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THE ROLE OF ALTERNATIVE PATHWAY RESPLRATION IN PLANT CELLS GROWING UNDER PHOSPHORUS LIMITATrON:
A S m Y USINO TRANSGENIC NICOTIANA TABACUM CELLS LACKING THE ALTERNATIVE OXIDASE
Hannah Leah Parsons
A thesis submitted in confonnity with the requirements for the degree of Master of Science
Graduate Department of Botany University of Toronto
O Copyright by Hannah Leah Parsons 1998
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The role of alternative pathway respiration in plant cells growing under phosphorus limitation:
a study using transgenic Nicotiana tabacum cells lacking the alternative oxidase.
Hannah Leah Parsons Master of Science
Department of Botany University of Toronto
Abstract
Plants and heterotrophic suspension cells of Nicofiana tabacum were used to
study the role of the mitochonàrial alternative oxidase (AOX) during respiration under
phosphate (P) limited growth conditions. A transgenic line of suspension cells (ASB),
expressing an antisense construct of the AOX gene, was also used. Growth of wild type
(WT) plants and suspension cells under P-limitation induced AOX protein and activity as
determined by gel blot and oxygen electrode analysis. This induction did not occur in
AS8 cells. 1) Thus, WT and AS8 cells were compared to evaluate what role AOX may
play during P-limitation. 2) The lack of AOX induction in AS8 resulted in a lower
respiration rate d u h g P-limitation. 3) Also, increased rates of cellular hydrogen peroxide
generation in AS8 indicated that components of the mitochoncûiai electron transport
chah were more resmcted. 4) 1 discuss the potentiai role of the non-phosphorylating
AOX pathway in allowing respiration to continue under P-limitation, a condition in
which adenylate restriction might otherwise become severe.
ii
Acknowledpents
It has been a packed two years. My seams are bursting fiom the amount of
knowledge 1 have gained fiom my thne with Dr. Greg Vaderberghe, who has k e n an
excellent supervisor. 1 thank him for his mong direction, interest and highly organized
laboratory. Justine Yip, as part of the lab, has been a good fnend and labmate and 1 wish
her the best in life. 1 thank my supe~sory cornmittee, Dr. Dan Riggs and Dr. Czesia
Nalwajko. 1 would also like to thank Dr. Eduardo Blumwdd and Dr. Dan Riggs for use of
laboratory equiprnent, their students for help over several months of experirnentation and
Pamela Noldner for help using the fluorescent probe.
Outside the lab and sometimes inside, 1 have appreciated aad enjoyed t h e spent
with many of the Botany Department graduate students, including Carolyn Hutcheon and
Jacki Wolfe. Long live canoe trips in Algonquin, skiing in Quebec, Botany soccer and
Hamish MacBeth. Carolyn, as a fellow West Coaster and fiiend, helped immensely in
adapting to Toronto and the east and 1 look forward to our funue adventures. My final
thanks and love go to my family and Thael Hill for their letters, phone calls and visits.
iii
Contents
C ha p ter 1 - General Introduction: Plant Respiration and the Alternative Oxidase. (section 1.0) --~----------------------------.Io-L-..-~L-------I-..-œ--------------- 1
List of Figures
Figure Page
Figure 1.1: Diagram of plant respiratory pathways; glycolysis, the TCA cycle
and the elecbon m s p o e chh.----------------------. 2
Figure 1.2: Diagram of plant mitochondrial electron transport chah.---------------- 5
Figure 1.3: Diagram of AOX structure with regdatory features.------------- 9
Figure 1.4: Diagram of regulatory factors controlling AOX activity-------------- 12
Figure 2.1: Diagram of oxygen electrode analysis of respiratory characteristics
of suspension cells. ----------------------------I..-I---I---------~.~~- 21
Figure 3.1 : Representative wild type shoot growth curve.-----------------------O-- 35
Figure 3.2: Representative wild type root growth curve.--------------------------..--- 37
Figure 3.3: Leaf total phosphorus content during wild type plant growth.------- 39
Figure 3.4: Leaf inorganic phosphoms content during wild type plant growth.------- -42
Figure 3.5: Immunoblot analysis of AOX protein in wild type shoots.---------- 4
Fipre 4.1 : Diagram of plant respiratory pathways under phosphorus limited
conditions; glycolysis, the TCA cycle and the mitochondrial electron
transport ~hain.-----------------------~-------------~-----------------~---~~-~--. 50
Figure 4.2: Growth of wild type suspension tells,---------------------..-------- 53
Figure 4.3: Ce11 total phosphorus content of wild type suspension celis.-------- 56
Figure 4.4: Ce11 inorganic phosphorus content of wild type suspension cel1s.----- 58
Figure 4.5: Respiratory capacity of wild type suspension ce1ls.----------------------- 61
Figure 4.6: Alternative oxidase capacity of wild type suspension ce1ls.----------- 63
Figure 4.7: Immunoblot adysis of AOX protein in wild type suspension ce1ls.------- 66
Fipre 4.8: Alternative oxidase capacity during manipulation of sucrose supply . . in wld type suspension tells.-------------------------------- 68
Figure 5.1: Diagram of arnino acid synthesis nom respiratory substrates.------- 74
Figure 5.2: Growth of wild type and antisense suspension cells (dry weight;
protein).---------~--------------~--..------~----iII..-------H-------II- 78
vii
viii
List of Appendices
Appendix A- Suspension Cell Growth
Appendk B Suspension Ce11 Respirab y Characteristics
B-3 Figure of respiratory rates of wild type and antisense suspension cells on &y 3
d e r subcult~e.--------------------~H~--~HNI--~----HH--H- 1 58
B-4 Table of respiratory capacities of wild type and antisense suspension ce1ls.---- 159
B-5 Table of unpaired t-test P-values for comparisons of respiratory capacities of wild
type and antisense suspension tells.------------------------------------- 160
B-6 Figure of respiratory capacities of wild type and antisense suspension cells on
&y 3 d e r su~~~e.----------------~--~---------------------------------- 161
B-7 Table of alternative oxidase capacities (measured using KCN) of wild type and
antisense suspension cells,---------------~*-~-------œ--.H--U-----------------o- 162
B-8 Table of unpaired t-test P-values for comparisons of alternative oxidase capacities
(measured using KCN) of wild type and antisense suspension cells. -------------O- 163
B-9 Figure of alternative oxidase capacities (measured using KCN) of wild type and
antisense suspension cells on day 3 after subculture.------------------------------- 164
B-10 Table of alternative oxidase capacities (measured using sodium azide) of
wild type and &sense suspension cells.--------------v----*--I--I---- 1 65
B-11 Table of unpaired t-test P-values for comparisons of alternative oxidase capacities
(measured using sodium azide) of wild type and antisense suspension cel1s.----166
B-12 Figure of alternative oxidase capacities (measured using sodium &de) of
wild type and antisew suspension cells on day 3 d e r subcu1ture.-------- 167
B-13 Figure of alternative oxidase capacities (measured using sodium &de) of
wild type and antisense suspension cells on &y 5 after subcu1ture.--------- 168
Appendix C- Suspension Ce11 Amho Acid Composition
C-1 Table of free amino acid composition of wild type and antisense suspension cells.169
C-2 Table of t-test P-values for comparisons of fiee amino acid composition of wild
type a d antisense suspension tells.----------------------- 170
Appendix D- Suspension CeU E202 Produdon and Morphology
List of Abbreviations
-o-----o---"H1--W~-~-~---~---HII--~-~-LI--------~~~ nitrogen
xii
Chaoter 1- General Introduction: Plant Res~iration and Aiternative
Oxidase.
Plant Respiration
Aerobic respiration, a process found in ail plants, involves the controlled
oxidation of metabolites containhg reduced carbon to produce carbon dioxide and water
(Taiz and Zeiger, 1991). Aerobic respiration is an important source of ATP used in
energy-requiring reactions for plant growth and development. Carbohydrate (CH20) is the
most common substrate used by plant tissues for respiration although fatty acids, organic
acids and amino acids cm also serve as substrate. The bbcontrolled oxidation" of
carbohydrate is accomplished through three biochemical pathways (Fig. 1.1): glycolysis,
the tricarboxylic acid (TCA) or Kreb's cycle and the mitochondrial electron transport
chah
Glycolysis occurs in the cytosol and generates a net yield of 4 moles of ATP and 4
moles of NADH per mole of sucrose consumed. NADH can be oxidized in the
mitochondrion thus M e r contributhg to ATP production through coupled oxidative
phosphorylation. in its most simple form, the h a i product of glycolysis is pynivate. This
three carbon compound can cross the outer and inner mitochondrial membrane and enter
the TCA cycle.
The enzymes of the TCA cycle are located in the mitochondrial matrix. Plant
mitochondna are small organelles (0.5 pm x 1-2 pm) enclosed by two lipid bilayers
(Newcomb, 1997). Between the outer and imer mitochondnal membrane is an
intermembrane space. Within the inner mitochondnal membrane is the mitochondnal
matrix space. The TCA cycle produces carbon dioxide as well as intemediates important
in other metabolic pathways such as amino acid synthesis (Hill, 1997). A limited amount
of ATP (one mole ATP per mole pynivate oxidized) and the electron donors NADH (4
moles per mole pynivate) and FADH2 (one mole per mole pyruvate) are also formed in
the TCA cycle (Salisbury and Ross, 1978). NADH and FADH2 cm be oxidued by the
mitochondnal electron transport chab to produce ATP via coupled oxidative
phosphorylation.
Figure 1.1: An outline of the primary respiratory pathways in plants: glycolysis, the
ûicarboxylic acid (TCA) cycle and the electron transport chah (modified fiom
Theodorou and Plaxton, 1993). For simplicity, not al1 reactions or electron transport chah
components have been included. Abbreviatioas: fni-6-P (hctose 6-phosphate), fni 1,6-
bisP (fhctose 1,6-bisphosphate), g-3-P (glyceraldehyde 3-phosphate), PEP
(phosphoenolpynivate). For o k abbreviations see List of Abbreviations.
(inner membrane)
The mitochondrial electron transport chah components are located within the
inner mitochondrial membrane (Fig. 1.2). In the simplest fonn of electron transport,
electrons (e.g. donated by NADH and FADH2) enter the electron transport chah through
complex 1 (NADH dehydrogenase complex) (Heldt, 1997). At this site there is the
potential for ATP production via generation of a proton gradient. Protons moved from the
matrix to the intermembrane space, during reductiocdoxidation of complex 1, generate a
proton potential. ATP formation is dnven by the proton-motive force in coupled
oxidative phosphorylation. Cornplex I is the first of a series of sites in the mitochondrial
electron transport chah that are sequentially reduced and oxidized to generate a proton
motive force. From complex 1, electrons are passed to the ubiquinone pool.
Complex II (not shown in Fig. 1.2) oxidizes succinate (fkom the TCA cycle) thus
contributing electrons to the ubiquinone pool. However, complex II is not c o ~ e c t e d to
translocation of protons across the inner mitochondrid membrane.
Electroos fiom the ubiquinone pool proceed ultimately to one of two terminal
oxidases in the electron transport c h a h cytochrome oxidase and alternative oxidase.
Acceptance of electrons by complex iIi (cytochrome b/ct complex) of the
cytochrome pathway is part of coupled oxidative phosphorylation. Complex III is the
second site in the mitochondrial electron transport ch& that contributes to the generation
of a proton potential. The final site contributing to generation of a proton potential is
complex N (cytochrome ah3 complex or the cytochrome oxidase, COX) which is the
terminal oxidase. This complex accepts electrons from complex III via cytochrome c.
Extrusion of protons by COX is coupled to ATP generation and reduction of molecular
oxygen to water. Most of the ATP derived fiom respiration is produced in the
mitochondna by coupled oxidative phosphorylation (Lambers, 1997b).
In contrast, acceptance of electrons fiom the ubiquinone pool by alternative
oxidase (AOX) bypasses two sites of ATP generation, complex III and IV. The alternative
(cyanide-resistant) oxidase retains the ability to reduce molecular oxygen to water (like
cytochrome oxidase), however, it does not contribute to generation of a proton potential
Figure 1.2: Plant mitochondrial electron traasport chah (modified fiom Labers,
1997b). Sites of stimulation or inhibition by various compounds are Uidicated. 1-complex
1, U-ubiquinone, AOX-alternative oxidase, III-complex IiI, cyt c-cytochrome c, N-
cornplex IV (COXcytochrome oxidase), FCCP-carbonyl cyanide p(tûifluoromethoxy)
phenyihydrazone, myxo-myxothiazol, AA-antimycin A, KCN-cyanide, &de-sodium
aide, SHAM-salicylhydroxamic acid, H'-protons. For other abbreviations used see List
of Abbreviations.
FCC
water
and ATP. The difference in ATP production between the cytochrorne and the alternative
pathway has k e n the driving force behind characterization of the alternative oxidase.
Except in the case of thermogenic infiuorescences in voodoo liiy (Meeuse, 1975), the
f'unction of AOX remaias largely imknown.
Occurrence of AOX
The phenomenon of alternative pathway respiration has intrigwd plant scientists
since the beginning of this cenhny (Lambers, 1997a). Al1 higher plants as well as some
protists, fungi and algae contain an alternative, cyanide-resistant pathway in the inner
membrane of their mitochondna (Vanlerberghe and Mcintosh, 1997). Some fùngi which
have been characterized as having alternative pathway respiration include Neurospora
crassa and Hansenula anornola. The green alga Chlumydomonas also contains this
respiratory pathway (Moore and Siedow, 1991). In higher plants, members of the
Araceae such as Sauromattum guttatum (voodoo lily) use high rates of AOX respiration
during flowe~g/anthesis to volatize aromatic compounds which attract pollinators
(Meeuse, 1975). Besides this very specialized fûnction, the occurrence of AOX in al1
higher plants is suggestive of an important general function to the plant kingdom. This
has been M e r supported by the finding that the AOX gene sequence is highly
conserved amongst diverse plant species (Mcintosh, 1994).
Molecular Biology of AOX
AOX is encoded by a nuclear gene (Mcintosh, 1994). AOX cDNA clones have
been isolated fiom Sauromatum guttufum (Rhoads and Mchtosh, 1991). Arabidopsis
thdiana (Kumar and Soll, 1992), Glycine m a L. (Whelan et al., L996a), H. anomala
(Sakajo et al., 1991) and N. tabacum (Vanlerberghe and Mchtosh, 1994). The plant
sequences show high similarity, sharîng a cleavable mitochondrial targeting presequence
and some conserved regions which are possibly involved in metai binding (Lambers,
1997a). However, the sequence of the yeast cDNA is quite dissimilar. In S. guttatum, the
sequence for the genomic clone contains four exons (coding regions of the gene)
separated by introns (non-coding regions) encoding the alternative oxidase gene (Rhoads
and Mchtosh, 1993). Exon 3 encodes the proposed a-helical regions of the mitochondrial
inner membrane spanning protein.
