34 (± 1.55) ab 1097 (± 25.17) abcd 383 (± 15.28) abcd 191 (± 7.57)

Warra bcde 192 (± 9.17) b 152 (± 6.56) bcd 20.48 (± 4.20) fgh 903 (± 23.09) abc 403 (± 5.77) abc 196 (± 4.04)

Kingaroy cdef 165 (± 1.08) fg 124 (± 2.10) de 8.31 (± 0.32) gh 857 (± 6.08) k 186 (± 3.54) fg 161 (± 1.79)

CICA0912

Billa Billa def 159 (± 6.24) b 152 (± 5.00) de 7.32 (± 1.24) abc 1057 (± 45.09) abc 397 (± 11.55) cdef 181 (± 2.52)

Warra abc 210 (± 35.50) bcd 149 (± 10.15) cde 10.82 (± 1.73) fgh 890 (± 40.00) ab 413 (± 15.28) abc 196 (± 5.13)

Kingaroy bcde 185 (± 6.80) g 118 (± 4.18) e 4.00 (± 0.11) gh 871 (± 19.68) k 229 (± 17.65) def 178 (± 6.94)

HatTrick

Billa Billa bcd 196 (± 16.37) bc 151 (± 2.00) cde 16.02 (± 6.22) cdef 977 (± 55.08) cde 363 (± 25.17) cdef 180 (± 2.08)

Roma a 257 (± 15.28) bcde 144 (± 0.58) de 7.10 (± 1.45) efg 930 (± 10.00) fg 303 (± 5.77) efg 168 (± 3.06)

Warra bcd 196 (± 31.53) b 153 (± 8.50) cde 16.56 (± 1.49) fgh 903 (± 5.77) a 420 (± 10.00) abcd 190 (± 1.00)

Kingaroy bcdef 167 (± 12.96) fg 124 (± 4.78) e 5.15 (± 0.04) h 836 (± 6.00) k 199 (± 4.40) fg 165 (± 3.05)

NSW a 242 (± 10.95) a 180 (± 4.62) e 6.34 (± 0.63) bcd 1016 (± 23.02) ab 408 (± 8.37) cde 182 (± 2.79)

Appendices 135

Table 8.3. Concentration of micro-elements in dry chickpea seeds. Data is presented as a mean ± SD (n= >3). Values sharing the same

superscript letters indicate groups that are not significantly different at p<0.05.

Fe

(mg/100g)

Zn

(mg/100g)

Mn

(mg/100g)

B

(mg/100g)

Cu

(mg/100g)

Genesis090

Billa Billa abcd 4.60 (± 0.07) fgh 3.33 (± 0.04) defgh 2.98 (± 0.14) bcdef 1.01 (± 0.01) fg 0.74 (± 0.00)

Roma a 5.20 (± 0.02) a 4.35 (± 0.07) fgh 2.73 (± 0.09) bcd 1.07 (± 0.05) abc 0.99 (± 0.03)

Warra ab 5.00 (± 0.46) bcd 3.92 (± 0.21) cdefg 3.25 (± 0.18) b 1.11 (± 0.11) abcd 0.95 (± 0.08)

Kalkee

Billa Billa cdef 4.22 (± 0.11) fghi 3.25 (± 0.04) cdefg 3.23 (± 0.09) bcd 1.06 (± 0.04) efg 0.79 (± 0.01)

Roma abc 4.83 (± 0.04) ab 4.22 (± 0.12) efgh 2.85 (± 0.10) bcd 1.05 (± 0.05) a 1.01 (± 0.01)

Warra bcde 4.42 (± 0.49) efg 3.50 (± 0.35) bcd 3.68 (± 0.35) bcd 1.06 (± 0.04) abcde 0.89 (± 0.08)

Monarch

Billa Billa bcde 4.43 (± 0.09) ghij 3.09 (± 0.09) bcdef 3.42 (± 0.09) fghi 0.86 (± 0.06) g 0.69 (± 0.02)

Roma abcd 4.58 (± 0.17) abc 3.98 (± 0.20) gh 2.66 (± 0.18) bcde 1.04 (± 0.05) abcd 0.95 (± 0.05)

Warra bcd 4.47 (± 0.10) fgh 3.40 (± 0.19) abc 3.86 (± 0.19) bc 1.10 (± 0.10) abcde 0.90 (± 0.06)

Boundary

Billa Billa def 4.15 (± 0.08) ghi 3.12 (± 0.11) bcdef 3.41 (± 0.41) defghi 0.91 (± 0.02) fg 0.76 (± 0.02)

Roma abcd 4.66 (± 0.25) ab 4.14 (± 0.16) defgh 3.09 (± 0.09) bcd 1.05 (± 0.01) ab 1.01 (± 0.05)

Warra bcd 4.53 (± 0.06) bcde 3.85 (± 0.04) abc 3.88 (± 0.23) bcdefg 0.95 (± 0.03) def 0.85 (± 0.03)

Kingaroy g 3.36 (± 0.06) jk 2.70 (± 0.05) ab 4.11 (± 0.11) hi 0.84 (± 0.01) gh 0.67 (± 0.01)

CICA0912

Billa Billa cdef 4.32 (± 0.18) ijk 2.89 (± 0.10) bcd 3.61 (± 0.21) efghi 0.88 (± 0.01) efg 0.79 (± 0.02)

Warra bcd 4.55 (± 0.11) bcd 3.93 (± 0.03) bcd 3.66 (± 0.49) bc 1.10 (± 0.13) abcde 0.90 (± 0.03)

Kingaroy fg 3.70 (± 0.15) cdef 3.58 (± 0.15) ab 4.04 (± 0.05) gc0.78 (± 0.02) hi 0.55 (± 0.03)

HatTrick

Billa Billa def 4.14 (± 0.18) hij 3.06 (± 0.04) ab 4.12 (± 0.29) bcdefg 0.96 (± 0.07) fg 0.76 (± 0.11)

Roma cdef 4.23 (± 0.11) bcde 3.90 (± 0.04) bcd 3.60 (± 0.06) bcdefg 0.97 (± 0.03) bcdef 0.88 (± 0.01)

Warra cdef 4.31 (± 0.07) bcde 3.86 (± 0.02) bcde 3.54 (± 0.48) cdefgh 0.94 (± 0.05) cdef 0.86 (± 0.01)

Kingaroy efg 3.82 (± 0.36) k 2.60 (± 0.08) a 4.44 (± 0.20) i 0.76 (± 0.01) i 0.45 (± 0.01)

NSW def 4.23 (± 0.12) def 3.49 (± 0.09) h 2.54 (± 0.07) a 1.41 (± 0.02) i 0.44 (± 0.03)

136 Appendices

Appendix B


Table 8.4. List of proteins used in the phylogenetic analysis.

Organism Gene Accession no.

Sequence

length

(aa)

Thale cress

(Arabidopsis thaliana)

AtNAS1 NP_196114.1 320

AtNAS2 NP_200419.1 320

AtNAS3 NP_176038 320

AtNAS4 NP_176038 324

Arabidopsis halleri AhNAS2 AFH08366.1 320

Barley

(Hordeum vulgare)

HvNAS1 Q9ZQV9 328

HvNAS2 Q9ZQV7.1 335

HvNAS3 Q9ZQV8.1 335

HvNAS4 Q9ZQV6.1 329

HvNAS5 BAA74584.1 267

HvNAS6 Q9ZQV3.1 328

HvNAS7 Q9ZWH8.1 329

HvNAS8 Q9XFB6.1 329

HvNAS9 Q9XFB7.1 340

Chickpea

(Cicer arietinum)

CaNAS2 XP_004495658.1 306

CaNAS XP_004487761.1 311

CaNAS XP_004488704.1 285

CaNAS XP_004494544.1 318

Lotus japonicas LjNAS1 BAH22562.1 318

LjNAS2 BAH22563.1 312

Barrel medic

(Medicago truncatula)

MtNAS XP_003591220.1 341

MtNAS XP_003594753.1 282

MtNAS XP_013450461.1 320

MtNAS XP_013464357.1 284

Rice

(Oryza sativa)

OsNAS1 BAA74588.2 332

OsNAS2 BAB17823.1 325

OsNAS3 BAB17824.1 343

Alpine pennygrass

(Thlaspi caerulescens) TcNAS CAC82913.1 321

Tomato

(Solanum lycopersicum) SlNAS NP_001296307.1 317

Maize

(Zea mays)

ZmNAS1 AFW88604.1 327

ZmNAS2 DAA45018.1 601

ZmNAS3 XP_008665178.1 359

Appendices 137

Figure 8.1. Representative photos of T1 tobacco screening via PCR using gene-

specific primers. The following genes were screened: A) GmFER, B) CaNAS2, and

C) OsNAS2.




NT = Untransformed chickpea

138 Appendices

Figure 8.2. Concentrations of A) iron, and B) zinc in T1 transgenic tobacco

leaves. Each bar represents an individual plant.

Appendices 139

Appendix C


Figure 8.3. GUS staining of transiently transformed chickpea A) after co-

cultivation and B) after the first selection round.

Table 8.5. Germination rates and segregation of transgenic chickpea lines.

LINES

GmFER-OsNAS2 GmFER-

CaNAS2

Generation 7.1 7.2 7.15 1.1 6.6 6.14

T1 Germination rate (%) 74.82 84.21 80.65 - 74.29 73.68

PCR + progeny (%) 69.23 75.00 72.00 - 17.31 85.71

T2 Germination rate (%) 46.74 15.38 8.99 72.73 46.58 27.52

PCR + progeny (%) 93.02 75.00 100.00 87.50 67.65 68.42

T3 Germination rate (%) 96.15 59.38 - 26.42 38.10 21.43

PCR + progeny (%) 100 57.89 - 100.00 87.50 100.00

T4 Germination rate (%) - - - 80.65 - -

PCR + progeny (%) - - - 100.00 - -

140 Appendices

Figure 8.4. Iron, zinc, manganese and phosphorus content of GM chickpea

seeds.

Bibliography 141

Bibliography

Acharjee, S., Sarmah, B. K., Kumar, P. A., Olsen, K., Mahon, R., Moar, W. J.,

Moore, A., Higgins, T. J. V. (2010) Transgenic chickpeas (Cicer arietinum

L.) expressing a sequence-modified cry2Aa gene. Plant Science 178: 333-339

Addinsoft (2016) XLSTAT. In,

Agbola, F. W., Bent, M. J., Rao, P., Kelley, T. (2000) Factors influencing the

demand for chickpea in India: Implications for marketing and promotion in

the Indian chickpea market. In Proceedings of the 43rd Conference of

Australian Agricultural and Resource Economics Society, Sydney, January,

Vol 23. Citeseer, p 25

Agegnehu, G., Ghizaw, A., Sinebo, W. (2006) Yield performance and land-use

efficiency of barley and faba bean mixed cropping in Ethiopian highlands.