The Aoxl gene of S. guttatum encodes a 35 kDa to 37 kDa protein as detemiiwd
by immunoblots probed with a monoclonal antibody raised against S. guttatum alternative
oxidase (Rhoads and Mchtosh, 1993). However, a similar study also in S. guttatum
observed a 42 kDa product of Aoxl (Elthon et al., 1989a). in A. thaliana, an AOX gene
(cloned by complimenthg an E. coli mutant deficient in cytochrome mediated
respiration) had a 3 1 kDa product ( b a r and Soll, 1992). These examples of variation in
AOX protein size could be due to: variation between species, multiple AOX genes,
dflerences in antibody affinity andor posttranslationd modifications.
It is now apparent that there are multiple alternative oxidase genes in several
species (multi-gene families). Differential expression of these genes have k e n observed.
Whelan et al. (1 996b), using PCR techniques, observed differential expression of Aoxl-3
between cotyledons and leaves in soybean. Li et al. (1996) cloned two genes in
Neurospora crassa (aod-1, structural and aud-2, regulatory) that are both required for
alternative oxidase activity. in tobacco, iwo cDNA clones have been identified by
researchers fiom different cultivars leading to the suggestion that tobacco plants contain
more than one alternative oxidase gene (Whelan et al., 1995b).
Affuiity of the AOX gene products to the commonly used monoclonal antibody
raised against S. guttatum can Vary fiom species to species despite the ability of this
antibody to cross react quite readily (Elthon et al,. 1989a). This may lead to variation in
the number of bands observed on hmunoblots.
Posttranslational processing a d o r modification of the Aox gene transcript cm
lead to the appearance of 2 or 3 AOX proteins in immunoblots (Wagner and Krab, 1995;
Vanierberghe and Mchtosh, 1997).
Structure of AOX
AOX is proposed to contain three a-helical regions in its structure (Fig. 1.3). Two
of the helices may be membrane spoianing while the third rnay be a d a c e helix in the
intermembrane space. Apparently common to ail AOX proteins are N- and C-temiinal
Figure 1.3: Structural mode1 of AOX with regulatory feahires (modifïed 60m
Vanlerberghe et ai., 1998). The AOX dimer, when covaiently linked by an intermolecular
d i s a d e bond at cys 126 (-S-S-), is a less active fom of AOX. Reduction of this
regulatory disulfide to its component sulfbydryls (-SH HS-) produces the more active
fom. The protein exists as a dimer regardless of whether the disulfide bond in present or
not present (Umbach and Siedow, 1993). The mechanism of reduction of the regulatory
disulfide may be mediated by a thioredoxin systern requiring NADPH. The reduced more
active form is more responsive to stimulation by pyruvate. The mechanism of stimulation
by pyruvate remah largely unknown. AOX features are not h w n to scale.
hydrophilic regions. The presence of six absolutely conse~ed histidine residues
encouraged speculation that ùon is a metal cofactor (Moore and Siedow 1991, Siedow et
al 1995). Two conserved cysteine residues have been proposed to be involved in AOX
regulation (Umbach et al., 1994). The conserved cys 126 residue in tobacco AOX has
been identified to be involved in disulfide linkage of the AOX monomers (Vanlerberghe
et al., 1998).
Replation of AOX
Control of respiratory electron partitionhg between the cytochrome pathway and
the non-energy conserving alternative pathway is only partially understood. AOX activity
can be stimuiated at a number of levels (Fig. 1.4). (1) Increases in the level of alternative
oxidase protein are determined by regdation of gene expression. Stimuli may upregulate
transcription of the AOX gene to increase AOX activity. Once the AOX protein is
incorporated into the inner mitochondrial membrane, its activity is then M e r regulated.
(2) The occurrence of alternative oxidase in its more active monomer form is determined
by the redox state of the mitochonària. Umbach and Siedow (1993) used soybean to show
that AOX exists as either a covaiently or non-covalently linked dimer. Reduction of the
AOX dimer covalent bond to monomers leaves the more active protein susceptible to
M e r (allosteric) activation by pynivate (Umbach et al., 1994). (3) The ailostenc
activation of the reduced AOX monomer is determined by the level of AOX activators
(dependent on carbon metabolism). Certain a-keto acids (e.g. pyruvate) may interact with
a cys sdfhdryl to activate the AOX monomer (Vanlerberghe et al., 1998). (4) Finally,
AOX activity is dependent on the electron supply which is detemined by the level of
reduced ubiquinone. Stimuli that affect AOX activity can be directed at one or more of
these levels of regulation. Study of the stimuli and their effect on AOX activity may lead
to a better understanding of the possible bction(s) of alternative pathway respiration.
Figure 1.4: Su- of major factors that may control partitionhg of respiratory
electrons to the non-energy consenhg alternative pathway in higher plant mitochondria
(fiom Vanlerberghe and McIntosh, 1997). Some factors "coarsely" control the amount of
AOX protein whereas others "fioely" control the activity of AOX once it has been
incoprated into the innet mitochondrial membrane.
gene expression
level of AOX protein
AOX Aetivity . activators . of <-)
Levei of reduced ubiquinaie
electron transport
level of ' reduced (active* fom of AOX
mitoc hondrial reQx state
Function of AOX
AOX induction by stress stimuli such as cold stress, pathogen attack and nutrient
limitation have al1 been reported (Lambers, 1997a). This also includes conditions that
restrict electron flow through the electron transpott chah Until recently, the widely held
view was that the alternative pathway acts as an overflow of the cytochrome pathway
only becoming engaged when the cytochrome pathway was inhibited or saturated with
electrons (Lambers, 1997a). Recent hdings indicate that the alternative pathway cm
share electrons, donated by their cornmon substrate, ubiquinol, with the cytochrome
pathway (Hoefhagel et al., 1995) even when the cytochrome pathway is not saturated.
Other research has indicated a variety of metabolic conditions can lead to saturation of the
cytochtome pathway and "electton overflow" in addition to excess carbohydrate that has
traditionally been thought to lead to electron overfiow of the cytochrome pathway.
Downstream products of carbohydrate oxidation in the glycolytic pathway and TCA cycle
such as pynivate, citrate and other organic acids are likely to accumulate if their rate of
production is not matched by oxidation in the mitochondria. Accumulation of TCA cycle
intermediates is likely to affect the reduction of the AOX (Vanlerberghe et al., 1995) and,
therefore, its activity. It has been suggested that the continuous oxidation of substrate via
a non-phosphorylating electron transport pathway allows the plant to increase the
availability of carbon for sinks which suddenly arise (Lambers, 1985).
The significance of an energy overflow via the alternative pathway may dso be in
preventing production of harmfbl levels of superoxide andor hydrogen peroxide should
electron flow become restricted (Wagner and Moore, 1997; Vanlerberghe and Mchtosh,
1997). It has been recognized that adenylate supply could play a vital role in the ability of
the cytochrome pathway to accept electrons. Decreases in adenylate supply could lead to
a decrease in the capacity of the cytochrome pathway to accept electrons. Under these
conditions, the activity of the alternative pathway may increase to allow continued
electron flow through the mitochondnal electron transport chah (and prevent oxidative
damage). Phosphorus (P) limitation is a stress stimuli that has been hypothesized to create
just such metabolic conditions. During P limitation, pools of adenylates decrease
(Theodorou and Plaxton, 1993) resuiting in decreased cytochrome pathway capacity. The
adenylate non-requiring activity of AOX could then become important in aliowing
continued electron flow.
The tenninology sunouncihg the study of AOX has recently become problematic.
Measurement of AOX activity was formerly determined (solely) through the use of
inhibitors due to the general acceptance of the electron overflow paradigm. Under this
model, AOX engagement, Le. a measurement of AOX activity under ambient conditions,
could be determined by inhibition of AOX (SHAM) then cytochrome oxidase (KCN,
myxtothiazol, sodium azide). AOX capacity, Le. a measurement of maximum AOX
activity, could be determined by inhibition of cytochrome oxidase then AOX (a reversal
of inhibitor addition). Observation of the sharing of electrons by the alternative and
cytochrome pathways under non-saturathg conditions has made the measurement of
alternative oxidase engagement by inhibitors questionable. However, determination of
alternative oxidase capacity by inhibitors remains a useful tool. Therefore, the terni AOX
activity has been used in this study as inclusive of both AOX engagement and AOX
capacity measurements.
The oxygen isotope fiactionation method has emerged as a usefbl tool in
measurement of alternative oxidase engagement (Guy et al., 1 989). Oxygen isotope
fiactionation avoids the use of inhibitors by taking advantage of the observation that
cytochrorne oxidase and alternative oxidase have different affinities for "O when
reducing oxygen to water. This fiactionation c m be measured with a mass spectrometer
in isolated mitochondria and intact tissues. Measurement of AOX activity by this method
appears to provide the f ~ s t ûue measurements of AOX engagement.
Generation of transgenic tobacco plants and suspension cells with antisense
constnicts of tobacco Aoxl by Vanlerberghe et al. (1994) were an important tool in our
study of AOX activity during P limitation. Severe adenylate restriction of respiratory
pathways during P limitation appears to be alleviated by increases in AOX activity.
Objectives:
1) To determine whether growth of tobacco plants and suspension cells under low P
conditions results in an induction of the AOX respiration pathway, as has been noted in
previous studies with other plant species.
2) To determine whether AOX induction by low P conditions would be suppressed in
transgenic tobacco suspenison cells contalliing an antisense AOX gene.
3) To determine the consequences to the plant ce11 of being unable to induce the AOX
respiration pathway under low P conditions.
Cha~ter 2 Materials and Methods
2.1 Plant Materials and Growth Conditions
2.1.1 Suspension Ceii Cultures
Vanlerberghe et ai. (1994) used antisense consûucts of the tobacco Aoxl gene to
generate a transgenic tobacco (Nicotiana tabacum cv Petit Havana SRI) line (AS8) with
decreased levels of AOX protein. From wild type and AS8 tobacco plants, suspension ce11
cultures were generated and are now approaching 5 years in culture. These suspension
ce11 cultures have been used in my study and the AS8 celis are abbreviated as AS. They
were grown on a Innova 4300 incubator shaker (New Brunswick Scientific, Edison, NJ,
USA) at 2S°C, 140 rpm. Axenic conditions were maintallied for al1 procedures. Every 7
days, 15 ml of the week old cells were subcultured to 200 ml of k s h , autoclaved
medium in a 500 ml Erlenmeyer flask.
Cells were grown in a standard Murishige and Skoog medium (Linsmaier and
Skoog, 1965) containhg MS salts [2 1 rnM W N 0 3 , 19 mM W03, 1.5 m M MgS04, 1 13
pM MnS04, 37 pM ZnS04, 0.1 pM CuS04, 3 mM CaC12,S pM KI, 0.1 1 pM CoC12, 2.57
mM KH2P04, 100 phi H3B03, 1 pM Na2Mo04, 100 pM FeS04, LOO pM sodium
ethylenediaminetetra-acetic acid (EDTA)], 1.5 pM thiarninehydrochloride, 1 pM 2,4-
dichlorophenoxyacetic acid, 555 pM myo-inositol and 3% [wlv] sucrose. The pH was
adjusted to 5.7 with KOH before autoclave sterilization of the medium. AS suspension
cells were aiways grown in medium supplemented with sterile kanamycin (75 pg active
Kn/ml), added just prior to the culture period.
The above complete (C) medium contains 2500 pM KH2P04, 21 m M W N 0 3
and 19 m M IWO3. A Ion phosphorus (LowP) medium was prepared to contain 250 pli4
KH2P04 or one tenth the concentration of phosphorus in the complete medium. In
phosphorus add back experiments, the low P medium was mpplemented with complete
medium levels of phosphorus. In sorne experiments, a low aitrogen (LowN) medium
was prepared to contain 700 pM NH4N03, 633 pM KN03 and supplemented with KCl to
maintain complete medium potassium levels. Sucrose manipulations of the medium
were also conducted for a few experiments, in the range of 1% to 6% [wlv] sucrose, in
combination with complete and low phosphorus medium.
2.1.2 Plants
Wild type Nicotiana tabacum (cv Petit Havana SRI) plants were grown in
magenta boxes for up to 40 days. Surface sterilized wild type tobacco seeds (surface
sterilized as described below) were sown in magenta boxes on 100 ml [w/v] 0.7%
phytagar containing a standard Murashige and Skoog medium (Linsmaier and Skoog,
1965): MS salts [21 mM NH4N03, 19 m M IWO3, 1.5 m M MgS04, 1 13 pM MnS04, 37
pM ZnSQ, 0.1 pM CuS04, 3 m M CaC12, 5 FM KI, 0.1 1 ph4 CoC12, 100 pM &BO3,
1pM Na2Mo04, 100 pM FeS04, 100 pM sodium ethylene-diaminetetra-acetic acid
(EDTA)], 3% [wh] sucrose, 8.1 pM nicotinic acid, 4.9 pM pyridoxine-HCI, 29.6 pM
thiamine-HCl, and 0.001% [w/v] myo-inositol. The level of phosphorus in the medium
was altered afler autoclaving via addition of filter sterilized phosphate (M2P04) stock,
before solidification of the agar. The complete medium provided 1.25 mM KH2P04. The
low phosphorus medium contained 0.05 m M KH2POs. A phosphonw add back (+P)
txeatment to low P grown plants was made by addition of sterile K&P04 solution to the
surface of solidified medium to bring phosphate concentration up to complete medium
levels (1 -25 m M m2Po4).
Seeds (approximately 50 pl volume) were surface sterilized in a sterile eppendod
tube. A solution of 30% bleach, 0.1% SDS (500 pl) was allowed to sit for 5-10 min
(occasionally agitated) with the seeds. Removal of the bleach1SDS solution was done by
three dH20 washes of the seeds. The seeds were resuspended in 500 pl sterile dH20 and a
sterile loop was used to spread out five seeds per box. Afier 7 days the seedlings were
thinned to three per box. Plants were grown under continuous fluorescent light at
approximately 28OC in the closed magenta box.
2.3 Phosphoms Determination
A modified Ames (1966) methoci was used to determine total and inorga.uk
phosphate content of fkeze dried suspension cells and oven-dried shoots obtained as
described in section 4.6.1. Total phosphorus refers to both free and bound phosphorus. In
this case an ashing step cleaves phosphate fiom organic molecules. Inorganic phosphorus
refen to oniy the fiee phosphorus which was often an important component of the total
phosphorus.