European Journal of Agronomy 25: 202-207

Aisen, P., Enns, C., Wessling-Resnick, M. (2001) Chemistry and biology of

eukaryotic iron metabolism. The International Journal of Biochemistry &

Cell Biology 33: 940-959

Akay, A. (2011) Effect of zinc fertilizer applications on yield and element contents

of some registered chickpeas varieties. African Journal of Biotechnology 10:

13090-13096

Akibode, S., Maredia, M. K. (2012) Global and regional trends in production, trade

and consumption of food legume crops. In, Michigan, USA

Alvarez-Tinaut, M. C., Leal, A., Martínez, L. R. (1980) Iron-manganese interaction

and its relation to boron levels in tomato plants. Plant and Soil 55: 377-388

Alvarez-Uria, G., Naik, P. K., Midde, M., Yalla, P. S., Pakam, R. (2014) Prevalence

and severity of anaemia stratified by age and gender in rural India. Anemia

2014

Antileo, E., Garri, C., Tapia, V., Munoz, J. P., Chiong, M., Nualart, F., Lavandero,

S., Fernandez, J., Nunez, M. T. (2013) Endocytic pathway of exogenous iron-

loaded ferritin in intestinal epithelial (Caco-2) cells. Am J Physiol

Gastrointest Liver Physiol 304: G655-661

Anuradha, K., Agarwal, S., Rao, Y. V., Rao, K. V., Viraktamath, B. C., Sarla, N.

(2012) Mapping QTLs and candidate genes for iron and zinc concentrations

in unpolished rice of Madhukar × Swarna RILs. Gene 508: 233-240

Anwar, F., Sharmila, P., Saradhi, P. P. (2010) No more recalcitrant: Chickpea

regeneration and genetic transformation. African Journal of Biotechnology 9:

782-797

Apel, K., Hirt, H. (2004) Reactive oxygen species: Metabolism, oxidative stress, and

signal transduction. Annual Review of Plant Biology 55: 373-399

Arend, D. N. (2010) Minitab 17 Statistical Software. In. Minitab, Inc., State College,

PA

Bakshi, S., Sadhukhan, A., Mishra, S., Sahoo, L. (2011) Improved Agrobacterium-

mediated transformation of cowpea via sonication and vacuum infiltration.

Plant Cell Reports 30: 2281-2292

Banana21 (2016) Biofortification

Banik, P., Midya, A., Sarkar, B. K., Ghose, S. S. (2006) Wheat and chickpea

intercropping systems in an additive series experiment: Advantages and weed

smothering. European Journal of Agronomy 24: 325-332

142 Bibliography

Bannai, H., Tamada, Y., Maruyama, O., Nakai, K., Miyano, S. (2001) Views:

Fundamental building blocks in the process of knowledge discovery. In

FLAIRS Conference, pp 233-238

Bannai, H., Tamada, Y., Maruyama, O., Nakai, K., Miyano, S. (2002) Extensive

feature detection of N-terminal protein sorting signals. Bioinformatics 18:

298-305

Barna, K. S., Wakhlu, A. K. (1993) Somatic embryogenesis and plant regeneration

from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Reports 12:

521-524

Bashir, K., Inoue, H., Nagasaka, S., Takahashi, M., Nakanishi, H., Mori, S.,

Nishizawa, N. K. (2006) Cloning and characterization of deoxymugineic acid

synthase genes from graminaceous plants. Journal of Biological Chemistry

281: 32395-32402

Batool, R., Butt, M. S., Sultan, M. T., Saeed, F., Naz, R. (2013) Protein-energy

malnutrition; A risk factor for various ailments. Critical Reviews in Food

Science and Nutrition 55: 242-253

Bauer, P., Ling, H. Q., Guerinot, M. L. (2007) FIT, the FER-LIKE IRON

DEFICIENCY INDUCED TRANSCRIPTION FACTOR in Arabidopsis.

Plant Physiology and Biochemistry 45: 260-261

Beard, J. L., Burton, J. W., Theil, E. C. (1996) Purified ferritin and soybean meal can

be sources of iron for treating iron deficiency in rats. The Journal of Nutrition

126: 154-160

Becker, R., Fritz, E., Manteuffel, R. (1995) Subcellular localization and

characterization of excessive iron in the nicotianamine-less tomato mutant

chloronerva. Plant Physiology 108: 269-275

Bejjani, S., Pullakhandam, R., Punjal, R., Nair, K. M. (2007) Gastric digestion of pea

ferritin and modulation of its iron bioavailability by ascorbic and phytic acids

in Caco-2 cells. World J Gastroenterol 13: 2083-2088

Bereczky, Z., Wang, H.-Y., Schubert, V., Ganal, M., Bauer, P. (2003) Differential

regulation of nramp and irt metal transporter genes in wild type and iron

uptake mutants of tomato. Journal of Biological Chemistry 278: 24697-24704

Bergersen, F. (1963) Short communications: Iron in the developing soybean nodule.

Australian Journal of Biological Sciences 16: 916-919

Bhatnagar-Mathur, P., Vadez, V., Jyostna Devi, M., Lavanya, M., Vani, G., Sharma,

K. K. (2009) Genetic engineering of chickpea (Cicer arietinum L.) with the

P5CSF129A gene for osmoregulation with implications on drought tolerance.

Molecular Breeding 23: 591-606

Bhattacharya, A., Sood, P., Citovsky, V. (2010) The roles of plant phenolics in

defence and communication during Agrobacterium and Rhizobium infection.

Molecular Plant Pathology 11: 705-719

Bhatty, R. S., Slinkard, A. E. (1989) Relationship between phytic acid and cooking

quality in lentil. Canadian Institute of Food Science and Technology Journal

22: 137-142

Bjellqvist, B., Basse, B., Olsen, E., Celis, J. E. (1994) Reference points for

comparisons of two-dimensional maps of proteins from different human cell

types defined in a pH scale where isoelectric points correlate with

polypeptide compositions. Electrophoresis 15: 529-539

Bjellqvist, B., Hughes, G. J., Pasquali, C., Paquet, N., Ravier, F., Sanchez, J. C.,

Frutiger, S., Hochstrasser, D. (1993) The focusing positions of polypeptides

Bibliography 143

in immobilized pH gradients can be predicted from their amino acid

sequences. Electrophoresis 14: 1023-1031

Björn-Rasmussen, E., Hallberg, L., Isaksson, B., Arvidsson, B. (1974) Food iron

absorption in man. Applications of the two-pool extrinsic tag method to

measure heme and nonheme iron absorption from the whole diet. Journal of

Clinical Investigation 53: 247

Blair, M. W., Astudillo, C., Grusak, M. A., Graham, R., Beebe, S. E. (2009)

Inheritance of seed iron and zinc concentrations in common bean (Phaseolus

vulgaris L.). Molecular Breeding 23: 197-207

Blair, M. W., Izquierdo, P., Astudillo, C., Grusak, M. A. (2013) A legume

biofortification quandary: Variability and genetic control of seed coat

micronutrient accumulation in common beans. Frontiers in plant science 4

Blair, M. W., Knewtson, S. J., Astudillo, C., Li, C.-M., Fernandez, A. C., Grusak, M.

A. (2010) Variation and inheritance of iron reductase activity in the roots of

common bean (Phaseolus vulgaris L.) and association with seed iron

accumulation QTL. BMC Plant Biology 10: 1-12

Bonneau, J., Baumann, U., Beasley, J., Li, Y., Johnson, A. A. T. (2016)

Identification and molecular characterization of the nicotianamine synthase

gene family in bread wheat. Plant Biotechnology Journal 14: 2228-2239.

Bouis, H. E., Welch, R. M. (2010) Biofortification — A sustainable agricultural

strategy for reducing micronutrient malnutrition in the global south. Crop

Science 50: S20-32

Brandt, E. B., Hess, D. (1994) In vitro regeneration and propagation of chickpea

(Cicer arietinum L.) from meristem tips and cotyledonary nodes. In Vitro

Cellular & Developmental Biology-Plant 30: 75-80

Briat, J.-F., Fobis-Loisy, I., Grignon, N., Lobréaux, S., Pascal, N., Savino, G.,

Thoiron, S., von Wirén, N., Van Wuytswinkel, O. (1995) Cellular and

molecular aspects of iron metabolism in plants. Biology of the Cell 84: 69-81

Briat, J.-F., Lobréaux, S. (1997) Iron transport and storage in plants. Trends in Plant

Science 2: 187-193

Briat, J. F., Duc, C., Ravet, K., Gaymard, F. (2010) Ferritins and iron storage in

plants. Biochim Biophys Acta 1800: 806-814

Briat, J. F., Lobréaux, S., Grignon, N., Vansuyt, G. (1999) Regulation of plant

ferritin synthesis: How and why. Cellular and Molecular Life Sciences CMLS

56: 155-166

Brown, J. C., Chaney, R. L. (1971) Effect of iron on the transport of citrate into the

xylem of soybeans and tomatoes. Plant Physiology 47: 836-840

Brüggemann, W., Maas-Kantel, K., Moog, P. R. (1993) Iron uptake by leaf

mesophyll cells: The role of the plasma membrane-bound ferric-chelate

reductase. Planta 190: 151-155

Brumbarova, T., Bauer, P. (2005) Iron-mediated control of the basic helix-loop-helix

protein FER, a regulator of iron uptake in tomato. Plant Physiology 137:

1018-1026

Bueckert, R., Thavarajah, D., Thavarajah, P., Pritchard, J. (2011) Phytic acid and

mineral micronutrients in field-grown chickpea (Cicer arietinum L.) cultivars

from western Canada. European Food Research and Technology 233: 203-

212

Burton, J. W., Harlow, C., Theil, E. C. (1998) Evidence for reutilization of nodule

iron in soybean seed development. Journal of Plant Nutrition 21: 913-927

144 Bibliography

Campion, B., Sparvoli, F., Doria, E., Tagliabue, G., Galasso, I., Fileppi, M., Bollini,

R., Nielsen, E. (2009) Isolation and characterisation of an lpa (low phytic

acid) mutant in common bean (Phaseolus vulgaris L.). Theoretical and

Applied Genetics 118: 1211-1221

Caracuta, V., Barzilai, O., Khalaily, H., Milevski, I., Paz, Y., Vardi, J., Regev, L.,

Boaretto, E. (2015) The onset of faba bean farming in the Southern Levant.

Scientific Reports 5

Carpenter, C. E., Mahoney, A. W. (1992) Contributions of heme and nonheme iron

to human nutrition. Critical Reviews in Food Science and Nutrition 31: 333-

367

Carvalhais, L. C., Dennis, P. G., Fedoseyenko, D., Hajirezaei, M. R., Borriss, R., von

Wirén, N. (2011) Root exudation of sugars, amino acids, and organic acids by

maize as affected by nitrogen, phosphorus, potassium, and iron deficiency.

Journal of Plant Nutrition and Soil Science 174: 3-11

Cassin, G., Mari, S., Curie, C., Briat, J.-F., Czernic, P. (2009) Increased sensitivity to

iron deficiency in Arabidopsis thaliana overaccumulating nicotianamine.

Journal of Experimental Botany 60: 1249-1259

Cercamondi, C. I., Egli, I. M., Mitchikpe, E., Tossou, F., Zeder, C., Hounhouigan, J.