Toiid phosphorus determinution used 1.5-2.5 mg of lyophilized cells or oven-
dried shoots. Duplicate samples were tested. The material was placed in glas culture
tubes (1 0x75 mm) with 100 pl 10% [wlv] Mg(N03)2 and ashed for 15 s in a flarne. The
ashes were ailowed to cool briefly before addition of 300 pl 0.5 N HCI. This mixture was
vortexed for 15 s and then covered tubes were boiled for 15 min. During the boiling step,
loose-fitting glass stoppers covered the tubes in order to prevent volume changes due to
condensation or evaporation. Nonetheless, any change in volume was noted and corrected
for. The liquid was transferred to an eppendorf tube and centrifuged for 10 minutes to
remove ashed particles. The supernatant was then assayed for phosphorus. First, the
phosphorus extnict was diluted 25-fold with 0.5 N HCl. Then 3 parts extract was aàded to
7 parts reaction solution. The reaction solution was a mixture of I part 10% [w/v]
ascorbic acid (made the day of use) to 6 parts molybdate solution (0.42% [wlv] -0,
28.6 ml [vlv] H2S04 in àH20). The mixture was incubated 20 min at 45' C to allow
colour formation. This was followed by measurements of absorbance ot 830 nm using a
diode array spectrophotometer (Hewlett Packard 8453). A standard curve was made using
0,25,50, 100, 150 pM KH2P04 in 0.5 N HCl.
Inoganic phspIIiocus ddermination required 1.5-2.5 mg starting material
(fieezedried suspension cells or ovendned shoots) to which was added 350 pl of 1%
[v/v] acetic acid. This mixture was vortexed for 10 s and dowed to sit for 5 min.
Particdate matter was removed by a 5 min centrifugation at 16 000 g.
An appropriate dilution (7-18 fold) of the extract with 1% acetic acid was
necessary before mixture of 3 parts extract to 7 parts reaction solution (as previously
described). This mixture was incubated 20 min at 45' C and then absorbance at 830 nm
was determineci using a diode array spectrophotometer (Hewlett Packard 8453). The
standard curve was made using 0,25,50, 100, 150 pM KH2P04 in 1% acetic acid.
2.5 Respiratory Anrlysis
Oxygn EZecmde Analysis of Suspension Cells. Respiratory characteristics of
suspension cells were analyzed using a Clark-type oxygen electrode at 28 O C (Hansatech
Ltd., England). It was assumed that the oxygen concentration in air-saturated water at
28 O C was 253 pM as caiculated.
Suspension cells were diluted to 0.5-1.5 mg DW * ml-' in their growth medium
before 1 ml of the suspension culture was placed in the oxygen electrode chamber. An
initial respiration rate (a) was established within a few minutes as illustrated in Fig. 2.1.
Addition of 1 pM carbonyl cyanide p-(üifluoromethoxy)phenyIhydrazone (FCCP), an
uncoupler of oxidative phosphorylation which makes the inner mitochondrial membrane
permeable to protons (Fig. 1.2), imrnediately established a new rate, respiratory capacity
(b), which was followed for a few minutes. Addition of either cyanide (1 m M KCN) or
sodium &de (10 mM N d 3 ) (inhibitors of complex iV or cytochrome oxidase) resulted
in the establishment of a new rate (c). A fuiai addition of 2 mM salicyihydroxamic acid
(SHAM) inhibited AOX. This residual rate (d) was followed for several minutes. The
residual rate had a range of 0-8 % of rate b. Alternative oxidase capacity was detemiuied
by a subtraction (rate c-rate d). In total, establishment of rates a-d took 10-1 5 min.
Figure 2.1: An idealized trace of oxygen consumption by suspension cells in the chamber
of an oxygen electrode comected to a chart recorder. Relevant respiratory characteristics
inchde control rate of oxygen uptake (rate a), respiratory capacity (rate b), AOX capacity
(rate cd) and residuai oxygen uptake (rate d).
Sites of stimulation or inhibition by FCCP, KCNy NaN3 and SHAM are indicated
in Fig. 1.2. In chapter 3 (section 3.0), we used N a 3 to inhibit the cytochrome pathway
whereas in chapter 4 (section 4.0) we used KCN. I found that the use of KCN to inhibit
the cytochrome pathway was preferable because it established a new steady-state rate
much more quickly than Na&. 1 fouad that the degree of inhibition of oxygen uptake by
NaNs increased with thne indicating that some non-specific effects might have k e n
taking place. Rates of oxygen consumption were expressed on a dry weight basis (nrnol
O2 * min-' * mgo1 DW) and on a protein basis ( m o l O2 * mi18 * mg'' protein). Dry
weight was determined as described in section 2.6.1. Protein content was detemiined as
described in section 2.6.2.
2.4 Mitochondrial Isolations
2.4.1 Suspension Cell Cultures
Washed mitochondna were isolated fiom suspension ce11 cultures as described in
Vanlerberghe and McIntosh (1992). Al1 steps were performed on ice and al1
centrifugation was done at 4' C. Mitochondria were isolated fiom cultures at 3, 4 and 5
days after subculture. In the earlier days, and especially under nutrient limitation, larger
volumes had to be used for a single isolation in order to obtain enough material. The
range of volumes required was 1-3 flash (200-600 ml). Cells were gently pelleted at 1
800 g and washed twice with 200 ml ice cold growth medium (section 2.1.1). The cells
were then added to 300 ml ice cold grinding medium (350 rnM mannitol, 30 mM MOI'S,
1 mM EDTA, pH 7.5 and added just prior to use 0.2 % [wh] BSA, 0.6 % [w/v] PVPP,
0.126 % [w/v] L-cysteine) in a blender and homogenized for 2 X 3 S. The homogenate
was fdtered through 4 layers of cheesecloth and centrifuged 2 min at 5 050 g. The
supematant was then centrifuged 5 min at 20 201 g and the resulting pellets were
resuspended in 160 mls washing medium (300 mM mannitol, 20 m M MOPS, 1 m .
EDTA-Na2, pH 7.2 and 0.1% [w/v] BSA added just prior to use) and centrifiiged 2 min at
5 050 g. The supematant was centrifuged 5 min at 20 201 g and this final pellet was
resuspended in 500 pl resuspension medium (250 m M sucrose, 30 mM MOPS, pH 6.8)
for storage at -80°C.
2.4.2 Plants
Mitochondria were isolated h m tobacco leaves usiag a miniprep method
adapted fiom Bout~y et al. (1984). Al1 steps were perfonned on ice and ail centrifbgation
was done at 4 O C. Leaves (1 g fresh weight) were ground for 20 s in a chilled mortar and
pestle with 2 g sand and 10 mls homogenization medium (0.3 M sucrose, 50 m M T ~ s , pH
7.6, 1 mM ethylene-dioxy-diethyl-enedinitrilo-tes acid (EGTA), 10 mM KH2P04,
0.2% [w/v] bovine semm albumin (BSA), 0.6% [w/v] polyvlnylpolypyrrolidone (PVPP)
and 0.4% [wfv] 2-mercaptoethanol). BSA, PVPP and 2-mercaptoethanol were added just
prior to use.
The homogenate was filtered through four layers of Miracloth (Calbiochem, La
Jolla, USA) in a 60 cc disposable syringe. A 30 s centrifugation at 3 000 g (4°C) removed
larger particles and the supernatant was then centrifuged at 26 895 g for 10 min to pellet
the mitochondna. The pellet was gently resuspended using a brush in 250 pl suspension
buffer (0.4 M mannitol, 10 m M KH2PO4, 0.2% BSA, pH 7.2, the BSA was added just
prior to use) and laid over a Percoll gradient in an eppendorf tube. The Percoll gradient
was made using Percoll gradient buffets that contain 0.25 M sucrose, 0.2% BSA and 13-
50% Percoll. The bottom Iayer of the gradient was 250 pl of the 50% Percoll buffer. The
second layer was 500 p1 26% Percoll buffer gently pipetted on top of the fint layer
followed by 250 pl 13.5% Percoll buffer placed on top of the previous two layers. The
250 p1 of resuspended rnitochondria in suspension buffet was gently pipeîied ont0 the top
of these three layers.
A centrifugation at 16 000 g for 15 min at 4OC causd the mitochondria to
accumulate at the interface of the 50% and 26% Percoll while chloroplasts remained at
the interface of the 26 % and 13.5 % Percoll. Approximately 200 pL of the concentnited
mitochondria were removed using a 1.0 cc disposable syringe with a bent tip and the
PercoWmitochondria were then diluted with approximateiy 1 ml suspension buffer. A
finai centrifiigation was performed for 20 min at 4OC of 16 000 g. Most of the supernatant
was removed by aspiration and the pellet was resuspended in the rernaining suspension
b&er (50 pi) and fiozen at -80°C.
In some cases, isolations in the presence of 5 mM pyruvate were perfomed. Al1
solutions described for the mitochondna mini preparations were supplemented prior to
use with 5 rnM pyruvate @repas& on the day of the isolation).
Mitochoncûial protein was quantified as described in section 2.63 and aaalyzed
as described in section 2.5.
2.5 Protein Analysis of Mitochoadria
Separution of Milochondriril Proteins. Equivaient amounts of protein were
analyzed. For reducing SDS-PAGE, 100-200 pg of isolated mitochondrial protein were
prepared in dH20 and combined with sample bufler (6% [wh] SDS, 6% [v/v] 2-
mercaptoethanol, 30% [vh] glycerol, 125 mM Tris, pH 6.8) in a 1 to 2 ratio. Non-
reducing SDS-PAGE was performed by omitting 2-mercaptoethanol fiom the sample
b&er and replacing it with an equal amount of dH20. The sample was boiled for 2 min
(to denature the proteins), cooled briefly on ice and rnixed with 0.08% [wh]
bromophenol blue tracking dye. The sample was centrifbged at 16 000 g for 2 min. The
SDS-PAGE analysis was perfomed with a SE 600 electrophoresis unit (Hoefer
Pharxnacia Biotech, San Francisco, USA) and the b d e r system of L a e d i (1970). The
running bufTer contained 0.025 M Tris, pH 8.3,0.192 M glycine, 0.1% SDS. A 5% [w/v]
polyacrylamide stacking gel and a 1047.5% polyacrylamide gradient resolving gel were
used. The prepared sample was loaded into the gel lanes and the system was comected to
a power supply. Mit0chondria.i proteins were separated by a constant cunent of 25 rnA for
-6 ht or 15 mA overnight. Separation was considered complete upon ninning off of dye
fkont fiom the gel.
Transfer of Mifochondriai Proteins to NitrocelUulse The resolved proteins were
tramferrd fiom the gel to nitrocellulose using a TE 50X electro-transfer unit (Hoefer
Pharmica Biotech, San Francisco, USA) and Towbin buffw (25 m M Tris-base, pH 8.3,
192 m M glycine, 20% methanol). M e r transfer ovemight at 0.1 mA, the Western blot
was washed 2X 15 min in PBS-Tween (10 m M NaH2P04, 150 m M NaCl, 0.3% Tween 20,
a pinch of NaN3) and allowed to dry ovemight.
Imniunoblof Anabsis. The Western blot was then incubated for 1 hr in PBS-
Tween containing a 1:200 dilution of a monoclonal antibody (AOA) raised against
Sauromatun guttatum AOX (Elthon et al., 1989). Then, after 2x5 min washes with PBS-
Tween, the Western blot was incubated with PBS-Tween containing a 1 :2000 dilution of
phosphatase labeled aanity purified antibody to mouse IgG(H+L) (Kirkegard and Perry
Laboratories, Gaithersburg, USA). This was followed by 2x5 min washes in PBS-Tween
and 2x5 min washes in Colour buffer-Tween (100 m M Tris, pH 9.5, 100 rnM NaCl, 5
rnM MgCl*, 0.3% Tween 20). The western blot was developed by reaction with Colour
buffer -Tween containing 0.4 mM p-nitroblue tetrazolium choride (nBT) and 0.4 m M
5-bromo-4-chloro-indolyl phosphate (BCIP). The colour development was stopped by
2x5 min washes in PBS-Tween.
Additional primary antibodies that were used in the study included an antibody
raised against yeast subunit II of COX (obtained from Dr. A. Tzagaloff, Columbia
University, New York, NY) and an antibody (386) raised against tobacco catalase 2 (a
gift fiom Daniel Klessig, Rutgers University, Waksman institute, New Jersey).
Irnrnunoblot analysis with the COX antibody followed the protocol for AOX immunoblot
analysis except a 1: 500 dilution of the primary antibody in PBS-Tween was used in the
initial incubation. Immunoblot andysis with the catalase antibody followed the AOX
protocol except for a 1 : 10 000 dilution of the primary antibody in PBS-Tween and a 3 hr
incubation of the blot with the primary antibody.
2.6 D y Weight and Protein Meagurement
2.6.1 D y Weight Measunment
Suspension tell cultures Samples for dry weight growth curves were taken daily.
Cells in culture (4 mi) were removed from their flask and placed in a preweighed
centrifuge tube. The sample was centrifbged for 5 min at 1 870 g at 4OC. Then the
supematant was removed and the cells were washed twice with 10 ml d&O. The cells
were then fiozen and subsequentiy lyophilized ovemight using the Labconco freeze dry
system, Lyph-lock 4.5.
Plan& Samples were taken for dry weight growth curves every 2-3 days. Boxes
containhg 3 plants each were chosen randomly and the shoots and roots were separated
(removal of roots fiom agar required one thorough rinse in dI-120), placed in foi1 and oven
dried at 1 SOO F for 48 hrs. Plants were harvested up to day 39.
2.4.2 Total Protein Extraction
A modified Pascal and Douce (1993) method was utilized to extract protein fiom
lyophilized suspension cells. An aliquot of lyophilized cells (2.5-3.5 mg) was placed in
100 jd extraction solution (2% [w/v] Na2C03 in 0.1 N NaOH, 0.0 1% [v/v] Triton X-100)
in an eppendorf tube. The mixture was vortexed for 15 s and left to stand for 6 hrs at 4
OC. At the end of the extraction period the mixture was vortexed for 15 s and centrifuged
at 16 000 g for 5 min. The supematant was then diluted 50 fold in dH20 to a final volume
of 200 pl for protein quantification by the Lowry assay (section 2.6.1). Protein standards
used in the assay also contaiwd a 50 fold dilution of the extraction solution to account for
any absocbance changes resulting fiom the presence of TritonX-100.
2h.3 Soluble Protein Extraction
A modified method h m Millar et al. (1998) was used to extract soluble protein
fiom suspension ceus. Appmximately 75 mg DW of suspension cells was filtered h m
their growth medium through Whatmaa glass fibre filters (GC/F) and washed 2X with ice
cold W20. Cells were fiozen in tiquid nitrogen and thawed in 1 ml extraction bufEer (100
mM Tris-Cl, pH 7.5,2 mM EDTA and added just prior to use 10 mM DIT and 1 mM
PMSF) before king ground for 60 s in a mortar. The homogenate was centrifuged at
1 6 000 g for 15 min and the supernatant was stored at -80 O C until protein quantification
(section 2.6.4) and SDS-PAGE anaiysis (section 2.5).