D., Hurrell, R. F. (2013) Total iron absorption by young women from iron-

biofortified pearl millet composite meals is double that from regular millet

meals but less than that from post-harvest iron-fortified millet meals. The

Journal of Nutrition 143: 1376-1382

Chaney, R. L., Brown, J. C., Tiffin, L. O. (1972) Obligatory reduction of ferric

chelates in iron uptake by soybeans. Plant Physiology 50: 208-213

Chauhan, Y. (2015) Kingaroy soil profile. In,

Chitra, U., Singh, U., Rao, P. V. (1997) Effect of varieties and processing methods

on the total and ionizable iron contents of grain legumes. Journal of

Agricultural and Food Chemistry 45: 3859-3862

Chitra, U., Vimala, V., Singh, U., Geervani, P. (1995) Variability in phytic acid

content and protein digestibility of grain legumes. Plant Foods for Human

Nutrition 47: 163-172

Colangelo, E. P., Guerinot, M. L. (2004) The essential basic helix-loop-helix protein

FIT1 is required for the iron deficiency response. Plant Cell 16: 3400-3412

Connolly, E. L., Campbell, N. H., Grotz, N., Prichard, C. L., Guerinot, M. L. (2003)

Overexpression of the FRO2 ferric chelate reductase confers tolerance to

growth on low iron and uncovers posttranscriptional control. Plant

Physiology 133: 1102-1110

Cornu, J.-Y., Deinlein, U., Höreth, S., Braun, M., Schmidt, H., Weber, M., Persson,

D. P., Husted, S., Schjoerring, J. K., Clemens, S. (2014) Contrasting effects

of nicotianamine synthase knockdown on zinc and nickel tolerance and

accumulation in the zinc/cadmium hyperaccumulator Arabidopsis halleri.

New Phytologist 206: 738–750

Curie, C., Panaviene, Z., Loulergue, C., Dellaporta, S. L., Briat, J. F., Walker, E. L.

(2001) Maize yellow stripe1 encodes a membrane protein directly involved in

Fe(III) uptake. Nature 409: 346-349

Dakora, F. D., Phillips, D. A. (2002) Root exudates as mediators of mineral

acquisition in low-nutrient environments. Plant and Soil 245: 35-47

Davila-Hicks, P., Theil, E. C., Lönnerdal, B. (2004) Iron in ferritin or in salts (ferrous

sulfate) is equally bioavailable in nonanemic women. The American Journal

of Clinical Nutrition 80: 936-940

Bibliography 145

De Castro, E., Sigrist, C. J., Gattiker, A., Bulliard, V., Langendijk-Genevaux, P. S.,

Gasteiger, E., Bairoch, A., Hulo, N. (2006) ScanProsite: detection of

PROSITE signature matches and ProRule-associated functional and structural

residues in proteins. Nucleic acids research 34: W362-W365

De Moura, F. F., Palmer, A. C., Finkelstein, J. L., Haas, J. D., Murray-Kolb, L. E.,

Wenger, M. J., Birol, E., Boy, E., Peña-Rosas, J. P. (2014) Are biofortified

staple food crops improving Vitamin A and iron status in women and

children? New evidence from efficacy trials. Advances in Nutrition: An

International Review Journal 5: 568-570

Deák, M., Horváth, G. V., Davletova, S., Török, K., Sass, L., Vass, I., Barna, B.,

Király, Z., Dudits, D. (1999) Plants ectopically expressing the ironbinding

protein, ferritin, are tolerant to oxidative damage and pathogens. Nature

Biotechnology 17: 192-196

Deinlein, U., Weber, M., Schmidt, H., Rensch, S., Trampczynska, A., Hansen, T. H.,

Husted, S., Schjoerring, J. K., Talke, I. N., Krämer, U., Clemens, S. (2012)

Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in

zinc hyperaccumulation. The Plant Cell 24: 708-723

Dell'Orto, M., Santi, S., De Nisi, P., Cesco, S., Varanini, Z., Zocchi, G., Pinton, R.

(2000) Development of Fe‐deficiency responses in cucumber (Cucumis

sativus L.) roots: Involvement of plasma membrane H+‐ATPase activity.


Diapari, M., Sindhu, A., Bett, K., Deokar, A., Warkentin, T. D., Tar'an, B. (2014)

Genetic diversity and association mapping of iron and zinc concentrations in

chickpea (Cicer arietinum L.). Genome 57: 459-468

Douchkov, D., Gryczka, C., Stephan, U. W., Hell, R., BÄUmlein, H. (2005) Ectopic

expression of nicotianamine synthase genes results in improved iron

accumulation and increased nickel tolerance in transgenic tobacco. Plant, Cell

& Environment 28: 365-374

Drakakaki, G., Christou, P., Stöger, E. (2000) Constitutive expression of soybean

ferritin cDNA in transgenic wheat and rice results in increased iron levels in

vegetative tissues but not in seeds. Transgenic Research 9: 445-452

Eide, D., Broderius, M., Fett, J., Guerinot, M. L. (1996) A novel iron-regulated metal

transporter from plants identified by functional expression in yeast.

Proceedings of the National Academy of Sciences 93: 5624-5628

Fan, T. W., Lane, A. N., Pedler, J., Crowley, D., Higashi, R. M. (1997)

Comprehensive analysis of organic ligands in whole root exudates using

nuclear magnetic resonance and gas chromatography-mass spectrometry.

Analytical Biochemistry 251: 57-68

FAO (1994) Pulses and derived products

FAO (2012) The state of food insecurity in the world. FAO Economic and Social

Development Department, Rome

FAO (2014) Food Outlook: Biannual report on global food markets. In Food

Outlook. FAO

FAO (2016a) Annual growth rates of total pulse production from 1961 to 2013

FAO (2016b) FAO Statistical Database (FAOSTAT)

Ficco, D., Riefolo, C., Nicastro, G., De Simone, V., Di Gesu, A., Beleggia, R.,

Platani, C., Cattivelli, L., De Vita, P. (2009) Phytate and mineral elements

concentration in a collection of Italian durum wheat cultivars. Field Crops

Research 111: 235-242

146 Bibliography

Filipe de Carvalho, V., Clauber Mateus Priebe, B., da Maia, L. C., de Sousa, R. O.,

Panaud, O., de Oliveira, A. C. (2012) Phylogenetic relationships and selective

pressure on gene families related to iron homeostasis in land plants. Genome

55: 883-900

Finkelstein, J. L., Mehta, S., Udipi, S. A., Ghugre, P. S., Luna, S. V., Wenger, M. J.,

Murray-Kolb, L. E., Przybyszewski, E. M., Haas, J. D. (2015) A randomized

trial of iron-biofortified pearl millet in school children in India. The Journal

of Nutrition 145: 1576-1581

Fontana, G., Santini, L., Caretto, S., Frugis, G., Mariotti, D. (1993) Genetic

transformation in the grain legume Cicer arietinum L. (chickpea). Plant Cell

Reports 12: 194-198

Fuller, D. Q., Korisettar, R., Venkatasubbaiah, P. C. (2001) Southern Neolithic

cultivation systems: A reconstruction based on archaeobotanical evidence.

South Asian Studies 17: 171-187

Ganguly, M., Molla, K. A., Karmakar, S., Datta, K., Datta, S. K. (2014)

Development of pod borer-resistant transgenic chickpea using a pod-specific

and a constitutive promoter-driven fused cry1Ab/Ac gene. Theoretical and

Applied Genetics 127: 2555-2565

Garcia, M. J., Lucena, C., Romera, F. J., Alcantara, E., Perez-Vicente, R. (2010)

Ethylene and nitric oxide involvement in the up-regulation of key genes

related to iron acquisition and homeostasis in Arabidopsis. Journal of

Experimental Botany 61: 3885-3899

Garg, R., Sahoo, A., Tyagi, A. K., Jain, M. (2010) Validation of internal control

genes for quantitative gene expression studies in chickpea (Cicer arietinum

L.). Biochemical and Biophysical Research Communications 396: 283-288

Garnett, T. P., Graham, R. D. (2005) Distribution and remobilization of iron and

copper in wheat. Annals of Botany 95: 817-826

Garvin, D. F., Welch, R. M., Finley, J. W. (2006) Historical shifts in the seed mineral

micronutrient concentration of US hard red winter wheat germplasm. Journal

of the Science of Food and Agriculture 86: 2213-2220

Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S. e., Wilkins, M. R., Appel, R.

D., Bairoch, A. (2005a) Protein identification and analysis tools on the

ExPASy server. Springer

Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S. e., Wilkins, M. R., Appel, R.

D., Bairoch, A. (2005b) Protein identification and analysis tools on the

ExPASy server. In The Proteomics Protocol Handbook. Humana Press, New

Jersey, USA, pp 571-607

Gaur, P. M., Samineni, S., Sajja, S., Chibbar, R. N. (2015) Achievements and

challenges in improving nutritional quality of chickpea. Legume

Perspectives: 31-33

GenomeNet. In,

Ghavidel, R. A., Prakash, J. (2007) The impact of germination and dehulling on

nutrients, antinutrients, in vitro iron and calcium bioavailability and in vitro

starch and protein digestibility of some legume seeds. LWT - Food Science

and Technology 40: 1292-1299

Gooding, M. J., Kasyanova, E., Ruske, R., Hauggaard-Nielsen, H., Jensen, E. S.,

Dahlmann, C., Von Fragstein, P., Dibet, A., Corre-Hellou, G., Crozat, Y.,

Pristeri, A., Romeo, M., Monti, M., Launay, M. (2007) Intercropping with

pulses to concentrate nitrogen and sulphur in wheat. The Journal of

Agricultural Science 145: 469-479

Bibliography 147

Goto, F., Yoshihara, T., Saiki, H. (2000) Iron accumulation and enhanced growth in

transgenic lettuce plants expressing the ironbinding protein ferritin.

Theoretical and Applied Genetics 100: 658-664

Goto, F., Yoshihara, T., Shigemoto, N., Toki, S., Takaiwa, F. (1999) Iron

fortification of rice seed by the soybean ferritin gene. Nature Biotechnology

17: 282-286

Graziano, M., Lamattina, L. (2005) Nitric oxide and iron in plants: An emerging and

converging story. Trends in Plant Science 10: 4-8

Griffiths, D. W., Thomas, T. A. (1981) Phytate and total phosphorus content of field

beans (Vicia faba L.). Journal of the Science of Food and Agriculture 32:

187-192

Grillet, L., Ouerdane, L., Flis, P., Hoang, M. T. T., Isaure, M.-P., Lobinski, R., Curie,

C., Mari, S. (2014) Ascorbate efflux as a new strategy for iron reduction and

transport in plants. Journal of Biological Chemistry 289: 2515-2525

Guelke, M., Von Blanckenburg, F. (2007) Fractionation of stable iron isotopes in

higher plants. Environmental Science & Technology 41: 1896-1901

Guelke, M., von Blanckenburg, F., Schoenberg, R., Staubwasser, M., Stuetzel, H.