2.6.4 Protein Measunment
A modified Lowry assay (Larson et al., 1986) was used to quantifi protein from
suspension ce11 extracts, plant shoots and roots and isolated mitochondria
Protein standards were made up in W20 or the appropriate solution from which
protein was being quantified using a BSA solution such that 0, 2, 5, 10, 20 pg protein
were in 200 pl volume. The extract to be quantified was aiso made up to 200 pl usually
by a 100 fold dilution, Le. 2 pl extract and 198 pl dH20. The prepared standards and
extracts were then mixed with 200 pl stock A-C solution (0.045% [wh] CuS04 5Hz0,
0.1% [w/v] NaKC&l&*4H20, 45 pM NaN3, 9 % [w/v] Na2C03, 0.45 N NaOH). This
mixture was in tum mixed with 600 pl phenol reagent (1 1 fold dilution of Folin-
Ciocalteau phenol reagent in â.H20) and allowed to sit for 3 min before addition of 100 pl
20 m M dithiothreitol @TT).
The absorbance at 750 nm was measured with a diode array spectrophotometer
(Hewlett Packard HP8584). The concentration of protein in the samples was determined
using the standard curve.
2.7 Starch and Amho Acid Analysis
2.7.1 Starch Measurement
Starch quantification of lyophilized celis (obtained as described is section 2.6.1)
was performed according to Vanlerberghe et al. (1996). Samples were hcubated for 2 hrs
at 95' C with 1 ml 0.02 N NaOH to solubilize the starch and degrade any fiee glucose in
the sample. The samples were cooled briefiy and then hcubated with amyloglucosidase
and a-amylase at 55OC overnight to hydrolyze the starch to glucose. Starch hydrolyzing
enzymes (200 units amyloglucosidase and 2 000 units a-amylase in 100 ml 0.2 M sodium
acetate, pH 5.0) were pdfied of contaminating glucose by dialysis. Spectrum molecular
porous membrane tubing containhg the hydrolyzing enzymes was washed for 3 X 1 L in
0.2 M sodium acetate, pH 5.0, for 6 lu periods. The hydrolyzed sample was centrifuged 2
min at 16 000 g. The supernatant was stored at -80 O C until quantification of glucose.
The assay
temperature in the
for glucose monitored
following reaction:
absorbance of NADP(H) at 340 nm at room
NADP+ NADPH
glucose \glucose &phosphate 6-phosphoglycerate
The sample (glucose) was mixed with 1.1 m M ATP, 0.5 mM NADP' and 2 units of
G6PDH (glucose 6-P dehydrogenase) in 100 m M Tris bufKer, pH 8.1 with 5 mM MgC12
and left to stand for several minutes before initiation of the reaction by 0.5 units of HK
(hexokinase). The increase in absorbance at 340 nm by conversion of NADP+ to NADPH
over the following 10-15 minutes was corrected for background by subtraction of the
average absorbance over the range of 400-410 nm and used to calculate the concentration
of glucose in the sample.
2.7.2 Amho Acid AnaïysW
Metabolite extraction fiom suspension cells was performed by a method modified
fiom Vanlerberghe and McIntosh (1996) at 5 days after subculture. Suspension cells
growing on a shaker at 140 rpm, 28 O C were sampled (800 pi). The sample was added to
133 pl 70 % perchloric acid in a preweighed eppendorf tube. The mixture was
immediately mixed and fiozen in liquid nitrogen followed by a one hour thaw on ice. The
mixture was then centrifuged 6 min at 16 O00 g. The pellet was washed two times with 1
ml M20, fiozen at -20 O C and then lyophilized to determine DW.
The supematant (extract) was hsuisfened to a fiesh tube and neutraiized with 5 M
KOH (approximately 350 pl). The resultant KCI04 precipitate was removed by
centrifugation (16 000 g, 5 min) and the volume of the extract was measured. The extract
was stored at -80 OC. Derivatization and HPLC analysis of the amino acids was
performed at the Amino Acid Analysis Facility, Biotechnology Service Centre,
Department of Lab Medicine and Pathobiology, University of Toronto.
2.8 Hydrogen Peroxide Analysis
Quantification of hydrogen peroxide (Hz02) production was performed through
use of the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (HIDCFDA)
(Royall and Ischiropouios, 1993). This probe readily crosses cellular membranes and
requires cleavage of a diecetate group by intracellular esterases for activation of the
probe. In the presence of cellular peroxidases and H202, the activated probe is oxidized to
DCFH, a compound which will fluoresce (emission wavelength 525 nm) upon excitation
by short wavelength blue light (excitation wavelength 488 nrn)
Suspension ce11 cultures were grown until day 3. A small volume of culture (10-
30 ml) was harvested and centrifuged 2 min at 200 g. The supematant was removed and
the ceiis were washed 2 times with approximately 40 ml modified growth medium ( 1 0
strength Murasbige and Skoog medium as descRbed in section 2.1.1, but with no
phosphorus and adjustment to pX 5.0). The cells were then resuspended in modified
growth medium to a final density of approximately 4 g DWIL. An aliquot of cells was
subsequently used to detemine the exact DW.
The suspension cells were incubated for 20 min under normal growth conditions
(28 OC, 140 rpm) before addition of 60 pM H2DCFDA (using 1 @/ml of a 20 mM stock
made up in anhydrous ethanol). A sample was immediately taken and mixed with KCN
(made day of use) to a h a l concentration of 5 mM to inhibit peroxidase activity. This
sample was stored in liquid nitrogen for one to two hours before fluorescence was
measured with a fluorometer (Hitachi F-4000, Tokyo, Japan). 1 found that samples could
be stored for up to 24 houn without significant changes in fluorescence. Samples were
thawed, cenûifuged 2 min at 16 000 g and the supernatant was diluted 10 fold with dH20
before measurement of fluorescence. An excitation wavelength (488 nm) was used on the
oxidized probe and the resultant fluorescent emission (525 nm) was quantified. In some
cases, other cornpounds were added to cells 2 min pnor to probe addition. These
included: 1 pM FCCP (added from a 1 mM stock in 95 % ethanol), 10pM Antimycin A
(added fiom a 70 mM stock in propanol) and 8 pM myxothiazol (added fkom a 4 m M
stock in DMSO).
2.9 Measurement of Ce11 Dimensions
Cellular dimensions were measured based on a method outlined in Winicur et al.
(1998). Randomly chosen cells (i.e., cells at the centre of particular fields) and were
magnified 400 X in a Nikon light microscope. Length was measured using a calibrated
micrometer in the ocular lens. In the case of individual cells, the longest dimension was
measured as the ce11 length. In the case of clumped cells or celis in a file, the ceil length
was defhed as the dimension perpendicular to the plane of ce11 division. Photography of
ceiis at 400 X magnification was performed with a Axiophot photomicroscope (Zeiss,
West Germany).
Chanter 3- Phos~horus Limitation in Whole Plants Increases
Alternative Oxidase Protein
3.1 Introduction
Phosphonis (P) is the most limiting nutrient to plant growth in many aquatic and
terrestrial environments (Bieleski and Ferguson, 1989) yet it is very important to plant
growth and metabolism. Phosphonis is a major structural constituent of many
biomolecules (nucleic acids, phospholipids, sugar phosphates, catalytic cofactors) and
plays a functional role in energy transfer (ATP) and metabolic regdation
(phosphorylation) (Becker and Deamer, 1991; Bosse and Koch, 1998). Part of this
unavailability is due to its occurrence in nature in insoluble forms of organic phosphorus
and bound mineral phosphorus. Billions of dollars are spent every year in North America
on phosphorus fertilizers (Lynch and Beebe, 1995). While the occurrence of phosphorus
in soi1 can be in the rnicromolar concentration and lower as acidity increases, plants
manage to concentrate phosphorus to millimolar concentrations. Plants exhibit many
rnorphological, physiological and metabolic adaptations to phosphorus limitation.
Study of whole plants growing under P limitation has both the advantages and
disadvantages of studying a complete (shoots and roots), complex system. Under P
limited conditions, plants reduce growth and alter allocation of biomass between shoot
and root (Paul and Stitt, 1993). Root growth is usually maintained or increased in plants
under nutrient stress resulting in increases in root:shoot ratios (Gutschick and Kay, 1995).
Plant uptake of inorganic phosphorus is enhanced several fold by P deficiency (Dong et
al., 1998). Plants increase secretion of acid phosphatases @ e W e and Randall, 1995).
The phosphorus is then transported fiom the roots throughout the plant in an effort to
maintain cytoplasmic P concentration. During this process vacuolar pools of P are
depleted (Dong et al., 1998). The vacuole is a major storage area for P in plant cells,
which c m be drawn upon if cytosolic concentrations fall below optimal levels.
The interactions between photosynthesis, carbon metabolism and respiration in
plants during P limitation are not well understood whereas these processes are relatively
well understood individually at a cellular level (Thorstellisson and Tillberg, 1990).
Phosphonis deprivation has been shown to have detrimental effects on photosynthesis
resdting in reduced rates possibly due to decreased activities of essential enzymes (e.g.
Usada and Sbgawara, 1992). Alteration of carbon metabolism by P limitation
commonly induces accumulation of starch and soluble sugars in leaves (e.g. Paul aad
Stitt, 1993). Respiratory adaptation to P limitation has led to decreases, maintenance and
increases in respiration (decreases in pea and barley leaves, Thorsteinsson and Tillberg,
1990; maintenance in bean leaves, Mikulska et al., 1998 and maintenance in roots of bean
seedlings, Rychter and Mikulska, 1990 and increases in unicellular green alga, Tillberg
and Rowley, 1989). These dBerent observations in respiratory rate adaptations to P
limitation could be due to many factors not the least of which are different species and
tissues and different experimentai techniques.
Plant respiration is linked to the rate of metabolism and growth due to
requirements for ATP, reductant and carbon skeletons during ce11 maintenance, division
and expansion (Millar et al., 1998). Plants adapt to P limitation by engagement of
metabolic bypasses that circumvent classical pathways when substrates for these
pathways become depleted (Murley et al., 1998). During P limitation, pools of adenylates
become depleted (Theodorou and Plaxton, 1994) and in plant respiratory pathways some
of the bypasses that have been best characterized occur in glycolysis. For example, PEP
phosphatase which catalyzes the conversion of PEP to pyruvate comptes with ADP-
dependent pyruvate kinase which is fiinctionally eliminated fiom cellular metabolism
during severe P stress (McHugh et al., 1995). Figure 4.1 in chapter 4 illustrates the-
reactions that convert PEP to pymvate as well as other reactions and their accompanying
bypasses.
In the rnitochondrial electron transport chah, respiratory O2 consumption may be
mediated via the phosphorylating cytochrome pathway or the non-phosphorylating
alternative pathway. It has been suggested that the alternative pathway may fhction as an
"energy overfiow" mechanism which becomes engaged only when the cytochrome
pathway is workhg at full capacity or when electron flow via the cytochrome pathway is
restricted, e.g., by low availability of adenylates andlor P. In the latter scemrio,
alternative oxidase rnay act as a bypass of ADP k t e d cytochrome oxidase under low P
conditions and rnay, therefore, increase its activity in response to P limitation.
Objective
1) Tobacco plants were grown under phosphorus limitation to quanti@ growth, P status
and alternative oxidase content. These preliminary experiments were used to determine
the suitability of this study in tobacco.
3.2.1 Plant Growth
Tobacco plants were grown as descnbed in section 2.1.2. Plants grown on
complete medium accumulated dry weight faster than plants grown on low P medium
throughout the 40 day growth period. This was due to greater shoot growth in complete
medium grown plants than in low P grown plants (Fig. 3.1). It was noted that the low P
grown shoots were a darker green in colouration than the complete nutrient grown shoots.
As well, the low P grown leaves appeared thicker and less delicate in comparison to the
complete medium grown leaves. Dry weight accumulation of roots was the saaie for both
complete medium and low P gown plants (Fig. 3.2).
Growth of low P medium plants responded quickly to supplementation of the
medium by additional phosphate through increased dry weight accumulation of the
shoots.
3.2.2 Leaf Phosphorus Content
Total phosphorus was extracted from oven dried leaves as described in section
2.6.1. Leaves from complete medium grown plants maintained a steady, average
phosphonis content of -40 mg P * g" DW (Fig. 33). In contrast, the low P grown leaves
gradually declined in total phosphorus content throughout the growth period. Early in the
growth period (Le. day 10) total phosphonis content of the Iow P grown leaves was on
Figure 3.1: A representative shoot growth curve. Wild type plants were grown on
complete or low P medium. The m w denotes the point at which the low P medium was
supplemented with additional phosphate (+P). Samples were taken every 3-5 days during
the growth period.
Figure 3.2: A representative root growth curve. Wild type plants were grown on
complete or low P medium. The arrow denotes the point at which the low P medium was
supplemented with additiod phosphate (+P). Samples were taken every 3-5 days during
the growth period.
Fi y r e 3.3: Leaf total phosphorus. Wid type plants were grown on complete or low P
medium. The arrow denotes the point at which the low P medium was supplemented with
additional phosphate (+P). Samples were taken every 3-5 days during the growth period.
Data are the average values fiom two independent experiments, each of which showed
s h d a r resdts.
average 20 mg P * g-l DW (2 fold less than that of cornplete medium grown leaves). By
day 35, low P grown leaves contained less than 5 mg P * g'l DW (8 fold less total
phosphoms content than complete medium grown leaves).
Addition of phosphoms to the low P medium quickly changed the P status of the
leaves. Within 72 hours of phosphorus supplementation of the low P medium, the total
phosphorus content of the leaves was as high as in complete medium grown leaves.
horganic phosphoms was extracted as described in section 2.2 fiom oven dried
leaves. Complete medium grown leaves contained approximately 30 mg P * g-' DW
inorganic phosphoms throughout the growth penod (Fig. 3.4). In contrast, low P grown
leaves had a 3-4 fold lower inorganic phosphorus content than complete medium grown
leaves early in the growth period and a 7-8 fold lower inorganic phosphorus content than
complete medium grown leaves late in the growth period (day 30). Supplementation 9f
low P medium with additional phosphate drarnatically increased inorganic P content of
the leaves to the level observed for complete medium grown leaves within 72 hrs.
inorganic P could be an important constituent of total P, especially at the beginning of the
culture period.
3.2.3 Immunoblot Analysis
Mitochondria were isolated from complete and low P grown leaves and analyzed
for alternative oxidase protein level with a monoclonal antibody raised against ' guttatum AOX (sections 2.4.2 and 2.5). Mitochondria fiom low P grown leaves had
higher levels of AOX protein than complete medium grown leaves (Fig. 3.5). Also, the
level of AOX in low P grown leaves decreased upon supplementation of the medium with
additional phosphate. M e r 72 hours, AOX protein level was discemibly lower although
still not as low as in complete medium grown leaves.