(2010) Determining the stable Fe isotope signature of plant-available iron in

soils. Chemical Geology 277: 269-280

Guttieri, M., Bowen, D., Dorsch, J. A., Raboy, V., Souza, E. (2004) Identification

and characterization of a low phytic acid wheat. Crop Science 44: 418-424

Haas, J. D., Beard, J. L., Murray-Kolb, L. E., del Mundo, A. M., Felix, A., Gregorio,

G. B. (2005) Iron-biofortified rice improves the iron stores of nonanemic

Filipino women. The Journal of Nutrition 135: 2823-2830

Haber, F., Weiss, J. (1934) The catalytic decomposition of hydrogen peroxide by

iron salts. In Proceedings of the Royal Society of London A: Mathematical,

Physical and Engineering Sciences, Vol 147. The Royal Society, pp 332-351

Hakoyama, T., Watanabe, H., Tomita, J., Yamamoto, A., Sato, S., Mori, Y., Kouchi,

H., Suganuma, N. (2009) Nicotianamine synthase specifically expressed in

root nodules of Lotus japonicus. Planta 230: 309-317

Harrison, P. M., Hoy, T. G., Macara, I. G., Hoare, R. J. (1974) Ferritin iron uptake

and release. Structure-function relationships. Biochem. J. 143: 445-451

HarvestPlus (2015a) Crops

HarvestPlus (2015b) FAQ about Biofortification

Hell, R., Stephan, U. (2003) Iron uptake, trafficking and homeostasis in plants.

Planta 216: 541-551

Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J. (2007)

qBase relative quantification framework and software for management and

automated analysis of real-time quantitative PCR data. GENOME BIOLOGY

8

Hemalatha, S., Platel, K., Srinivasan, K. (2007a) Influence of heat processing on the

bioaccessibility of zinc and iron from cereals and pulses consumed in India.

Journal of Trace Elements in Medicine and Biology 21: 1-7

Hemalatha, S., Platel, K., Srinivasan, K. (2007b) Zinc and iron contents and their

bioaccessibility in cereals and pulses consumed in India. Food Chemistry

102: 1328-1336

Herbik, A., Koch, G., Mock, H. P., Dushkov, D., Czihal, A., Thielmann, J., Stephan,

U. W. (1999) Isolation, characterization and cDNA cloning of nicotianamine

synthase from barley. European Journal of Biochemistry 265: 231-239

148 Bibliography

Higuchi, K., Kanazawa, K., Nishizawa, N.-K., Chino, M., Mori, S. (1994)

Purification and characterization of nicotianamine synthase from Fe-deficient

barley roots. Plant and Soil 165: 173-179

Higuchi, K., Kanazawa, K., Nishizawa, N.-K., Mori, S. (1996) The role of

nicotianamine synthase in response to Fe nutrition status in Gramineae. Plant

and Soil 178: 171-177

Higuchi, K., Nakanishi, H., Suzuki, K., Nishizawa, N. K., Mori, S. (1999a) Presence

of nicotianamine synthase isozymes and their homologues in the root of

graminaceous plants. Soil Science and Plant Nutrition 45: 681-691

Higuchi, K., Nishizawa, N.-K., Yamaguchi, H., Römheld, V., Marschner, H., Mori,

S. (1995) SHORT COMMUNICATION: Response of nicotianamine

synthase activity to Fe-deficiency in tobacco plants as compared with barley.


Higuchi, K., Suzuki, K., Nakanishi, H., Yamaguchi, H., Nishizawa, N.-K., Mori, S.

(1999b) Cloning of nicotianamine synthase genes, novel genes involved in

the biosynthesis of phytosiderophores. Plant Physiology 119: 471-480

Higuchi, K., Watanabe, S., Takahashi, M., Kawasaki, S., Nakanishi, H., Nishizawa,

N. K., Mori, S. (2001) Nicotianamine synthase gene expression differs in

barley and rice under Fe-deficient conditions. The Plant Journal 25: 159-167

Hindt, M. N., Guerinot, M. L. (2012) Getting a sense for signals: Regulation of the

plant iron deficiency response. Biochimica et Biophysica Acta (BBA) -

Molecular Cell Research 1823: 1521-1530

Hocking, P. J., Pate, J. S. (1977) Mobilization of minerals to developing seeds of

legumes. Annals of Botany 41: 1259-1278

Hoppler, M., Schönbächler, A., Meile, L., Hurrell, R. F., Walczyk, T. (2008)

Ferritin-iron is released during boiling and in vitro gastric digestion. The

Journal of Nutrition 138: 878-884

Horton, S., Mannar, V., Wesley, A. (2008) Micronutrient fortification (iron and salt

iodization). In Copenhagen Consensus 2008, Denmark

House, W. A. (1999) Trace element bioavailability as exemplified by iron and zinc.

Field Crops Research 60: 115-141

Ibrikci, H., Knewtson, S. J. B., Grusak, M. A. (2003) Chickpea leaves as a vegetable

green for humans: Evaluation of mineral composition. Journal of the Science

of Food and Agriculture 83: 945-950

Ihemere, U. E., Narayanan, N. N., Sayre, R. T. (2012) Iron biofortification and

homeostasis in transgenic cassava roots expressing the algal iron assimilatory

gene, FEA1. Frontiers in plant science 3

IMECHE (2013) Global Food - Waste not, Want not. In, Wales, England

Indurker, S., Misra, H. S., Eapen, S. (2010) Agrobacterium-mediated transformation

in chickpea (Cicer arietinum L.) with an insecticidal protein gene:

Optimisation of different factors. Physiology and Molecular Biology of

Plants 16: 273–284

Inoue, H., Higuchi, K., Takahashi, M., Nakanishi, H., Mori, S., Nishizawa, N. K.

(2003) Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and

OsNAS3 are expressed in cells involved in long-distance transport of iron and

differentially regulated by iron. The Plant Journal 36: 366-381

Inoue, H., Kobayashi, T., Nozoye, T., Takahashi, M., Kakei, Y., Suzuki, K.,

Nakazono, M., Nakanishi, H., Mori, S., Nishizawa, N. K. (2009) Rice

OsYSL15 is an iron-regulated iron (III)-deoxymugineic acid transporter

Bibliography 149

expressed in the roots and is essential for iron uptake in early growth of the

seedlings. Journal of Biological Chemistry 284: 3470-3479

Iqbal, A., Khalil, I. A., Ateeq, N., Sayyar Khan, M. (2006) Nutritional quality of

important food legumes. Food Chemistry 97: 331-335

Ishimaru, Y., Masuda, H., Bashir, K., Inoue, H., Tsukamoto, T., Takahashi, M.,

Nakanishi, H., Aoki, N., Hirose, T., Ohsugi, R., Nishizawa, N. K. (2010) Rice

metal-nicotianamine transporter, OsYSL2, is required for the long-distance

transport of iron and manganese. The Plant Journal 62: 379-390

Ishimaru, Y., Suzuki, M., Tsukamoto, T., Suzuki, K., Nakazono, M., Kobayashi, T.,

Wada, Y., Watanabe, S., Matsuhashi, S., Takahashi, M., Nakanishi, H., Mori,

S., Nishizawa, N. K. (2006) Rice plants take up iron as an Fe3+-

phytosiderophore and as Fe2+. The Plant Journal 45: 335-346

Itai, R. N., Ogo, Y., Kobayashi, T., Nakanishi, H., Nishizawa, N. K. (2013) Rice

genes involved in phytosiderophore biosynthesis are synchronously regulated

during the early stages of iron deficiency in roots. Rice 6: 1-13

Jambunathan, R., Singh, U. (1981) Studies on desi and kabuli chickpea (Cicer

arietinum L.) cultivars. 3. Mineral and trace elements composition. Journal of

Agricultural and Food Chemistry 29: 1091-1093

Jeong, J., Connolly, E. L. (2009) Iron uptake mechanisms in plants: Functions of the

FRO family of ferric reductases. Plant Science 176: 709-714

Jeong, J., Guerinot, M. L. (2009) Homing in on iron homeostasis in plants. Trends in

Plant Science 14: 280-285

Jin, C. W., He, Y. F., Tang, C. X., Wu, P., Zheng, S. J. (2006) Mechanisms of

microbially enhanced Fe acquisition in red clover (Trifolium pratense L.).

Plant, Cell & Environment 29: 888-897

Jin, C. W., You, G. Y., He, Y. F., Tang, C., Wu, P., Zheng, S. J. (2007) Iron

deficiency-induced secretion of phenolics facilitates the reutilization of root

apoplastic iron in red clover. Plant Physiology 144: 278-285

Jin, T., Zhou, J., Chen, J., Zhu, L., Zhao, Y., Huang, Y. (2013) The genetic

architecture of zinc and iron content in maize grains as revealed by QTL

mapping and meta-analysis. Breeding Science 63: 317–324

Johnson, A. A. T., Kyriacou, B., Callahan, D. L., Carruthers, L., Stangoulis, J.,

Lombi, E., Tester, M. (2011) Constitutive overexpression of the OsNAS gene

family reveals single-gene strategies for effective iron- and zinc-

biofortification of rice endosperm. PLoS ONE 6: e24476

Jones, D. L., Darah, P. R., Kochian, L. V. (1996) Critical evaluation of organic acid

mediated iron dissolution in the rhizosphere and its potential role in root iron

uptake. Plant and Soil 180: 57-66

Jukanti, A. K., Gaur, P. M., Gowda, C. L. L., Chibbar, R. N. (2012) Nutritional

quality and health benefits of chickpea (Cicer arietinum L.): A review.

British Journal of Nutrition 108: S11-S26

Käll, L., Krogh, A., Sonnhammer, E. L. L. (2004) A combined transmembrane

topology and signal peptide prediction method. Journal of Molecular Biology

338: 1027-1036

Kanazawa, K., Higuchi, K., Nishizawa, N.-K., Fushiya, S., Chino, M., Mori, S.

(1994) Nicotianamine aminotransferase activities are correlated to the

phytosiderophore secretions under Fe-deficient conditions in Gramineae.


150 Bibliography

Kar, S., Johnson, T. M., Nayak, P., Sen, S. K. (1996) Efficient transgenic plant

regeneration through Agrobacterium-mediated transformation of chickpea

(Cicer arietinum L.). Plant Cell Reports 16: 32-37

Kassebaum, N. J. (2016) The global burden of anemia. Hematology/Oncology

Clinics of North America 30: 247-308

Kataki, P. K. (2002) Shifts in cropping system and its effect on human nutrition:

Case study from India. Journal of Crop Production 6: 119-144

Katsvairo, L. (2015) Delivery of iron beans in Rwanda. In The 2nd Global

Conference on Biofortification: Getting Nutritious Foods to People, Kigali,

Rwanda

Kehrer, J. P. (2000) The Haber–Weiss reaction and mechanisms of toxicity.

Toxicology 149: 43-50

Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. E. (2015) The

Phyre2 web portal for protein modeling, prediction and analysis. Nature

Protocols 10: 845-858

Kennedy, E. T., Bouis, H. E. (1993) Linkages between agriculture and nutrition:

Implications for policy and research. International Food Policy Research

Institute, Washington, USA

Khan, M. A., Akhtar, N., Ullah, I., Jaffery, S. (1995) Nutritional evaluation of desi

and kabuli chickpeas and their products commonly consumed in Pakistan.

International journal of food sciences and nutrition 46: 215-223

Kim, S., Takahashi, M., Higuchi, K., Tsunoda, K., Nakanishi, H., Yoshimura, E.,

Mori, S., Nishizawa, N. K. (2005) Increased nicotianamine biosynthesis

confers enhanced tolerance of high levels of metals, in particular nickel, to

plants. Plant and Cell Physiology 46: 1809-1818

Kim, S. A., Guerinot, M. L. (2007) Mining iron: Iron uptake and transport in plants.

FEBS Letters 581: 2273-2280

Klatte, M., Schuler, M., Wirtz, M., Fink-Straube, C., Hell, R., Bauer, P. (2009) The

analysis of Arabidopsis nicotianamine synthase mutants reveals functions for

nicotianamine in seed iron loading and iron deficiency responses. Plant


Ko, M. P., Huang, P.-Y., Huang, J.-S., Barker, K. R. (1987) The occurrence of

phytoferritin and its relationship to effectiveness of soybean nodules. Plant


Kobayashi, T., Nakayama, Y., Itai, R. N., Nakanishi, H., Yoshihara, T., Mori, S.,

Nishizawa, N. K. (2003) Identification of novel cis-acting elements, IDE1

and IDE2, of the barley IDS2 gene promoter conferring iron-deficiency-

inducible, root-specific expression in heterogeneous tobacco plants. The Plant

Journal 36: 780-793

Kobayashi, T., Nishizawa, N. K. (2012) Iron uptake, translocation, and regulation in

higher plants. Annual Review of Plant Biology 63: 131-152

Kobayashi, T., Ogo, Y., Itai, R. N., Nakanishi, H., Takahashi, M., Mori, S.,

Nishizawa, N. K. (2007) The transcription factor IDEF1 regulates the

response to and tolerance of iron deficiency in plants. Proceedings of the

National Academy of Sciences of the United States of America 104: 19150-

19155

Kodkany, B. S., Bellad, R. M., Mahantshetti, N. S., Westcott, J. E., Krebs, N. F.,

Kemp, J. F., Hambidge, K. M. (2013) Biofortification of pearl millet with

iron and zinc in a randomized controlled trial increases absorption of these

Bibliography 151

minerals above physiologic requirements in young children. The Journal of

Nutrition 143: 1489-1493

Koen, E., Besson-Bard, A., Duc, C., Astier, J., Gravot, A., Richaud, P., Lamotte, O.,

Boucherez, J., Gaymard, F., Wendehenne, D. (2013) Arabidopsis thaliana

nicotianamine synthase 4 is required for proper response to iron deficiency

and to cadmium exposure. Plant Science 209: 1-11

Koike, S., Inoue, H., Mizuno, D., Takahashi, M., Nakanishi, H., Mori, S., Nishizawa,

N. K. (2004) OsYSL2 is a rice metal-nicotianamine transporter that is

regulated by iron and expressed in the phloem. The Plant Journal 39: 415-424

Kon, S., Sanchuck, D. W. (1981) Phytate content and its effect on cooking quality of

beans. Journal of Food Processing and Preservation 5: 169-178

Koressaar, T., Remm, M. (2007) Enhancements and modifications of primer design

program Primer3. Bioinformatics 23: 1289-1291

Krishnamurthy, K., Suhasini, K., Sagare, A., Meixner, M., De Kathen, A., Pickardt,

T., Schieder, O. (2000) Agrobacterium mediated transformation of chickpea

(Cicer arietinum L.) embryo axes. Plant Cell Reports 19: 235-240

Kumar, G. B. S., Srinivas, L., Ganapathi, T. R. (2011) Iron fortification of banana by

the expression of soybean ferritin. Biological Trace Element Research 142:

232-241

Lal, B. M., Prakash, V., Verma, S. C. (1963) The distribution of nutrients in the seed

parts of bengal gram. Experientia 19: 154-155

Larson, S. R., Rutger, J. N., Young, K. A., Raboy, V. (2000) Isolation and genetic

mapping of a non-lethal rice (Oryza sativa L.) low phytic acid 1 mutation.

Crop Science 40: 1397-1405

Larson, S. R., Young, K. A., Cook, A., Blake, T. K., Raboy, V. (1998) Linkage

mapping of two mutations that reduce phytic acid content of barley grain.

Theoretical and Applied Genetics 97: 141-146

Laulhere, J. P., Briat, J. F. (1993) Iron release and uptake by plant ferritin: Effects of

pH, reduction and chelation. Biochem. J. 290: 693-699

Lee, S., Jeon, U. S., Lee, S. J., Kim, Y.-K., Persson, D. P., Husted, S., Schjørring, J.

K., Kakei, Y., Masuda, H., Nishizawa, N. K., An, G. (2009) Iron fortification

of rice seeds through activation of the nicotianamine synthase gene.

Proceedings of the National Academy of Sciences 106: 22014-22019

Lee, S., Persson, D. P., Hansen, T. H., Husted, S., Schjoerring, J. K., Kim, Y.-S.,

Jeon, U. S., Kim, Y.-K., Kakei, Y., Masuda, H., Nishizawa, N. K., An, G.

(2011) Bio-available zinc in rice seeds is increased by activation tagging of

nicotianamine synthase. Plant Biotechnology Journal 9: 865-873

Li, L., Cheng, X., Ling, H.-q. (2004a) Isolation and characterization of Fe(III)-

chelate reductase gene LeFRO1 in tomato. Plant Molecular Biology 54: 125-

136

Li, L., Tang, C., Rengel, Z., Zhang, F. (2003) Chickpea facilitates phosphorus uptake

by intercropped wheat from an organic phosphorus source. Plant and Soil

248: 297-303

Li, S. M., Li, L., Zhang, F. S., Tang, C. (2004b) Acid phosphatase role in

chickpea/maize intercropping. Annals of Botany 94: 297-303

Lin, Y.-F., Liang, H.-M., Yang, S.-Y., Boch, A., Clemens, S., Chen, C.-C., Wu, J.-F.,

Huang, J.-L., Yeh, K.-C. (2009) Arabidopsis IRT3 is a zinc-regulated and

plasma membrane localized zinc/iron transporter. New Phytologist 182: 392-

404

152 Bibliography

Ling, H.-Q., Koch, G., Bäumlein, H., Ganal, M. W. (1999) Map-based cloning of

chloronerva, a gene involved in iron uptake of higher plants encoding

nicotianamine synthase. Proceedings of the National Academy of Sciences of

the United States of America 96: 7098-7103

Lobréaux, S., Hardy, T., Briat, J. F. (1993) Abscisic acid is involved in the iron-

induced synthesis of maize ferritin. The EMBO Journal 12: 651-657

Lolas, G., Palamidis, N., Markakis, P. (1976) The phytic acid-total phosphorus

relationship in barley, oats, soybeans, and wheat. Cereal Chemistry Journal

53: 867-871

Lönnerdal, B., Bryant, A., Liu, X., Theil, E. C. (2006) Iron absorption from soybean

ferritin in nonanemic women. The American Journal of Clinical Nutrition 83:

103-107

Lucena, C., Waters, B. M., Romera, F. J., Garcia, M. J., Morales, M., Alcantara, E.,

Perez-Vicente, R. (2006) Ethylene could influence ferric reductase, iron

transporter, and H+-ATPase gene expression by affecting FER (or FER-like)

gene activity. Journal of Experimental Botany 57: 4145-4154

Ma, J. F., Nomoto, K. (1993) Two related biosynthetic pathways of mugineic acids

in gramineous plants. Plant Physiology 102: 373-378

Mahmoudi, H., Labidi, N., Ksouri, R., Gharsalli, M., Abdelly, C. (2007) Differential

tolerance to iron deficiency of chickpea varieties and Fe resupply effects.

Comptes Rendus Biologies 330: 237-246

Maillard, A., Diquélou, S., Billard, V., Laîné, P., Garnica, M., Prudent, M., Garcia-

Mina, J.-M., Yvin, J.-C., Ourry, A. (2015) Leaf mineral nutrient

remobilization during leaf senescence and modulation by nutrient deficiency.

Frontiers in plant science 6: 317

Mari, S., Gendre, D., Pianelli, K., Ouerdane, L., Lobinski, R., Briat, J.-F., Lebrun,

M., Czernic, P. (2006) Root-to-shoot long-distance circulation of

nicotianamine and nicotianamine–nickel chelates in the metal

hyperaccumulator Thlaspi caerulescens. Journal of Experimental Botany 57:

4111-4122

Masuda, H., Aung, M., Nishizawa, N. (2013a) Iron biofortification of rice using

different transgenic approaches. Rice 6: 1-12

Masuda, H., Ishimaru, Y., Aung, M. S., Kobayshi, T., Kakei, Y., Takahashi, M.,

Kiguchi, K., Nakanishi, H., Nishizawa, N., K. (2012) Iron biofortification in

rice by the introduction of multiple genes involved in iron nutrition. Scientific

Reports 2

Masuda, H., Kobayshi, T., Ishimaru, Y., Takahashi, M., Aung, M. S., Nakanishi, H.,

Mori, S., Nishizawa, N. K. (2013b) Iron-biofortification in rice by the

introduction of three barley genes participated in mugineic acid biosynthesis

with soybean ferritin gene. Frontiers in plant science 4

Masuda, H., Usuda, K., Kobayashi, T., Ishimaru, Y., Kakei, Y., Takahashi, M.,

Higuchi, K., Nakanishi, H., Mori, S., Nishizawa, N. (2009) Overexpression of

the barley nicotianamine synthase gene HvNAS1 increases iron and zinc

concentrations in rice grains. Rice 2: 155-166

Matovu, M. (2016) Molecular and biochemical characterisation of transgenic banana

lines containing iron uptake and storage enhancing genes. PhD. Queensland

University of Technology

Medicago truncatula Gene Expression Atlas (2014). In. The Samuel Roberts Noble

Foundation

Bibliography 153

Meiners, C. R., Derise, N. L., Lau, H. C., Crews, M. G., Ritchey, S. J., Murphy, E.

W. (1976) The content of nine mineral elements in raw and cooked mature

dry legumes. Journal of Agricultural and Food Chemistry 24: 1126-1130

Mendoza, C., Viteri, F. E., Lönnerdal, B., Young, K. A., Raboy, V., Brown, K. H.

(1998) Effect of genetically modified, low-phytic acid maize on absorption of

iron from tortillas. The American Journal of Clinical Nutrition 68: 1123-1127

Mishra, S., Jha, S., Singh, R., Chaudhary, S., Sanyal, I., Amla, D. V. (2013)

Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor

(α1-PI) driven by a seed-specific promoters from the common bean Phaseolus

vulgaris (L.). Plant Cell, Tissue and Organ Culture (PCTOC) 115: 23-33

Mizuno, D., Higuchi, K., Sakamoto, T., Nakanishi, H., Mori, S., Nishizawa, N. K.

(2003) Three nicotianamine synthase genes isolated from maize are

differentially regulated by iron nutritional status. Plant Physiology 132: 1989-

1997

Mori, S., Nishizawa, N. (1989) Identification of barley chromosome no. 4, possible

encoder of genes of mugineic acid synthesis from 2′-deoxymugineic acid

using wheat-barley addition lines. Plant and Cell Physiology 30: 1057-1061

Mori, S., Nishizawa, N., Kawai, S., Sato, Y., Takagi, S. (1987) Dynamic state of

mugineic acid and analogous phytosiderophores in Fe‐deficient barley.