3.3 Discussion
The concentration of P chosen for our low P treatment (50 pM =O4) was used
as a balance between stressful conditions that would inhibit plant growth (and
metabolism) yet still allow enough growth that plant material for experimentation would
Figure 3.4: Leaf inorganic phosphorus. Wild type plants were grown on complete or low
P medium. The mow denotes the point at which the Low P medium was supplemented
with additional phosphate (+P). Sarnples were taken every 3-5 days during the growth
period. Data are the average values of two independent experiments, each of which
showed similar results.
Fipn 3.5: Representative immunoblot analysis of AOX. Mitochondria were isolated
from wild type leaves grown on complete or low P medium. Mitochondria were isolated
from plants of the approximate same size (i.e. mitochondria were isolated fiom complete
medium grown leaves on day 25 and fiom low P grown leaves on day 35). Additional
phosphate was added to low P grown plants on &y 35 and mitochondria were isolated
fiom the leaves 72 hrs later.
total P (mg ph3 DW)
+P C LowP72 hrs ---
be readily available. We also noted that whiie many previous experimenters (Theodorou
and Plaxton studying Brussica nigra suspension ceiis 1994, Johnson et al. studying
Lypinus albus 1996) had chosen to work with plants grown under zero phosphorus
conditions, plants are more likely to encounter low phosphorus conditions in their natural
environment. This was the case in our study of both whole plants and suspension cells
under zero phosphorus conditions (data not shown) that had such low levels of growth
and metabolism as to make useful data collection very difficdt.
Decreased shoot growth and unaffected root growth in the low P grown tobacco
plants resulted in a decreased shoot:root ratio, a commonly observeci response to nutrient
limitation (Gutschick and Kay, 1995). Growth of low P shoots was quick to respond to
increased P availability by rapid increases in dry weight. This response to P addition was
also reflected by quick changes in P status of the shoots. Complete medium grown shoots
maintained a total and inorganic leaf P content throughout the growth period of at least 2
fold greater than in low P grown shoots. This difference was abolished within 72 hr of P
addition.
in typical plant tissues, phosphorus is approximately 0.3 % of dry weight (Bieleski
and Ferguson, 1989) as was obsewed in our experiments. In mature leaf tissue, much of
leaf phosphorus is believed stored in the vacuole (Lauer et al., 1988). 31~-nuclear
magnetic resonance has been used to determine distribution of phosphorus. This led to the
finding that cytoplasmic Pi (metabolic) is usually maintained at the expense of
fluctuations in vacuolar Pi (storage) (Theodorou and Plaxton, 1994). P limitation in our
tobacco plants was severe enough to result in decreases in leaf inorganic (and total) P
despite possible reallocation of P. n ie cytoplasmic pool of Pi appears to be the hub of al1
cellular P metabolism and of the whole P economy of the plant (Bieleski and Ferguson,
1989). Our measurements of P status in tobacco leaves indicate that our P limitation was
severe enough to decrease cellular P pools.
Growth of plants containing an antisense constnict of the AOX gene (AS8
abbrevîated to AS) in complete and low P medium was measured without obseMog any
differences in shoot or mot dry weight accumulation from WT plants (data not shown).
Shidy of the AS plants was not pursued. However, suspension celis derived fiom AS
plants were reported extensively in chapters 5 and 6 and difierences were observed in
growth as measured by dry weight and protein accumulation.
AOX protein content was detemiined fiom mitochondria isolated fiom leaves of
plants of approximately the same size gmwn in complete and low P medium. AOX
protein was high in low P grown leaves compared to complete medium grown shoots and
declined rapidly in response to P addition. We observed two bands on out immunoblots
fiom reducing gels that represented the reduced and non-reduced form of the AOX
protein. Observation of AOX protein in other tissues was desirable, however isolation of
mitochondria fiom roots was difficult due to the small amount of material that could be
collected (for this reason, correspondhg P stanis data was not collected). There is
evidence that expression of AOX genes may be tissue specific in tobacco (Whelan et al.,
1995b). For example, in soybean, measurement of AOX protein expression in shoots,
roots and nodules detected one protein in roots, two proteins in shoots and none in the
nodules (Keams et al., 1992). Examination of AOX in soybean by Hilai et al. (1997) led
to the observation that AOX protein is Iocalized in the apical meristem and developing
xylem.
The activity of the AOX protein in low P grown shoots was measured
unsuccessfully with a leaf disc electrode. One of the problems with this method was
penetration of inhibitors into the leaf tissue. Other studies have measured AOX activity in
leaves. Keams et al. (1992) measured AOX in shoots, roots and nodules and found the
highest activity in shoots and the lowest in nodules. Under P limited conditions,
respiration was measured in bean leaves by measurement of CO2 evolution and O2 uptake
(Mikuiska et al., 1998). They found that within leaf tissue, total respiration was ody
slightly infiuenced by P limitation, however, there was an increased resistance of
respiration to KCN and higher inhibition by SHAM that suggested a higher engagement
of alternative pathway respiration and lower ATP production.
In conclusion, we observed that P limitation of our wild type tobacco plants
reduced growth and P content and increased AOX protein in leaves. In the next chapter,
we made similar observations for P limitation of wild type suspension ce11 cultures.
Cha~ter 4- Pbos~horus Limitation in Plant Sus~ension Cells Increases
Alternative Oxidase Protein and Activitv
4.1 Introduction
Phosphorus limitation is a common stress encountered by plants in nature. Plant
suspension ceil cultures are in many ways an ideal tool for studying the response of plants
to phosphorus stress. Suspension cells are usefid because they are undifferentiated,
uniform, easy to manipulate and in direct contact with their growth medium (van
Emmerik et al., 1992; Leifert et al., 1995; Hashimoto and Yamada, 1991). They also grow
very rapidly thus providing abundant material for experimentation in relatively short
periods of time.
The structural cornplexity of higher plants, which have a highiy differentiated
organization, formerly led to the use of singleîell green algae as a simple mode1 to study
regdation of growth and photosynthesis (Rebeille, 1988). The discovery and use of plant
growth regdators and high concentrations of mineral nutrients in growth medium, such as
Murashige and Skoog's (1 %Z), made growth of ceil cultures possible.
Photoautotrophic green plant ceil cultures have been used as experimental models
(Peel, 1982; Dalton, 1983), but are difficult to establish and maintain (LaRosa et al.,
1984). Addition of sucrose to extemal medium will inhibit photosynthetic activity and
chlorophyll production (Dalton and Street, 1977; Pamplin and Chapman, 1975), however,
use of sucrose as a carbon source appears to ease the establishment and maintenance of
ce11 cultures. Use of "non-green" cultures is now common and has the advantage of king
a highly simplified system biochemically. However, this simplification also necessitates
obsenhg responses of whole plants. For example, shoots and roots have been observed
to have different AOX expression patterns in soybean (Keams et al., 1992).
Under phosphorus limited conditions, pools of adenyiates and Pi become severeiy
depressed (Theodorou and Plaxton, 1993). There are t h e diîferent energy donor systems
that may operate in the cytosol of higher plants: denine nucleotides, uridine nucleotides
and PPi (pyrophosphate) (Dancer et al. 1990). Many enzymes in glycoiysis, the TCA
cycle and the electron transport chain require adenylates and Pi. For example, fnctose-
6-phosphate-1-phosphotransferase (PFK) in glycolysis and the ATP synthase complexes
of the electron transport chah require adenylates. Without alternate pathways that
fûnction independently of adenylate and Pi supply, carbon flow through these pathways
could become restricted thus increasing the stress of phosphorus limitation. Oxidative
stress resulting fiom restriction of electron flow through the mitochondrial electron
transport chain is discussed in chapter 6. AIso, many biochemical pathways associated
with respiration (e.g. nitrogen rnetabolism) require carbon skeletons, reductant and ATP
produced by respiratory pathways. Fig. 4.1 illustrates altemate pathways in glycolysis and
the electron transport chah that may be employed during phosphorus stress due to thek
use of energy donors other than ATP, ADP andior Pi and their use of compounds such as
PPi that remain relatively abundant under phosphorus limited conditions (Dancer et al.
1990).
Electron flow to AOX is not coupled to ATP production, making it an apparently
obvious alternate pathway for electron flow through the mitochondrial electron transport
chain during phosphorus limitation. Bingham and Farrar (1988) suggested that the AOX
pathway function may be related to ATP turnover. Whereas, Lambers (1982) suggested
that AOX rnay f'unction as an energy overfiow pathway in the presence of excess sugar. In
an effort to elucidate the function of AOX (adenylate control or sucrose supply), many
experimenters have studied the effects of sugar supply on respiration. Many of these
studies have combined sugar supply manipulations with nutrient supply manipulations to
observe how the respiratory responses change.
Hoefhagel et al. (1993), working with Catharantheus roseus suspension cells and
manipulating both sucrose and phosphorus supply, found that both the presence of
(perceived) excess sucrose and the absence of phosphorus were required to elicit an
induction of alternative pathway tespiration. If under low phosphorus conditions the
sucrose supply was decreased fiom control levels then the AOX induction was no longer
observed. However, an overabundance of sugar alone did not induce AOX pathway
respiration as ascertained fiom observing cells grown in complete nutrient medium with
elevated levels of sucrose.
Li and Ashihara (1989) also observed C. roseus suspension celis grown under low
Figure 4.1: Respiratory pathways in plants under 'nomiai' and phosphow limited
conditions (modified fkom 'ïheodorou and Plaxtoa, 1993). Black arrows indicate
pathways utilized under phosphom limited conditions. Grey arrows indicate pathways
used under 'normal' conditions which can be limited by ADP supply. Enzymes in the
pathways are iadicated by numben 1-10: 1- phosphofnictokinase (PFK), 2- PPi
dependent phosphofnictokinase (PFP), 3- phosphorylating NAD-glyceraldehyde 3-
phosphodehydrogenase (G3PDH), 4- 3-phosphoglycerate kinase, 5- non-phosphorylating
NAD-G3PDH, 6- phosphoenolpyruvate (PEP) phosphatase, 7- pyruvate kinase (PK), 8-
phosphoenolpyruvate carboxylase (PEPC), 9- rnalate dehydrogenase, 1 0 NAD malic
enzyme. ûther abbreviations: fm-6-P- hctose 6 phosphate, fm-l,6-bLP- hctose 1,6
bisphosphate, g-3-P- glyceraldehyde 3 phosphate, PEP- phosphoenolpyruvate, OAA-
oxaloacetic acid.
phosphorus conditions and observed that sugar uptake decreased. Whole plants with
phosphorus and nitrogen deficiencies show accumulation of chhydrate in mature
leaves and roots within hom of nutrient removal (Henry and Raper, 1991, Thorsteinsson
and Tilberg, 1990). These observations imply that under low phosphonas conditions
utilization of sucrose becomes more difficuit to the point that uptake becomes limited.
These results support the energy overflow fiinction of AOX suggested by Lambers
(1982).
Objectives:
1) Wild type suspension ceil cultures were grown under P limitation to quanti@ responses
in growth, P status, respiration and AOX activity in a manner similar to experiments with
whole plants.
2) The effect of sucrose supply on the induction of AOX activity by low P was exarnined.
4.2 Results
4.2.1 Growth
The representative DW growth curve in Fig. 4.2 clearly shows the differences in
dry weight accumulation brought about by differences in P supply. Cornplete medium
grown cells quickly entered an exponential growth phase (day 2) afler inoculation and dry
weight accumulation continued until cultures entered a stationary phase by days 6 and 7.
Complete medium grown suspension ce11 cultures obtained a maximum density of -15 g
* L*'. In contrast, low P grown cells may decrease dry weight accumulation throughout
the growth period The low P grown ce11 culture did not appear to enter an exponential
phase (as in the Complete medium grown celi cultures) and by day 7 the total dry weight
accumulation of the low P grown cultures was 2 fold less than the complete medium
grown ce11 culture (-7 g * ~ ' 3 .
Figure 43: A representative suspension ceU culture growth curve. Wild type N. tabacum
suspension cell cultures were grown in complete or low P medium. The arrow denotes the
point at which the low P medium was supplemented with additional P (+P).
P addition to low P p w n ce11 cultures on &y 3 appeared to cause the low P
grown cells to enter an exponential phase of growth. M e r 72 hrs, the low P cultures
supplemented with additional P had accumulated as much dry weight as the complete
medium grown cultures.
4.2.2 Phosphorus Content
Ce11 total phosphoms. Total P content as detemllned fiom lyophilized samples
from the suspension ce11 cultures (section 2.0) is shown in Fig. 4.3. Cell total phosphonis
content of complete medium grown cell cultures showed a sharp increase 24 hours a e r
inoculation into fresh medium. Total phosphorus content then gaduaîly declined fiom the
peak at 60 mg P * g-' DW until day 4 where total P content leveled out at 25 mg P * g' DW for the rest of the culture period.
The low P grown ceil cultures showed no increase in cell total P content upon
inoculation. Ce11 total P content declined graduaily throughout the growth period from 25
mg P * DW to 10 mg P * g-' DW. The ce11 total P of low P grown cells was thus
more than 2 fold lower than total P content of complete medium grown cultures
throughout the culture period.
P addition to low P grown cultures precipitated a rapid increase (24 hrs) in ce11
total P content to levels observed in complete medium grown cultures early in theû
culture period. Ce11 total P content in the "add back" ce11 cultures then declined steadily
until in matched complete medium grown culture levels on day 7.
Ce11 inorganic phosphorus. Ce11 inorganic P content of suspension ce11 cultures as
show in Fig. 4.4 shows the same trends as observed for ce11 totai P content. Ce11
inorganic P content was determined as described in section 4.0.
The cell inorganic P content of cornplete medium grown cultures increased
rapidly in the first 24 hours &er inoculation into fiesh medium. Early in the culture
period the ce11 inorganic P content made up the majority of the celi total P content. The
ce11 inorganic P content declined throughout the culture period and by day 7 made up only
a fifth of the cell total P content.
Figure 43: Ce11 total phosphorus. Wild type N. tobocum suspension ce11 cultures were
grown in complete or low P medium. The m w denotes the point at which the low P
medium was supplemented with additional P (+P). Average values from three
independent experiments have been plotted, each of which showed sirnilar results.
Figure 4.4: Cell inorganic phosphmus. Wild type N. tabacum suspension ce11 cultures
were grown in complete or low P medium. The arrow denotes the point at which the low
P medium was supplemented with additional P (+P). Average values fiom three
independent experiments have been plotted, each of which showed similar results.
Low P medium grown cultures xnaintained a low cell inorganic P content (5-10
mg P * g-' DW) throughout the culture period. There was not a rapid increase of
inorganic P content after inoculation into fksh medium as observed in complete medium
grown cultures. The low P grown cultures had 5 fold less ce11 inorganic P fiom days 0-3,
but for the rest of the culhue petiod there was no difference in ce11 iwrganic P content
between the complete and low P grown cultures due to the depletion of the inorganic P
pools of the complete medium grown cultures.
P addition to low P grown cultures on day 3 precipitated a rapid increase of
inorganic P content to complete medium grown culture levels followed by a graduai
decline for the rest of the culture period.
4.2.3 Respiratory Characteristics
Respiratory analysis (using a Clark type oxygen electrode as described in
Materials and Methods, section 2.5) was perforrned on wild type suspension ce11 cultures.
In Fig. 4.5, the respiratory capacity started high on day 1 for al1 of the culture treatments
(complete and low P). However, by day 5 after subculture, respiratory capacity had
decreased sl i ghtly.
Alternative oxidase capacity in Fig. 4.6 was determiwd in the presence of FCCP,
by successive addition of the inhibitors sodium azide and SHAM.
On day 1, complete medium and low P grown cultures had the same AOX
capacity. By day 3, the low phosphorus grown cultures had a 5 fold greater AOX capacity
than the complete medium grown cultures. This was due to a simultaneous increase in
AOX capacity in the low P grown cultures to approximately 2.5 nmol O2 * min*' * mgm1
DW and decrease in AOX capacity in the complete medium grown culhues to
approximately 0.5 nmol O2 * min" * mgœ' DW. Addition of P on day 3 to the 'add back'
cultures successfully reversed the induction of AOX capacity mch that 48 hours d e r P
addition, the AOX capacity was as low as control levels.
Figure 4.5: Respiratory capacity of wild type Al tabacum suspension cells grown in
complete or low P medium. The arrow denotes the point at which the low P medium was
supplemented with additionai phosphate (+P). Respiration was meamred on days 1, 3 ,4
and 5 of the culture pend in the presence of 1 pM FCCP, an uncoupler of oxidative
phosphorylation. Average values (i se) fiom three independent experiments are shown.
Figure 4.6: Alternative oxidase capacity. Wild type N. tabacum suspension cells were
grown in complete or low P medium. The m w denotes the point at which the low P
medium was supplemented with additional P (+P). AOX capacity was measured on days
1, 3, 4 and 5 of the culture peiod in the presence of 1 pM FCCP, an uncoupler of
oxidative phosphorylation followed by the sequential addition of the inhibitors sodium
azide (10 mM) and SHAM (2 mM). Average vaiues (& se) from three independent
experiments are show.
4.2.4 Inununoblot Analysis
Altemative oxidase protein was quantifiai from mitochondria isolated fiom wild
type suspension cells (section 2.0) in Fig. 4.7. On day 3 low P grown cultures had a
higher AOX protein content than complete medium grown cultures. This difference
persisted through &y 5 despite a now visible AOX protein content in the complete
medium grown cultures.
Addition of P on day 3 to low P grown cells rapidly brought about changes in
AOX protein content. Within 48 hours after P addition, AOX protein was only slightiy
detectable on the immunoblots.
4.2.5 Sucrose Treatments
Sucrose in the growth medium of the suspension cells was increased and
decreased fiom control levels of 3% sucrose. Fig. 4.8 shows that in complete nutrient
medium an increase in available sucrose fiom control levels to 6% sucrose on day 3 did
not result in an increase in AOX capacity. Neither did a decrease to 1% sucrose on day 3
in complete nutrient medium have an effect on AOX capacity which remained low at 0.6
nmol O2 * mino1 * mg" DW.
In low P medium at control sucrose levels (3%), AOX capacity was high on day 3
at 2.2 nmol O2 * min-' * mgs' DW. AOX capacity remained high in low P medium
when sucrose supply was decreased to 1% sucrose.
4 3 Discussion
Accordhg to Li and Ashihara (1990), inorganic P is one of the most important
factors in control of growth and metabolism of plant cells. Suspension ce11 growth was
highly restricted by P limitation. Cells inoculated into zero P medium grew very Little at
the beginning of the culture period (data not shown) whereas inoculation into low P
medium allowed several more celi divisions although far less dry weight was
accumulated than in complete medium grown cultures.
Figure 4.7: Immunoblot analysis of AOX. Mitochondria isolated fiom wild type Al
tabanun suspension ce11 c u b e s grown in cornplete or low P medium on days 3-5 after
subculhire. Some low P grown cultures were suppiemented with additional P on &y 3
and mitochondria were isolated fiom these cultures 24 and 48 hrs after P addition.
Immunoblot analysis of the mitochondrial proteins was perfonned using a monoclonal
antibody raised against S. guttatum AOX.
Day 3 Day 4 Day 5
LowP C LowP 24 Lowp 48 hrs
AOX . . , ,
total P 7.1 35.6 6.4 33.1 7.9 22.1 38.4
Figure 4.8: Alternative oxidase capacity during increased and decreased sucrose (S)
supply. Wild type N tabacum suspension ce11 cultures were grown in complete or low P
medium supplemented with 14% sucrose (3% sucrose supply under control conditions).
Alternative oxiàase capacity was measured on day 3 after subcuiture by sequential
addition of sodium azide (10 mM) and SHAM (2 mM). Average values fiom two
independent experiments are show with range.
P status of the suspension ceils was very different between complete and low
phosphorus grown cells. Total P content of low P grown ceiîs remained 2 fold lower than
in complete medium grown cells throughout the culture period whereas inorganic P
content of low P grown cells started at 5 fold lower than complete medium grown cells,
but ended up at the same level of inorganic P due to larger decreases in the inorganic P
content of complete medium grown ceus.
Cornparison of respiration of low P grown cells to complete medium grown cells
led us to conclude that respiratory capacity (+FCCP) was not altered by P limitation.
Other studies have observed increases, decreases and no change in respiration due to P
limitation. This variation may be due to different study species and/or experimental
conditions. Examples of variation in respiration are: higher respiration in C. roseus
suspension cells in complete medium than in P deficient (Li and Ashihara, 1990), 5 fold
higher respiration in nutrient sufficient S. minuîum than in P limitation (Theodorou et al.,
1991), 2 fold increase in P starvation respiration of Lycopersicon (tomato) suspension
cells than in complete medium (Goldstein et al., 1989).
AOX induction was determined through oxygen electrode and gel blot analysis.
Respiration via the alternative pathway was measured as AOX capacity by successive
addition of sodium azide (inhibits cytochrome oxidase) then SHAM (inhibits AOX) to
suspension cells. Induction of AOX in low P grown cells comprised approximately 15 %
of the total respiratory capacity or a 5 fold greater activity than in the complete medium
grown cells. This induction was readily reversible by P addition. These observations
were repeated for AOX activity in AOX protein content on immunoblots.
The specificity of the observed AOX induction by P limitation was tested in a
couple of ways. It has been hypothesized that nutrient limitation induction of the
alternative pahway may be due to a perceived alteration (excess) of sucrose supply rather
than the direct effects of the absence of the nutrient (Hoefhagel et al., 1993). They also
observed that alternative pathway induction in C. roseus suspension cells resulted fiom a
combination of P starvation and erceived) excess sugar. We did not observe this in our
A? tabacum suspension celis. cbArtificialy' reduction of sucrose supply (Fig. 4.8) did not
reduce AOX capacity. Also, naturd depletion of sucrose supply drning aging of complete
medium grown cultures achially results in increases in AOX capacity (Vderberghe et
al., 1994). However, our results did concur with the Hoefhagel study which reported that
excess sugar alone does not induce AOX. Excess sucrose was observed to lengthen the
lag phase of suspension cell growth possibly due to osmotic stress (data not shown).
Phosphmate Experiments. In another effort to determine if induction of AOX
expression by P limitation was due specifically to the absence of phosphorus or to
concurrent conditions, we used the cornpouad phosphonate (Phi). Carswell et al. (1997)
used phosphonate, an anti-hgal agent that is an analogue of phosphate, to disrupt the
phosphate starvation responses in oilseed rape suspension cells. According to their study,
the primary site of Phi action in higher plants is at the level of the signal transduction
chah by which plants perceive and respond to Pi stress at the molecuiar level. We
hypothesized that phosphonate might disnipt AOX induction by low phosphom if the
induction is part of a specific P limitation response. Complete and low phosphorus media
were supplemented with phosphonate, but it was found to have toxic effects on both
treatments. Experiments with Phi were, therefore, not pursued. In chapter 5, a nitrogen
limitation treatrnent was included in the experirnents as a cornparison for P limitation.
We observed that P limitation of wild type suspension cells reduced growth and P
content and increased AOX activity. In order to understand the fûnction of the induced
AOX during P limitation, we turned to transgenic suspension cells with antisense
constnicts of the AOX gene. These antisense (AS) suspension cells are discussed in
chapters 5 and 6.
Chai~ter 5- Transgenic Suswnsion Cells Lackiae Alternative Oxidase
Have Altered Growtb and Res~iratorv Remonses When Grown Under
Phos~horus Limitation
5.1 Introduction
Generation and Characteriution of Transgenic Lines
Vaderberghe et al. (1994) used sense and antisense consmcts of the tobacco
Aoxl gene to generate transgenic tobacco plants with both increased and decreased levels
of AOX protein. These transgenic plants have been used to study the AOX enzyme and
its regdatory properties (Vanlerberghe et al., 1995). From the sense and antisense
tobacco plants, suspension ce11 cultures were generated (Vanlerberghe et al. 1994). The
antisense suspension ce11 culture (AS) dong with the wild type (WT) suspension cells
(now 5 years in culture) have been used in this study to investigate the physiological
significance of AOX.
Metabolic Adaptations to Phosphoms Limitation
In the previous chapters, an increased AOX capacity and protein content under P
limited growth conditions was observed for both whole plants and suspension cells.
However, this does not conclusively indicate engagement of the alternative pathway
during P limitation. Engagement of AOX in the absence of inhibitors requins both an
abundance of AOX protein as well as metabolic conditions that up-regdate AOX activity
(Fig. 1.4 summarîzes these conditions). For example, the reduction level of the AOX
protein in P-limited cells cornpared to complete medium grown cells has not k e n
detennined or the level of AOX activators have not k e n measwed.
Some of the metabolic conditions arising during P limitation have been studied.
Theodorou and Plaxton (1993) found that under P lixnited conditions, the concentrations
of adenylates (ATP, ADP) and Pi decrease sharply whüe the concentrations of other high
energy P compounds such as pyrophosphate (PPi) remain stable. They suggest that this
could cestrict the activity of enzymes which are dependent on these substrates (e.g.
cytochrome oxidase). I M e r hypothesized that restriction of respiratory pathways
under this severe adenylate control might result in accumulation of intemediates of
glycolysis and the TCA cycle that play a role in AOX regdation. Fig. 5.1 illustrates how
respiratory pathway intermediates (PGA, PEP, PYR, 2-OG and OAA as descnbed in the
figure legend) provide carbon skeletons for amino acid synthesis. Restriction of these
pathways might lead to accumulation of certain intemediates and thus result in an
increase in amino acids derived fiom these intermediates.
Accumulation of a-keto acids (indicated by free amino acid accumulation),
particularly pyruvate, due to adenylate restriction of respiratory pathways d u h g P
limitation could stimulate AOX activity since pyruvate has been identified as an
important activator of AOX (Millar and Day, 1997). A study by Veith and Komor
(1993), using heterotrophic sugarcane suspension celis, ûbserved that under P limitecl
conditions there was an increased concentration of amino acids derived fiom pynivate
(e.g. alanine). This may indicate an abundance of pyruvate under low P conditions in
these cells. Furthemore, accumulation of isocitrate or malate in P limited cells could
generate metabolic conditions conducive to AOX engagement. Generation of
intramitochondrial NADPH by isocitrate or malate is important for AOX reduction (to its
more active fonn), putatively mediated by thioredoxin or glutathione which require
NADPH (Vanlerberghe and McIntosh, 1997). Observation of accumulation of these
intermediates (pynivate, isocitrate, malate) would indicate that AOX protein is not only
increased under low P conditions, but it is also active.
Nitmgen Limitation
Nitrogen is a major limiting nutrient to plants in many environments and nitrogen
metabolism is a significant process of cellular metabolism (Vance, 1997). As a
cornparison to results king seen with P limitation, 1 investigated the response of WT and
AS cells to nitrogen (N) limitation.
Figure 5.1: Diagram of amino acid families derived h m respiratory substrates. The
amino acid families are: PGA (ser, cys, gly, his), PEP (phe, tyr, trp), PYR ( a h val, leu),
2-OG (gin, glu, pro, arg), OAA (am, asp, ile, met, thr, lys).
asn asP ile met thr lys
Aceîyl CoA
The effects of nitmgen limitation can range fiom altered metabolic rates to altered
ce11 composition. Hoehagel et al. (1993), observing N limited C. roseils suspension
cells, found that respiration was decreased from control levels and that the activity of the
cyanide-resistant pathway remained low as in the controls. Only during prolonged
nitrogen starvation was some increase in AOX capacity observed. However, alternative
pathway respiration may play a role during N assimilation rather than during N limitation.
Plants primarily obtain nitrogen fiom the soi1 in the form of nitrate (NO,-) which
is subsequently reduced to NQi and then N&+ (ammonium) which is toxic to plants at
high concentrations. Assimilation of NH,' occurs via the GS-GOGAT pathway
invoiving the enzymes: glutamine synthetase (GS) and glutamate synthase (GOGAT)
(Dennis et ai., 1997). Assimilation of Nfi' involves the expression of many genes
involved in the primary assimilation of nitrogen and is closely associated with carbon
metabolism (Makino and Osmond, 1991 ; Vanlerberghe et al., 1990).
During N assimilation, contribution of the alternative pathway to respiration
appears to vary. Bameix et al. (1984), working with wheat (Triticun, aestivicum),
observed that the contribution of the alternative pathway was high at over 40% of total
respiration in roots (determined by the difference in O2 consumption in the absence and
presence of SHAM, an inhibitor of AOX) during NH,+ assimilation. In contrast, Visser
and Lamben (1983) found that in pea (Pisum sativum), the efficiency of N2-fixing plants
was high during mf assimilation due to the low activity of AOX (measured in the same
manner as in Barnieux et al., 1984). Furthennore, assimilation of NO< did not result in
increased AOX activity (Barnieux et al., 1984). Thus the role of AOX during N
assimilation remains unclear.
Some of the other effects of N limitation include increased allocation of total
Mtrogen to mitochondna (Makino and Osmond, 1991), starch accumulation in leaves of
soybean plants exposed to nitrogen free medium (Rufly et al., 1988) and translocation of
carbohydrate fiom leaves to the mot system resulting in a decline of the shoot to root
weight ratio (Ingestad, 1979). These responses imply a general decline in carbohydrate
utilization due to nitrogen limitation.