Journal of Plant Nutrition 10: 1003-1011

Mukherjee, I., Campbell, N. H., Ash, J. S., Connolly, E. L. (2006) Expression

profiling of the Arabidopsis ferric chelate reductase (FRO) gene family

reveals differential regulation by iron and copper. Planta 223: 1178-1190

Murata, Y., Ma, J. F., Yamaji, N., Ueno, D., Nomoto, K., Iwashita, T. (2006) A

specific transporter for iron(III)-phytosiderophore in barley roots. The Plant

Journal 46: 563-572

Murgia, I., Delledonne, M., Soave, C. (2002) Nitric oxide mediates iron-induced

ferritin accumulation in Arabidopsis. The Plant Journal 30: 521-528

Murray-Kolb, L. E., Welch, R., Theil, E. C., Beard, J. L. (2003) Women with low

iron stores absorb iron from soybeans. The American Journal of Clinical


Namanya, P. (2011) Towards the biofortification of banana fruit for enhanced

micronutrient content. PhD. Queensland University of Technology

Nelson, R. L., Yoo, S. J., Tanure, J. C., Andrianopoulos, G., Misumi, A. (1988) The

effect of iron on experimental colorectal carcinogenesis. Anticancer Res 9:

1477-1482

Nestel, P., Bouis, H. E., Meenakshi, J. V., Pfeiffer, W. (2006) Biofortification of

staple food crops. The Journal of Nutrition 136: 1064-1067

Nobile, C. G. M., Carreras, J., Grosso, R., Inga, M., Silva, M., Aguilar, R., Allende,

M. J., Badini, R., Martinez, M. J. (2013) Proximate composition and seed

lipid components of “kabuli”-type chickpea (Cicer arietinum L.) from

Argentina. Agricultural Sciences 4: 729-737

Norton, G. J., Deacon, C. M., Xiong, L., Huang, S., Meharg, A. A., Price, A. H.

(2010) Genetic mapping of the rice ionome in leaves and grain: Identification

of QTLs for 17 elements including arsenic, cadmium, iron and selenium.

Plant and Soil 329: 139-153

Norton, G. J., Douglas, A., Lahner, B., Yakubova, E., Guerinot, M. L., Pinson, S. R.,

Tarpley, L., Eizenga, G. C., McGrath, S. P., Zhao, F.-J. (2014) Genome wide

association mapping of grain arsenic, copper, molybdenum and zinc in rice

154 Bibliography

(Oryza sativa L.) grown at four international field sites. PLoS ONE 9:

e89685

Norton, R. (2013) Micronutrient Survey – GRDC Project Report #16, More Profit

from Crop Nutrition II. In, Horsham, Victoria, Australia

Nozoye, T., Nagasaka, S., Bashir, K., Takahashi, M., Kobayashi, T., Nakanishi, H.,

Nishizawa, N. K. (2014a) Nicotianamine synthase 2 localizes to the vesicles

of iron-deficient rice roots, and its mutation in the YXXφ or LL motif causes

the disruption of vesicle formation or movement in rice. The Plant Journal

77: 246-260

Nozoye, T., Nagasaka, S., Kobayashi, T., Takahashi, M., Sato, Y., Sato, Y., Uozumi,

N., Nakanishi, H., Nishizawa, N. K. (2011) Phytosiderophore efflux

transporters are crucial for iron acquisition in graminaceous plants. Journal of

Biological Chemistry 286: 5446-5454

Nozoye, T., Tsunoda, K., Nagasaka, S., Bashir, K., Takahashi, M., Kobayashi, T.,

Nakanishi, H., Nishizawa, N. K. (2014b) Rice nicotianamine synthase

localizes to particular vesicles for proper function. Plant Signalling and

Behaviour 9

O'Hara, G. W., Dilworth, M. J., Boonkerd, N., Parkpian, P. (1988) Iron-deficiency

specifically limits nodule development in peanut inoculated with

Bradyrhizobium sp. New Phytologist 108: 51-57

Ogo, Y., Itai, R. N., Kobayashi, T., Aung, M. S., Nakanishi, H., Nishizawa, N. K.

(2011) OsIRO2 is responsible for iron utilization in rice and improves growth

and yield in calcareous soil. Plant Molecular Biology 75: 593-605

Ogo, Y., Itai, R. N., Nakanishi, H., Inoue, H., Kobayashi, T., Suzuki, M., Takahashi,

M., Mori, S., Nishizawa, N. K. (2006) Isolation and characterization of IRO2,

a novel iron-regulated bHLH transcription factor in graminaceous plants.


Ogo, Y., Itai, R. N., Nakanishi, H., Kobayashi, T., Takahashi, M., Mori, S.,

Nishizawa, N. K. (2007) The rice bHLH protein OsIRO2 is an essential

regulator of the genes involved in Fe uptake under Fe-deficient conditions.

The Plant Journal 51: 366-377

Pande, S., Galloway, J., Gaur, P. M., Siddique, K. H. M., Tripathi, H. S., Taylor, P.,

MacLeod, M. W. J., Basandrai, A. K., Bakr, A., Joshi, S. (2006) Botrytis grey

mould of chickpea: A review of biology, epidemiology, and disease

management. Crop and Pasture Science 57: 1137-1150

Papanikolaou, G., Pantopoulos, K. (2005) Iron metabolism and toxicity. Toxicology

and Applied Pharmacology 202: 199-211

Peng, A. H., Liu, X. F., He, Y. R., Xu, L. Z., Lei, T. G., Yao, L. X., Cao, L., Chen, S.

C. (2015) Characterization of transgenic Poncirus trifoliata overexpressing

the ferric chelate reductase gene CjFRO2 from Citrus junos. Biologia

Plantarum 59: 654-660

Peters, N., Verma, D. (1990) Phenolic compounds as regulators of gene expression in

plant-microbe interactions. Molecular Plant-Microbe Interactions Journal 3:

4-8

Petry, N., Egli, I., Campion, B., Nielsen, E., Hurrell, R. (2013) Genetic reduction of

phytate in common bean (Phaseolus vulgaris L.) seeds increases iron

absorption in young women. The Journal of Nutrition 143: 1219-1224

Petry, N., Egli, I., Gahutu, J. B., Tugirimana, P. L., Boy, E., Hurrell, R. (2012) Stable

iron isotope studies in Rwandese women indicate that the common bean has

Bibliography 155

limited potential as a vehicle for iron biofortification. The Journal of


Petry, N., Egli, I., Gahutu, J. B., Tugirimana, P. L., Boy, E., Hurrell, R. (2014) Phytic

acid concentration influences iron bioavailability from biofortified beans in

Rwandese women with low iron status. The Journal of Nutrition 144: 1681-

1687

Petry, N., Rohner, F., Gahutu, J. B., Campion, B., Boy, E., Tugirimana, P. L.,

Zimmerman, M. B., Zwahlen, C., Wirth, J. P., Moretti, D. (2016) In

Rwandese women with low iron status, iron absorption from low-phytic acid

beans and biofortified beans is comparable, but low-phytic acid beans cause

adverse gastrointestinal symptoms. The Journal of Nutrition 146: 970-975

Petterson, D. S., Mackintosh, J. B. (1994) The chemical composition and nutritive

value of Australian grain legumes. In. Grains Research and Development

Corporation, Canberra, Australia

Phillips, R. D. (1993) Starchy legumes in human nutrition, health and culture. Plant

Foods for Human Nutrition 44: 195-211

Pianelli, K., Mari, S., Marquès, L., Lebrun, M., Czernic, P. (2005) Nicotianamine

over-accumulation confers resistance to nickel in Arabidopsis thaliana.

Transgenic Research 14: 739-748

Pich, A., Manteuffel, R., Hillmer, S., Scholz, G., Schmidt, W. (2001) Fe homeostasis

in plant cells: Does nicotianamine play multiple roles in the regulation of

cytoplasmic Fe concentration? Planta 213: 967-976

Pich, A., Scholz, G. (1993) The relationship between the activity of various iron-

containing and iron-free enzymes and the presence of nicotianamine in

tomato seedlings. Physiologia Plantarum 88: 172-178

Pich, A., Scholz, G. (1996) Translocation of copper and other micronutrients in

tomato plants (Lycopersicon esculentum Mill.): Nicotianamine-stimulated

copper transport in the xylem. Journal of Experimental Botany 47: 41-47

Pich, A., Scholz, G., Stephan, U. (1994) Iron-dependent changes of heavy metals,

nicotianamine, and citrate in different plant organs and in the xylem exudate

of two tomato genotypes. Nicotianamine as possible copper translocator.


Pietrangelo, A. (2003) Haemochromatosis. Gut 52: ii23-ii30

Pingali, P. L. (2012) Green Revolution: Impacts, limits, and the path ahead.

Proceedings of the National Academy of Sciences of the United States of

America 109: 12302-12308

Pinstrup‐Andersen, P., Hazell, P. B. (1985) The impact of the Green Revolution and

prospects for the future. Food Reviews International 1: 1-25

Platel, K., Srinivasan, K. (2016) Bioavailability of micronutrients from plant foods:

An update. Critical Reviews in Food Science and Nutrition 56: 1608-1619

Polowick, P. L., Baliski, D. S., Mahon, J. D. (2004) Agrobacterium tumefaciens-

mediated transformation of chickpea (Cicer arietinum L.): Gene integration,

expression and inheritance. Plant Cell Reports 23: 485-491

Poltronieri, F., Arêas, J. A. G., Colli, C. (2000) Extrusion and iron bioavailability in

chickpea (Cicer arietinum L.). Food Chemistry 70: 175-180

Pulse Australia (2016) Best Management Guide. Chickpea Production: Northern

Region

Pulse Canada (2016) Protein quality of cooked pulses (PDCAAS method)

156 Bibliography

Qu, L. Q., Yoshihara, T., Ooyama, A., Goto, F., Takaiwa, F. (2005) Iron

accumulation does not parallel the high expression level of ferritin in

transgenic rice seeds. Planta 222: 225-233

Queensland Department of Agriculture and Fisheries (2015) Varieties of chickpea

Rabotti, G., Zocchi, G. (1994) Plasma membrane-bound H+-ATPase and reductase

activities in Fe-deficient cucumber roots. Physiologia Plantarum 90: 779-785

Raboy, V., Gerbasi, P. F., Young, K. A., Stoneberg, S. D., Pickett, S. G., Bauman, A.

T., Murthy, P. P. N., Sheridan, W. F., Ertl, D. S. (2000) Origin and seed

phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant


Rao, M., Willey, R. (1980) Evaluation of yield stability in intercropping: Studies on

sorghum/pigeonpea. Experimental Agriculture 16: 105-116

Rao, P. P., Birthal, P., Bhagavatula, S., Bantilan, M. (2010) Chickpea and pigeonpea

economies in Asia: Facts, trends and outlook. In. International Crops

Research Institute for the Semi-Arid Tropics, Patancheru

Ravet, K., Touraine, B., Boucherez, J., Briat, J.-F., Gaymard, F., Cellier, F. (2009)

Ferritins control interaction between iron homeostasis and oxidative stress in

Arabidopsis. The Plant Journal 57: 400-412

Ravindran, V., Ravindran, G., Sivalogan, S. (1994) Total and phytate phosphorus

contents of various foods and feedstuffs of plant origin. Food Chemistry 50:

133-136

Ray, H., Bett, K., Tar’an, B., Vandenberg, A., Thavarajah, D., Warkentin, T. (2014)

Mineral micronutrient content of cultivars of field pea, chickpea, common

bean, and lentil grown in Saskatchewan, Canada. Crop Science 54: 1698-

1708

Rincón, F., Martínez, B., Ibáñez, M. V. (1998) Proximate composition and

antinutritive substances in chickpea (Cicer arietinum L) as affected by the

biotype factor. Journal of the Science of Food and Agriculture 78: 382-388

Rizkalla, S. W., Bellisle, F., Slama, G. (2002) Health benefits of low glycaemic

index foods, such as pulses, in diabetic patients and healthy individuals.