Objectives:
1) Transgenic suspension ce11 cultures lacking the alternative oxidase (AS) and wild type
(WT) suspension cells were grown under P limitation to quanti@ changes in growth,
respiratory characteristics and alternative oxidase activity.
2) WT and AS suspension ce11 cultures were grown under nitrogen limitation for
cornparison with suspension cells grown under P limitation.
3) The metabolic consequences of growth under P limitation were investigated. Free
amino acid composition of WT and AS suspension cells was measured.
5.2 Results
Data was collected from suspension cells on days 3 and 5 afler subculture. For
simplification of presentation, only figures representing data from day 5 have been
included in this section. Al1 other data, including tables of values and statistical results,
are available in Appendices A-C.
5.2.1 Growth
Ce11 growth was expressed on either a dry weight @W) basis or a protein basis
(section 2.6). Fig. 5.2A shows that the accumulation of DW by wild type and antisense
cultures grown in complete medium (WT C and AS C) was not significantly different
(- 14 g DW * L-'). Wild type and antisense low nitrogen grown cultures (\NT LowN and
AS LowN) also did not differ fiom each other in dry weight accumulation (approximately
8 g DW * L-'). However, there was a difference in DW accumulation between wild type
and antisense low P grown cultures (WT LowP and AS LowP). AS LowP cultures had
significantly highet DW (8.9 f 0.8 g DW * L") than WT LowP cultures (- 5.2 f 0.3 g
DW * L-') (student unpaired t-test, P= 0.01). Growth of both WT and AS suspension
cells in low phosphorus and low nitrogen medium was signincantly lower than in
complete medium.
Figure 5.2: Gmwth of WT and AS N tabanun suspension ceiis in complete, low P or
low N medium after 5 days in cdture. Growth is expressed on either a DW (A) or protein
(B) basis. Data are the average values (I se) fiom 6 independent experiments.
WT LowP
AS LowP
LowN -r
AS LowN
Protein accumulation in Fig. 5.2B shows that both WT C and AS C cultutes
accumuiated approximately 1400 mg protein * L-' by day 5 which was significantly
higher than al1 of the low nutrient treatments except AS LowP. However, WT LowP and
AS LowP cultures did not significantly diffet tiom each other when growth was
expressed on a protein basis (student unpaired t-test, P= 0.22). AS LowP culaires
accumulated 1103 f 199 mg protein * L" and wild type low phosphorus grown cultures
accumulated 829 f 59 mg protein * L-'. The low nitrogen grown cultures had the Ieast
growth expressed on a protein basis (3 fold less than complete medium grown cultures)
with no significant difference between the WT and AS treatments (approximately 425 mg
protein * L"). Given the differences seen between expression of growth on a DW basis versus
expression of growth on a protein basis, 1 examined the protein content per g DW of WT
and AS cells in different growth media (Fig. 53). This analysis indicated that AS LowP
cells had significantiy lower protein per g DW than did WT LowP cells (P- 0.05). No
difference was observed between WT LowN and AS LowN cells.
Given the difference seen in composition @rotein/g DW) of WT LowP versus AS
LowP cells, 1 examined whether the difference in DW growth between the WT LowP
and AS LowP cells was the result of excessive accumulation of starch in AS LowP cells
(Fig. 5.4). While WT LowP and AS LowP cells tended to accumulate higher levels of
starch than WT C and AS C, the differences were not significant. No difference was
observed between WT LowN and AS LowN cells. Also, the absolute level of starch (1- 4
% of total dry weight) was low in dl cells and hence codd not explain the diflerences
seen in DW growth.
Figure 5.3: Proteidg DW in WT and AS N. tabanrm suspension ceiis grown in
complete, low P or low N medium for 5 days. Data are the average values (k se) from 6
independent experiments.
Figure 5.4: Starch content (rneasured as glucose) of WT and AS N tc~bacurn suspension
cells grown in complete, low P or low N medium for 5 days. Data are the average values
(k se) fiom 6 independent experiments.
AS LowP
LowN
AS LowN
5.2.2 Respiratory Cbaracteristics
Given the difTerences seen in composition of WT and AS ceUs @rotein/g DW), I
decided to express various respiratory characteristics being compared between these cell
types on both a DW bais and a protein basis (section 2.3). Respiratory characteristics
were deterrnined as outlined in Fig. 2.1.
Respiration Rate. Rates of tesphtory oxygen consumption by WT and AS
suspension cells (day 5 after subcuihire into complete, low P or low N growth medium)
were quantified. WT C and AS C cells did not differ sipnificantly in respiration rate
when expressed on either a DW (Fig. 5S.A) or protein bais (Fig. 5.SB). WT LowP and
AS LowP cells also did not dBer significantly in respiration on a dry weight or protein
basis fiom each other although WT LowP grown cells tended to respire at a higher rate
(6.8 nmol 0.2 * mid * mg-' DW) than AS LowP cells (4.0 nmol 02 * min-' * mge'
DW). Finally, WT LowN and AS LowN cells aiso did not differ significantly £tom each
other,
Cornparison of respiration between nutrient treatments on a dry weight and
protein basis showed that WT C cells did not have a significantly different respiration rate
than WT LowP cells on a DW basis. In contrast, AS C cultures had a significantly higher
respiration than in AS LowP cultures when expressed on a DW (unpaired t-test, P= 0.02)
indicating different responses by WT and AS cells to P limitation. Respiration in
complete medium grown cells was significantly higher than in low nitrogen grow cells
when expressed on a DW basis, but not when expressed on a protein basis.
Respiratory Capacity. An uncoupler of oxidative phosphorylation (FCCP) was
added to cells and respiratory O2 consumption in its presence was considered to represent
the maximum flow of electrons through the respiratory electron transport chain (Le.
capacity of both the cytochrome and alternative pathway). In al1 cases, FCCP addition
increased the rate of O2 uptake of cells, indicating that respiration capacity always
exceeded control respiration rates.
Figure 5.5: Respiration rate of WT and AS N. tubucwn suspension cells grown in
complete, low P and low N medium for 5 days. Respiration is expressed on either a DW
(A) or protein (B) bais. Data are the average values (i se) fiom 6 independent
experiments.
LowP
T
AS wT LowN LowN
On &y 5, WT C and AS C cells did not differ in respiratory capacity when
expressed on either a DW or protein basis (Fig. 5.6A and B). However, \NT LowP
cuitmes had a significantly higher respiratory capacity than AS LowP cultures when
expressed on a DW basis (unpaired t-test, P= 0.005) although this difference was not as
apparent when expressed on a protein basis (P= 0.1 1). WT LowN and AS LowN cells
also differed significantly in respiratory capacity expressed on a dry weight basis although
this ciifference was minor compared to that seen in low P grown cells. WT LowN and AS
LowN ceils did not ciiffer fiom each other in respiratory capacity on a protein basis.
Cornparison of WT C and WT LowP grown cultures showed that respiratory
capacities did not differ significantly on a DW basis although this did not continue when
expressed on a protein basis. In conhast, AS C cells had higher respiratory capacities
than AS LowP cells on both a DW and protein basis. Respiratory capacity of WT LowN
and AS LowN cells was significantly reduced when compared to complete medium
grown cells.
AOX Capacity. The capacity of the AOX pathway was determined in the presence
of FCCP and with the use of the respiratory inhibitors KCN and SHAM (as outlined in
Fig. 2.1). When expressed on a DW basis (Fig. 5.7A), al1 cells had very low AOX
capacity except for WT LowP cells which showed a dramatic induction of AOX capacity.
This indicates that AOX induction by growth in low P was completely suppressed in the
AS cells. When expressed on a protein basis (Fig. WB), WT LowP grown cultures
retained a high AOX capacity compared to al1 other cells although AOX induction was
detectable in WT LowN cells.
5.23 Immunoblot Analysis
Mitochondria were isolated fiom suspension cells on day 3 and 5 after subcuiture
and the levels of AOX protein and cytochrome oxidase subunit Il protein were
detemiiwd through immunoblot analysis (section 2.5). In the WT C cells, the presence
of AOX was slightly visible on the immunoblot whereas in the WT LowP and WT LowN
cells the expression of the AOX protein was strong (Fig. 5.8). No immunoreactive AOX
band was visible in any of the AS mitochondria. Expression of COX was similar in di
Figure 5.6: Respiratory capacity of WT and AS N. tabacum suspension ce11 cultures
grown in complete, low P or low N medium for 5 days. Respiratory capacity was
detemhed in the presence of 1 plbf FCCP and bas been expressed on either a DW (A) or
protein (B) basis. Data are the average values (f se) fiom 6 independent experiments.
WT LowP +
T
AS LowP
WT LowN
AS LowN
1
Figure 5.7: Alternative oxidase capacity of WT and AS A? tabacum suspension cell
cultures grown in complete, low P or low N medium for 5 days. Alternative oxidase
capacity was measured by sequential addition of I m M KCN followed by 2 mM SHAM.
AOX capacity is expressed on either a DW (A) or protein (B) basis. Data are the average
values (k se) fiom 6 independent experiments.
Figure 5.8: Representative immunoblot d y s i s of rnitochondrial proteins isolated from
WT and AS N. tabacum suspension ceii cultures grown in complete, low P or low N
medium for 3 or 5 days. Immunoblot analysis was performed using monoclonal
antibodies to AOX and COX.
Day 3 Day 5
cases and acted as a convenient control that similar levels of protein were loaded for ail
treatments.
5.2.4 Amino Acid AnalysU
Measurement of fiee amino acid level was perfonned on metabolite extracts fiom
5 day old WT and AS suspension cells grown in complete or low P media (Materials and
Methods section 2.7). In Fig. 5.9, the total amino acid content of WT C and AS C cells
did not significantiy dEer from each other. However, WT LowP and AS LowP cells did
differ significantly from each other (P= 0.01) due to higher total amino acid content of
WT LowP cells. Both WT LowP and AS LowP cells had significantly higher total amino
acid content than complete medium grown cells.
Individual amino acids were pooled into five farnilies based upon whether their
carbon skeletons are denved fiom 2-OG, OAA, PYR, PEP or PGA (Fig. 5.1). In Fig.
5.10 (and Appendix C-2), WT C cells accumulated a significantly larger pool of amino
acids in the PYR family than any of the other treatments. The 2-OG and OAA family
pools in WT LowP and AS LowP cells were significantly higher than in WT C and AS C
cells. Also, the AS LowP cells had significantly larger pools of arnino acids in the PEP
and PGA families than al! the other treatments.
To examine these differences more closely, the level of select individual amino
acids is show in Fig. 5.11. Detection of high levels of alanine in WT C cells accounted
for the significantly high pool of PYR family arnino acids compared to al1 other
treatments. The large increase in the size of the 2-OG family in low P grown cells was
entuely due to a massive accumulation of glutamine. In fact, accumulation of glutamine
accounts for most of the increase in total fiee amino acid pool of low P cells. Significant
accumulation of tyrosine in AS LowP cells accounted for the increase in the size of the
PEP family and significant accumulation of serine in AS LowP cells accouated for the
hcrease in size of the PGA family.
Figure 5.9: Total amino acid content of WT and AS suspension ce11 cultures grown in
complete and low P medium on day 5 &er subcuiture. Data are the average (2 se) fiom 3
independent experiments.
. .- .. . . - - -
O WTC ASC WT LowP AS LowP
Figure 5.10: Total content of amino acid families of WT and AS suspension ceM culnires
grown in complete or low P medium on day 5 &ter subcuiture. The amino acid familes
are: PGA (sa, cys, gly, his), PEP (phe, tyr, trp), PYR (da, val, leu), 2-OG (gin, glu, pro,
mg), OAA (am, asp, ile, met, thr, lys). Data are the average (* se) fiom 3 independent
experiments.
Figure 5.11: Free amino acid content (pnol * g-' DW) of WT and AS suspension ceil
cultures grown in complete or low P medium on &y 5 afler subculture. Data are the
average (k se) h m 3 independent experiments.
LowP - amino acids WT (derived from)
d a (PYR) 115.7 i 15.9 glu(KG) 21.7 * 5.8 asp (OAA) 3.0 0.9 gln(KG) 56.5 * 8.1 tyr (PEP) 3.4 * 0.3 ser (PGA) 6.0 * 0.9
total 264.8 * 40.0
53 Discussion
Growth (Fig. 52-5.4)
Antisense suspension cells responded differently to P limitation than did wild type
cells. WT LowP suspension cells had reduced dry weight and protein accumulation
compared to complete medium grown cultures. This concurs with work by Goldstein et
ai. (1989). They found that P stress inhibited biomass accumulation in tomato suspension
cells. WT LowP cells also increased protein content on a dry weight basis compared to
complete medium grown cells which concurs with fmdings by Li and Ashihara (1990) in
C. roseus suspension cells and Paul and Stitt (1993) in tobacco seedlings. However,
Nielsen et al. (1998) found that P limited tobacco seedlings decreased in growth
expressed on a protein bais unlike nitrogen limited seedlings when compared to nutrient-
unlirnited seedlings. Finally, WT LowP suspension cells accumulated starch (as
previously observed e.g. Thorsteinsson and Tillberg, 1990) compared to complete
medium grown cells.
Different responses to P limitation were observed in antisense cells. AS LowP
cells accumulated significantly more dry weight than WT LowP cells and protein
accumulation was equivdent. Also, protein per g DW of AS LowP cells was not
significantly higher than complete medium grown cells unlike WT LowP cells. Starch
content of AS LowP cells was not significantly higher than complete medium grown cells
unlike WT LowP cells. The altered responses of AS cells to P limitation hdicate that
AOX rnay play an important role in metabolisrn during P limitation.
In contrast to P limitation of WT and AS suspension ceils, nitrogen limitation did
not result in different growth responses by the WT and AS cells. Both WT and AS LowN
cells had reduced growth expressed on a dry weight and protein basis which concurs with
findings in tobacco seedlings by Paul and Stitt (1990). Protein per g DW of WT and AS
LowN cells was decreased and starch content was iacmased which also concurs with the
literatwe (Veith and Komor, 1993). These results indicate that AOX may not play a role
in growth responses duriag N limitation. Hence, AOX may not have a general role in
nutrient limitation per se, but rather a role specific to P limitation.
Respiratory Characteristics (Fig. 5.5-5.7)
Differences between WT and AS suspension ceils grown under P limitation
peaist when respiratory characteristics are compared. Respiration and respiratory
capacity of WT LowP cells, when expressed on a dry weight basis, did not differ fiom
complete medium grown cells. In contrast, AS LowP cells had significantly reduced
respiration and resphtory capacity when compared to complete medium grown cells.
Expression of respiratory characteristics on a protein basis resulted in lower respiration in
both WT and AS LowP cells compared to complete medium grown cells.