British Journal of Nutrition 88: 255-262

Robinson, N. J., Groom, S. J., Groom, Q. J. (1997) The froh gene family from

Arabidopsis thaliana: Putative iron-chelate reductases. Plant and Soil 196:

245-248

Robinson, N. J., Procter, C. M., Connolly, E. L., Guerinot, M. L. (1999) A ferric-

chelate reductase for iron uptake from soils. Nature 397: 694-697

Rochfort, S., Panozzo, J. (2007) Phytochemicals for health, the role of pulses.

Journal of Agricultural and Food Chemistry 55: 7981-7994

Römheld, V., Marschner, H. (1981) Iron deficiency stress induced morphological

and physiological changes in root tips of sunflower. Physiologia Plantarum

53: 354-360

Römheld, V., Marschner, H. (1983) Mechanism of iron uptake by peanut plants: I.

FeIII reduction, chelate splitting, and release of phenolics. Plant Physiology

71: 949-954

Römheld, V., Marschner, H. (1986) Evidence for a specific uptake system for iron

phytosiderophores in roots of grasses. Plant Physiology 80: 175-180

Roy, F., Boye, J. I., Simpson, B. K. (2010) Bioactive proteins and peptides in pulse

crops: Pea, chickpea and lentil. Food Research International 43: 432-442

Bibliography 157

Sagare, A. P., Suhasini, K., Krishnamurthy, K. V. (1995) Histology of somatic

embryo initiation and development in chickpea (Cicer arietinum L.). Plant

Science 109: 87-93

San Martin, C. D., Garri, C., Pizarro, F., Walter, T., Theil, E. C., Núñez, M. T.

(2008) Caco-2 intestinal epithelial cells absorb soybean ferritin by μ2 (AP2)-

dependent endocytosis. The Journal of Nutrition 138: 659-666

Sánchez-Rodríguez, A. R., del Campillo, M. C., Torrent, J., Jones, D. L. (2014)

Organic acids alleviate iron chlorosis in chickpea grown on two P-fertilized

soils. Journal of soil science and plant nutrition 14: 292-303

Sandberg, A. S., Carlsson, N. G., Svanberg, U. (1989) Effects of inositol tri‐, tetra‐, penta‐, and hexaphosphates on in vitro estimation of iron availability. Journal

of Food Science 54: 159-161

Santi, S., Cesco, S., Varanini, Z., Pinton, R. (2005) Two plasma membrane H+-

ATPase genes are differentially expressed in iron-deficient cucumber plants.

Plant Physiology and Biochemistry 43: 287-292

Santi, S., Schmidt, W. (2009) Dissecting iron deficiency‐induced proton extrusion in

Arabidopsis roots. New Phytologist 183: 1072-1084

Sanyal, I., Singh, A. K., Kaushik, M., Amla, D. V. (2005) Agrobacterium-mediated

transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis

cry1Ac gene for resistance against pod borer insect Helicoverpa armigera.

Plant Science 168: 1135-1146

Sarker, R., Ferdous, T., Hoque, M. (2005) In vitro direct regeneration of three

indigenous chickpea (Cicer arietinum L.) varieties of Bangladesh. Plant

Tissue Culture & Biotechnology 15: 135-144

Sarmah, B., Moore, A., Tate, W., Molvig, L., Morton, R., Rees, D., Chiaiese, P.,

Chrispeels, M., Tabe, L., Higgins, T. J. V. (2004) Transgenic chickpea seeds

expressing high levels of a bean α-amylase inhibitor. Molecular Breeding 14:

73-82

Saxena, M. C. (1987) Agronomy of chickpea. In Saxena, M. C., Singh, K. B., eds,

The Chickpea. CAB International, Wallingford, pp 207-232

Scholz, G., Becker, R., Pich, A., Stephan, U. W. (1992) Nicotianamine ‐ A common

constituent of strategies I and II of iron acquisition by plants: A review.

Journal of Plant Nutrition 15: 1647-1665

Schuler, M., Bauer, P. (2011) Heavy metals need assistance: The contribution of

nicotianamine to metal circulation throughout the plant and the Arabidopsis

NAS gene family. Frontiers in plant science 2

Schuler, M., Rellán-Álvarez, R., Fink-Straube, C., Abadía, J., Bauer, P. (2012a)

Nicotianamine functions in the phloem-based transport of iron to sink organs,

in pollen development and pollen tube growth in Arabidopsis. The Plant Cell

Online 24: 2380-2400

Schuler, M., Rellán-Álvarez, R., Fink-Straube, C., Abadía, J., Bauer, P. (2012b)

Nicotianamine functions in the phloem-based transport of iron to sink organs,

in pollen development and pollen tube growth in Arabidopsis. The Plant Cell

24: 2380-2400

Schwertmann, U. (1991) Solubility and dissolution of iron oxides. Plant and Soil

130: 1-25

Sebastiá, V., Barberá, R., Farré, R., Lagarda, M. J. (2001) Effects of legume

processing on calcium, iron and zinc contents and dialysabilities. Journal of

the Science of Food and Agriculture 81: 1180-1185

158 Bibliography

Seckback, J. (1982) Ferreting out the secrets of plant ferritin ‐ A review. Journal of

Plant Nutrition 5: 369-394

Senthil, G., Williamson, B., Dinkins, R. D., Ramsay, G. (2004) An efficient

transformation system for chickpea (Cicer arietinum L.). Plant Cell Reports

23: 297-303

Sharma, R. D. (1986) Phytate and the epidemiology of heart disease, renal calculi

and colon cancer. Phytic Acid Chemistry and Applications: 161-172

Sharma, S. S., Dietz, K.-J. (2006) The significance of amino acids and amino acid-

derived molecules in plant responses and adaptation to heavy metal stress.


Shi, R., Weber, G., Köster, J., Reza-Hajirezaei, M., Zou, C., Zhang, F., von Wirén,

N. (2012) Senescence-induced iron mobilization in source leaves of barley

(Hordeum vulgare) plants. New Phytologist 195: 372-383

Sievenpiper, J. L., Kendall, C. W. C., Esfahani, A., Wong, J. M. W., Carleton, A. J.,

Jiang, H. Y., Bazinet, R. P., Vidgen, E., Jenkins, D. J. A. (2009) Effect of

non-oil-seed pulses on glycaemic control: A systematic review and meta-

analysis of randomised controlled experimental trials in people with and

without diabetes. Diabetologia 52: 1479-1495

Sperotto, R. A., Ricachenevsky, F. K., Waldow, V. d. A., Fett, J. P. (2012) Iron

biofortification in rice: It's a long way to the top. Plant Science 190: 24-39

Stephan, U., Schmidke, I., Pich, A. (1994a) Phloem translocation of Fe, Cu, Mn, and

Zn in Ricinus seedlings in relation to the concentrations of nicotianamine, an

endogenous chelator of divalent metal ions, in different seedling parts. Plant

and Soil 165: 181-188

Stephan, U. W., Grün, M. (1989) Physiological disorders of the nicotianamine-

auxothroph tomato mutant chloronerva at different levels of iron nutrition II.

Iron deficiency response and heavy metal metabolism. Biochemie und

Physiologie der Pflanzen 185: 189-200

Stephan, U. W., Schmidke, I., Pich, A. (1994b) Phloem translocation of Fe, Cu, Mn,

and Zn in Ricinus seedlings in relation to the concentrations of nicotianamine,

an endogenous chelator of divalent metal ions, in different seedling parts.


Stephan, U. W., Schmidke, I., Stephan, V. W., Scholz, G. (1996) The nicotianamine

molecule is made-to-measure for complexation of metal micronutrients in

plants. BioMetals 9: 84-90

Stephan, U. W., Scholz, G. (1990) Nicotianamine concentrations in iron sufficient

and iron deficient sunflower and barley roots. Journal of Plant Physiology

136: 631-634

Stephan, U. W., Scholz, G. (1993) Nicotianamine: Mediator of transport of iron and

heavy metals in the phloem? Physiologia Plantarum 88: 522-529

Strozycki, P. M., Szczurek, A., Lotocka, B., Figlerowicz, M., Legocki, A. B. (2007)

Ferritins and nodulation in Lupinus luteus: Iron management in indeterminate

type nodules. Journal of Experimental Botany 58: 3145-3153

Takahashi, M., Terada, Y., Nakai, I., Nakanishi, H., Yoshimura, E., Mori, S.,

Nishizawa, N. K. (2003a) Role of nicotianamine in the intracellular delivery

of metals and plant reproductive development. The Plant Cell Online 15:

1263-1280

Takahashi, M., Terada, Y., Nakai, I., Nakanishi, H., Yoshimura, E., Mori, S.,

Nishizawa, N. K. (2003b) Role of nicotianamine in the intracellular delivery

of metals and plant reproductive development. The Plant Cell 15: 1263-1280

Bibliography 159

Takahashi, M., Yamaguchi, H., Nakanishi, H., Shioiri, T., Nishizawa, N. K., Mori, S.

(1999) Cloning two genes for nicotianamine aminotransferase, a critical

enzyme in iron acquisition (Strategy II) in graminaceous plants. Plant


Tanaka, A., Navasero, S. A. (1966) Interaction between iron and manganese in the

rice plant. Soil Science and Plant Nutrition 12: 29-33

Tang, C., Robson, A. D., Dilworth, M. J. (1990) The role of iron in nodulation and

nitrogen fixation in Lupinus angustifolius L. New Phytologist 114: 173-182

Tewari-Singh, N., Sen, J., Kiesecker, H., Reddy, V. S., Jacobsen, H. J., Guha-

Mukherjee, S. (2004) Use of a herbicide or lysine plus threonine for non-

antibiotic selection of transgenic chickpea. Plant Cell Reports 22: 576-583

Thavarajah, D., Thavarajah, P. (2012) Evaluation of chickpea (Cicer arietinum L.)

micronutrient composition: Biofortification opportunities to combat global

micronutrient malnutrition. Food Research International 49: 99-104

Thavarajah, D., Thavarajah, P., Gupta, D. S. (2014) Pulses biofortification in

genomic era: Multidisciplinary opportunities and challenges. In Gupta, S.,

Nadarajan, N., Gupta, S. D., eds, Legumes in the Omic Era. Springer New

York, New York, NY, pp 207-220

Thomas, C. E., Aust, S. D. (1986) Reductive release of iron from ferritin by cation

free radicals of paraquat and other bipyridyls. Journal of Biological

Chemistry 261: 13064-13070

Thomine, S., Lanquar, V. (2011) Iron transport and signaling in plants. In Geisler,

M., Venema, K., eds, Transporters and Pumps in Plant Signaling. Springer

Berlin Heidelberg, Berlin, Heidelberg, pp 99-131

Thomson, D., Henry, R. (1995) Single-step protocol for preparation of plant tissue

for analysis by PCR. Biotechniques 19: 394-397

Tiffin, L. O., Chaney, R., Ambler, J. (1973) Translocation of iron from soybean

cotyledons. Plant Physiology 52: 393-396

Trick, H. N., Finer, J. J. (1997) SAAT: Sonication-assisted Agrobacterium-mediated

transformation. Transgenic Research 6: 329-336

Trick, H. N., Finer, J. J. (1998) Sonication-assisted Agrobacterium-mediated

transformation of soybean [Glycine max (L.) Merrill] embryogenic

suspension culture tissue. Plant Cell Reports 17: 482-488

Trijatmiko, K. R., Dueñas, C., Tsakirpaloglou, N., Torrizo, L., Arines, F. M., Adeva,

C., Balindong, J., Oliva, N., Sapasap, M. V., Borrero, J., Rey, J., Francisco,

P., Nelson, A., Nakanishi, H., Lombi, E., Tako, E., Glahn, R. P., Stangoulis,

J., Chadha-Mohanty, P., Johnson, A. A. T., Tohme, J., Barry, G., Slamet-

Loedin, I. H. (2016) Biofortified indica rice attains iron and zinc nutrition

dietary targets in the field. Scientific Reports 6

Tripathi, L., Singh, A., Singh, S., Singh, R., Chaudhary, S., Sanyal, I., Amla, D. V.

(2013) Optimization of regeneration and Agrobacterium-mediated

transformation of immature cotyledons of chickpea (Cicer arietinum L.).

Plant Cell, Tissue and Organ Culture (PCTOC) 113: 513-527

Trumbo, P., Yates, A. A., Schlicker, S., Poos, M. (2001) Dietary reference intakes:

Vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron,

manganese, molybdenum, nickel, silicon, vanadium, and zinc. Journal of the

American Dietetic Association 101: 294-301

Tsednee, M., Yang, S.-C., Lee, D.-C., Yeh, K.-C. (2014) Root-secreted

nicotianamine from Arabidopsis halleri facilitates zinc hypertolerance by

regulating zinc bioavailability. Plant Physiology 166: 839-852

160 Bibliography

Twyman, E. S. (1951) The iron and manganese requirements of plants. New

Phytologist 50: 210-226

Umaid, U., Manohar, S., Singh, A. (1984) The anatomical structure of desi and

kabuli chickpea seed coats. International Chickpea Newsletter 10: 26-27

United Nations (2015) World population prospects, the 2015 revision. In. United

Nations Department of Economic and Social Affairs

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M.,

Rozen, S. G. (2012) Primer3 - New capabilities and interfaces. Nucleic acids

research 40: e115

Upadhyaya, H. D., Bajaj, D., Das, S., Kumar, V., Gowda, C. L. L., Sharma, S.,

Tyagi, A. K., Parida, S. K. (2016) Genetic dissection of seed-iron and zinc

concentrations in chickpea. Scientific Reports 6: 24050

USDA Basic Report: 16056, Chickpeas (garbanzo beans, bengal gram), mature

seeds, raw

USDA (2013). In National Nutrient Database for Standard Reference,

Van Wuytswinkel, O., Vansuyt, G., Grignon, N., Fourcroy, P., Briat, J.-F. (1999)

Iron homeostasis alteration in transgenic tobacco overexpressing ferritin. The

Plant Journal 17: 93-97

Varotto, C., Maiwald, D., Pesaresi, P., Jahns, P., Salamini, F., Leister, D. (2002) The

metal ion transporter IRT1 is necessary for iron homeostasis and efficient

photosynthesis in Arabidopsis thaliana. The Plant Journal 31: 589-599

Varshney, R. K., Song, C., Saxena, R. K., Azam, S., Yu, S., Sharpe, A. G., Cannon,

S., Baek, J., Rosen, B. D., Tar'an, B., Millan, T., Zhang, X., Ramsay, L. D.,

Iwata, A., Wang, Y., Nelson, W., Farmer, A. D., Gaur, P. M., Soderlund, C.,

Penmetsa, R. V., Xu, C., Bharti, A. K., He, W., Winter, P., Zhao, S., Hane, J.

K., Carrasquilla-Garcia, N., Condie, J. A., Upadhyaya, H. D., Luo, M.-C.,

Thudi, M., Gowda, C. L. L., Singh, N. P., Lichtenzveig, J., Gali, K. K.,

Rubio, J., Nadarajan, N., Dolezel, J., Bansal, K. C., Xu, X., Edwards, D.,

Zhang, G., Kahl, G., Gil, J., Singh, K. B., Datta, S. K., Jackson, S. A., Wang,

J., Cook, D. R. (2013) Draft genome sequence of chickpea (Cicer arietinum)

provides a resource for trait improvement. Nature Biotechnology 31: 240–

246

Vasconcelos, M., Datta, K., Oliva, N., Khalekuzzaman, M., Torrizo, L., Krishnan, S.,

Oliveira, M., Goto, F., Datta, S. K. (2003) Enhanced iron and zinc

accumulation in transgenic rice with the ferritin gene. Plant Science 164:

371-378

Vert, G., Barberon, M., Zelazny, E., Séguéla, M., Briat, J.-F., Curie, C. (2009)

Arabidopsis IRT2 cooperates with the high-affinity iron uptake system to

maintain iron homeostasis in root epidermal cells. Planta 229: 1171-1179

Vert, G., Grotz, N., Favienne, D., Gaymard, F., et al. (2002) IRT1, and Arabidopsis

transporter essential for iron uptake from the soil and for plant growth. The

Plant Cell 14: 1223-1233

Vert, G. A., Briat, J.-F., Curie, C. (2003) Dual regulation of the Arabidopsis high-

affinity root iron uptake system by local and long-distance signals. Plant


von Wirén, N., Khodr, H., Hider, R. C. (2000) Hydroxylated phytosiderophore

species possess an enhanced chelate stability and affinity for iron(III). Plant


Bibliography 161

von Wirén, N., Klair, S., Bansal, S., Briat, J.-F., Khodr, H., Shioiri, T., Leigh, R. A.,

Hider, R. C. (1999) Nicotianamine chelates both FeIII and FeII. Implications

for metal transport in plants. Plant Physiology 119: 1107-1114

Vucenik, I., Shamsuddin, A. M. (2003) Cancer inhibition by inositol hexaphosphate

(IP6) and inositol: From laboratory to clinic. The Journal of Nutrition 133:

3778S-3784S

Walter, A., Pich, A., Scholz, G., Marschner, H., Römheld, V. (1995) Diurnal

variations in release of phytosiderophores and in concentrations of

phytosiderophores and nicotianamine in roots and shoots of barley. Journal of

Plant Physiology 147: 191-196

Waters, B. M., Blevins, D. G., Eide, D. J. (2002) Characterization of FRO1, a pea

ferric-chelate reductase involved in root iron acquisition. Plant Physiology

129: 85-94

Waters, B. M., Troupe, G. C. (2012) Natural variation in iron use efficiency and

mineral remobilization in cucumber (Cucumis sativus). Plant and Soil 352:

185-197

Waters, B. M., Uauy, C., Dubcovsky, J., Grusak, M. A. (2009) Wheat (Triticum

aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen

compounds from vegetative tissues to grain. Journal of Experimental Botany

60: 4263-4274

Weber, M., Harada, E., Vess, C., Roepenack-Lahaye, E. v., Clemens, S. (2004)

Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis

halleri roots identifies nicotianamine synthase, a ZIP transporter and other

genes as potential metal hyperaccumulation factors. The Plant Journal 37:

269-281

Wei, L. C., Loeppert, R. H., Ocumpaugh, W. R. (1997) Fe-deficiency stress response

in Fe-deficiency resistant and susceptible subterranean clover: Importance of

induced H+ release. Journal of Experimental Botany 48: 239-246

Wells, H. F. (2016) Vegetables and pulses yearbook data. In. USDA, USA

WHO (2001) Iron deficiency anaemia: Assessment, prevention and control: A guide

for programme managers. In. Geneva : World Health Organization

WHO (2008) Worldwide prevalence of anaemia 1993-2005. World Health

Organisation

WHO (2016) Biofortification of staple crops

Wirth, J., Poletti, S., Aeschlimann, B., Yakandawala, N., Drosse, B., Osorio, S.,

Tohge, T., Fernie, A. R., Günther, D., Gruissem, W., Sautter, C. (2009) Rice

endosperm iron biofortification by targeted and synergistic action of

nicotianamine synthase and ferritin. Plant Biotechnology Journal 7: 631-644

Wood, J. A., Grusak, M. A. (2007) Nutritional value of chickpea. In Yadav, S. S.,

Redden, R. J., Chen, W., Sharma, B., eds, Chickpea breeding and

management. CAB International, pp 101-142

Wood, J. A., Knights, E. J., Choct, M. (2011) Morphology of chickpea seeds (Cicer

arietinum L.): Comparison of desi and kabuli types. International Journal of

Plant Sciences 172: 632-643

Wu, H., Li, L., Du, J., Yuan, Y., Cheng, X., Ling, H.-Q. (2005) Molecular and

biochemical characterization of the Fe (III) chelate reductase gene family in

Arabidopsis thaliana. Plant and Cell Physiology 46: 1505-1514

Xing, J., Wang, T., Ni, Z. (2015) Epigenetic regulation of iron homeostasis in

Arabidopsis. Plant Signalling and Behaviour 10: e1064574

162 Bibliography

Xu, Y., An, D., Liu, D., Zhang, A., Xu, H., Li, B. (2012) Molecular mapping of

QTLs for grain zinc, iron and protein concentration of wheat across two

environments. Field Crops Research 138: 57-62

Yadav, S. S., Longnecker, N., Dusunceli, F., Bejiga, G., Yadav, M., Rizvi, A. H.,

Manohar, M., Reddy, A. A., Xaxiao, Z., Chen, W. (2007) Uses, consumption

and utilisation. In Yadav, S. S., Redden, R. J., Chen, W., Sharma, B., eds,

Chickpea breeding and management. CAB International, pp 72-100

Yuan, Y., Wu, H., Wang, N., Li, J., Zhao, W., Du, J., Wang, D., Ling, H. Q. (2008)

FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene

expression for iron homeostasis in Arabidopsis. Cell Res 18: 385-397

Zheng, L., Cheng, Z., Ai, C., Jiang, X., Bei, X., Zheng, Y., Glahn, R. P., Welch, R.

M., Miller, D. D., Lei, X. G., Shou, H. (2010) Nicotianamine, a novel

enhancer of rice iron bioavailability to humans. PLoS ONE 5: e10190

Zhou, M.-L., Qi, L.-P., Pang, J.-F., Zhang, Q., Lei, Z., Tang, Y.-X., Zhu, X.-M.,

Shao, J.-R., Wu, Y.-M. (2013a) Nicotianamine synthase gene family as

central components in heavy metal and phytohormone response in maize.

Functional & Integrative Genomics 13: 229-239

Zhou, X., Li, S., Zhao, Q., Liu, X., Zhang, S., Sun, C., Fan, Y., Zhang, C., Chen, R.

(2013b) Genome-wide identification, classification and expression profiling

of nicotianamine synthase (NAS) gene family in maize. BMC Genomics 14:

238

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