When comparing the respiratory characteristics of my tobacco cells grown under P
limitation to respiratory characteristics in the literature, 1 found that various investigations
yielded conflicting resufts. Goldstein et al. (1989) found in tomato cells that P limitation
led to a 2 fold increase in respiration when expressed on a DW basis. Weger (1996)
found that P limitation of C. reinhardtii cells decreased respiration when expressed on a
chlorophyll basis. Finally, Hoehagel et al. (1 993) found that P limitation decreased
respiration on a DW basis. In general, lower rates of respiration have been measured
when P supply is low. Expression of respiration on a variety of basis may account for the
observation of contrasting responses to P limitation as well as species variation. This
concurs with my findings that P limitation decreases respiration of WT cells when
expressed on a protein basis and does not effect respiration expressed on a DW basis.
AOX capacity of WT LowP cells was dramatically high, especially when
expressed on a DW basis. AOX capacity of WT LowP cells was up to 50% of the
respiratory capacity. AS LowP cells were unable to induce AOX. Of particular
siWcance, the low respiratory capacity of AS LowP cells (in cornparison to WT LowP
cells) can be completely quantitatively accounted for by the difference in AOX capacity
between these two ce11 types. Respiratory capacity of AS LowP cells is - 10 nmol Oz * min" * mg-' DW lower than that of WT LowP cells, as is the AOX capacity.
AOX capacity of WT LowP cells, expressed on a protein basis, was also high.
However, induction of AOX in the WT LowN cells was now detectable although to a
significaotly iesser extent than in the WT LowP cells. This concurs with the findings of
Hoehgel et al. (1993) in C. roseus that only prolonged N starvation resulted in
alternative pathway respiration (expressed on a DW basis). WT LowN cells did not
induce AOX when measured on a dry weight basis nor did any of the antisense
treatments.
AOX capacity was measured in this chapter after successive addition of KCN then
SHATlI. Use of KCN to inhibit cytochrome oxidase rather than sodium &de as in the
previous chapter was due to the observation that inhibition by KCN occurred almost
instantaneously whereas inhibition by sodium aide took several minutes to complete.
~ u n o b l o t Analysis (Fig. 5.8)
The detection of high levels of AOX protein in WT LowP cells corresponds
extremely well with the respiratory analysis of high AOX capacity (expressed on both a
dry weight and protein basis). This is not the case for WT LowN cells which also
exhibited high levels of AOX protein on immunoblots, but did not have a
correspondingly high AOX respiratory capacity (when expressed on either a dry weight or
protein basis). This incongniity may be related to the different compositions of cells
grown in complete, low P and low N media: On day O of the culture period, AOX protein
is slightiy elevated in ail WT cells. The media into which the cells were inoculated
would determine the fate of that AOX protein e.g. degradation in complete medium,
m e r induction in low P medium and maintenance in low N. Maintenance of day O
levels of AOX protein in WT LowN cells which have decreased protein content wouid
result in the detection of high levels of AOX protein on immunoblots.
The detection of approximately equivalent levels of cytochrome oxidase (COX)
subunit II (this mitochondrially encoded subunit contains the binding site for cytochrome
c and a redox centre for the intermediate acceptance of electrons, Calhoun et al., 1994)
does not support my respiratory characteristic measurements (Fig. 5.5). Respiration in
AS LowP cells that had very low AOX capacity was 2 fold lower than AS C cells that
also had a very low AOX capacity. Therefore, the dflerences in respiration must be due
to different cytochrome pathway capacity and possibly a difference in COX level.
However, detection of diffiereaces in COX via an immunoblot may be dificuit as only
large ciifferences would be apparent, Two methods for measurement of cytochrome
oxidase activity were partially developed with variable success. A Palet (1991) method
was rejected after it was fouiid that high non-specific respiratory activity interfered with
accurate measurements of COX activity by oxygen electrode analysis. A
spectrophotometric method adapted fkom Wagner et al. (1995) was more promising, but
was not completed as initial immunoblots had indicated possible differences in COX
content (which were not supported by subsequent immunoblots).
Amino Acid Analysis (Fig. 5.1 and 5.9411)
Cornplete medium grown ceils. The amino acid composition of WT C and AS C
cells were essentially identical with one exception. AS C cells had a lower pool of amino
acids derived fiom pyruvate. This was almost entirely due to less alanine in AS C cells (3
% of total &O acid content) compared to WT C cells (44 % of total amino acid
content). This may indicate that in complete medium, the lower AOX in AS C cells than
WT C cells (see day 5 immunoblots, Fig. 5.8) resulted in increased adenylate restriction
of pyruvate kinase (PK) (Fig. LI), decreased formation of pyruvate and thus decreased
levels of alanine. Hence, AOX has a role even in complete medium grown cells in
prevention of adenylate restriction at the PK reaction of glycolysis. Significantly higher
levels of aspartate (derived fiom OAA) in AS C cells may also indicate increased
adenylate restriction at PK as bypassing of this reaction may result in increased formation
of OAA (Fig. 5.1).
Low P grown ceils. A major change in amino acid composition of low P grown
cells was a dramatic accumulation of glutamine and an increased glutamuie/glutamate
ratio suggesting that the provision of 2-OG by the TCA cycle is now compromised in
both WT LowP and AS LowP cells and hence glutamine is accurnulating over the . A
low supply of carbon skeletons (2-OG fiom the K A cycle) =stricts GS-GOGAT
pathway fixation of w4' and results in accumulation of glutamine (Vanlerberghe et al.,
1990).
Adenylate restriction at the PK step of glycolysis appears to be even more severe
in WT LowP and AS LowP cells. Despite a 5 fold increase in the total amino acid pool
of low P grow cells (mostly due to glutamine accumulation) fiom complete medium
grown cells, alanine comprised less than 3 % of the total amino acid pool for both WT
LowP and AS LowP cells. An apparent diversion of carbon to OAA via PEP
carboxylase, has led to an increase in the OAA family of both WT LowP and AS LowP
celis. In AS LowP cells, a more severe restriction of PK and the TCA cycle significantly
increased pools of PEP and PGA derived amino acids compared to WT LowP cells (PEP
and PGA are generally in equilibrium with one another, Demis et al., 1997) resulting in
significantly higher levels of tyrosine (denved from PEP) and serine (derived from PGA).
Several experiments were perfomied in this study aimed at determinhg if the
AOX protein in WT LowP cells was engaged. They have not been included in the results,
but merit some discussion. The first of these experirnents involved the use of pynivate in
mitochondrial isolation media. It was found by Vanlerberghe (unpublished) that pyruvate
would protect tobacco mitochonària fiom oxidation during isolation thus making it
possible to observe the reduction state of AOX in vivo. Mitochondria were isolated, in
the presence of pynivate, fiom ûansgenic cells that contained a sense constmct of the
Aoxl gene (B9) grown in complete or low P media. It was hypothesized that the B9 LowP
cells might have the same amount of AOX protein as B9 C cells, but a higher level of it
would be in the reduced (active) form. However, interpretation of the results was
complicated by higher levels of AOX protein in B9 LowP cells compared to B9 C cells
despite constitutive overexpression of AOX.
As well, metabolite assays for citrate and pyruvate (respiratory intermediates) as
well as ATP, ADP and AMP in WT and AS suspension cells grown in complete and low
P medium were attempted. It was hypothesized that P lirnited cells rnight contain higher
levels of citrate and pyruvate that rnight feed forward to activate AOX and that levels of
ATP, ADP and AMP would be low (Theodorou and Plaxton, 1993) compared to
complete medium grown cells. Detection of these metabolites proved difficult due to
their occurrence in concentrations as low as 1 p o V g DW. However, accumulation of
certain respiratory intemediates was suggested indirectfy by the amino acid results.
Cha~ter 6- Trans~enic Sus~cnsion Cells Lacking Alternative Oxidase
Growine Have Increased Rates of Hvdroeen Peroxide Production
When Grown Under Phos~horus Limitation
6.1 Introduction
Reactive oxygen species
Reactive oxygen species (ROS), such as superoxide (O2> and hydrogen peroxide
(H202), are unavoidable byproducts of cell metabolism. In particuiar, they are generated
by both the photosynthetic and respiratory electron transport chahs (vanden Hoek et al.,
1998; Purvis et ai., 1995; Shigenaga et al., 1994). ROS generation is intensified during
penods of stress induced by such things as chilling injury (Purvis and Shewfelt, 1993) or
pathogen attack (Allan and Fluhr, 1997).
Mitochondria generate ROS at several sites in the electron transport chain. Most
notably, the ubiquinone (Q) site generates ROS and is infiuenced by the redox state of the
mitochoadria in its ROS production (Chandel et al., 1998). In the Q cycle, reduction of
ubiquinone occurs by a single electron transfer (Q) to generate ubisemiquinone (QJ.
Ubisemiquinone is, in large part, re-oxidized by the cytochrome cornplex, but can react
directly with molecular oxygen to generate superoxide (which can be converted to H202
by superoxide dismutase) when normal electron transport is disrupted (Purvis and
Shewfelt, 1993). Further reduction of ubisemiquinone by a single electron plus two
protons results in formation of ubiquinol (QH2) which does not react with oxygen
(Larnbers, 1997a). As seen in Fig. 1.2, the ubiquinone pool is the site of partitioning of
electrons to either the cytochrome pathway or the alternative pathway. Restriction of
electron flow through the electron transport chah cm occur, as previously described,
under P limited conditions when the capacity of the cytocbrome pathway to accept
electrons is decreased due to reduced pools of adenylates. This dght result in a
prolonged Metirne of ubisemiquinone and increased ROS generation.
ROS cm have several roles in cellular metabolism. There is evidence that ROS
have a direct antimicrobial effect (Heath, 1998) during oxidative bursts following
pathogen attack. They may also have a role in signaling pathways, e.g., oxidative bursts
could lead to the direct or indirect oxidation of some cellular sulfhydryl groups (protein or
non-protein) and these oxidized products could be themselves responsible for the
transduction of an elicitor signal (Degousee et al., 1994). However, tissue damage is also
a consequence of high levels of ROS that are often only mildly reactive, but cm convert
to more reactive and darnaging species. For exarnple, H202 (derived fiorn superoxide via
superoxide dismutase) cm be converted, in the presence of ~ e ~ + , to the extremely toxic
hydroxyl free radical, OH; via the Fenton reaction (Allan and Fluhr, 1997). Hydrogen
peroxide is involved in modification of ce11 walls through peroxidase-cataiyzed cross-
linking of polymen such as proteins (Thordal-Christensen et al., 1997). Therefore, cells
must have mechanisms to reduce the level of ROS when necessary.
Cells contain antioxidant defense systems and under normal circumstances the
deleterious effects of reactive oxygen species are minimized. The defense systems
include reactive oxygen scavenging enzymes such as superoxide dismutase, catalase and
various peroxidases as well as reactive oxygen scavenging metabolites such as ascorbic
acid, a-tocopherol, glutathione and carotenoids (Purvis et al., 1995). However, the most
effective defense systems against oxygen free radicals are mechanisms to avoid the
generation of reactive oxygen species (e.g. photoinhibition, van Camp et al., 1996;
Mishra et al., 1995). For exarnple, when electron flow through the cytochrome pathway
is disrupted or restricted leading to conditions of increased ROS production, alternative
oxidase rnay function to limit the production of ROS (Purvis and Shewfelt, 1993). When
electron flow through the electron transport chain becomes restncted there is increased
formation of Of by-product (Shigenaga et ai., 1994). It has k e n hpthesized that under
low P conditions electron flow through the electron transport chain becomes restricted
due to reduced pools of ADP (Theudorou and Plaxton, 1993). 1 M e r hypothesize that
restriction of electron flow during P limitation might then lead to increased ROS
production and that alternative oxidase may have a role in allowing continued electroo
flow through the electron transport chah (as its activity is not limited by ADP supply)
and might, therefore, be involved in prevention of ROS generation.
Ca talase
Catalases are ROS scavenging enzymes that degrade H202 to H20 and 02. Thus
fa, plant catalases have only been found in peroxisomes (Willekens et al., 1994) and in a
single case, in mitochondria (Guan and Scandalios, 1995). Tbey appear to be involved in
cold acclimation to prevent excessive H202 production and are possibly part of the
salicylic acid-mediated signaling pathway which induces systemic acquired resistance in
plants (Willekens et al., 1994).
Use of Inhibitors
inhibition of alternative oxidase of plant mitochondria by disulfram led to
enhanced superoxide and hydrogen peroxide production in soybean and pea (Popov et al.,
1997). Altematively, conditions that enhanced electron flow resulted in reduced ROS
production. Addition of ADP and uncouplers of the electron transport chah reduced
superoxide production in green bel1 pepper rnitochondria (Purvis, 1997). Similarly,
addition of an uncoupler of oxidative phosphorylation, ADP and phosphate inhibited the
rate of H202 production by mitochondria isolated from rat heart (Kotshunov et al., 1997).
These experiments indicate that the importance of alternative pathway respiration
may be in prevention of ROS production due to the effects of compounds that dimpt
electron flow. However, these experiments are limited by their use of inhibitors that
themselves have properties that stimulate aad/or inhibit ROS generation. For example,
SHAM has k e n found to act as an antioxidant (MolIer et al., 1988; Purvis et al., 1995)
which might countenrt the eRects of its inhibition of AOX (leading to enhanced ROS
production). Inhibition of the cytochrome pathway has classically used cyanide which,
however, also inhibits certain peroxidases in many tissues (Mollet et al., 1988). Cyanide
inhibits at cytochrome a3 in complex N in the cytochrome pathway (see Fig. 13). This
is also the site of sodium azide inhibition, but without the M e r inhibition of peroxidase
activity. Therefore, the use of inhibitors in detection of ROS can be problematic and
experimenters must take into account the non-specinc effects of these compounds.
My use of the AS suspension cells lacking the alternative oxidase circumvents this
problem by allowing the non-invasive study of ROS production in the absence of AOX.
These experiments have been described in this chapter.
Detection of ROS
The use of fluorescent probes for studies of living plant cells bas become
important for a variety of purposes (Oparka and Read, 1994) from specific vital staining
of membranes and organelles to measurements of ca2+, pH and ROS. OAen the
measurement of a specific reactive oxygen species is necessary. For example, hyârogen
peroxide has been proposed as the most attractive candidate for signaling via reactive
oxygen species because of its relatively long life and high pemeability across membranes
(Allan and Fluhr, 1997). The fluorescent probe 2',7-dichlorodihydrofluorescein diacetate
(DCFH-DA) has been used for the specific detection of Hz02, primarily in neutrophils
(Royal1 and Ischiropoulas, 1993), but has more recently k e n used in plants (Carolyn
Hutcheon, Department of Botany, University of Toronto, personal cornmimication). This
study has made use of DCFH-DA detection of H202 to measure rates of Hz02 generation
in WT and AS suspension cells grown under complete or low P conditions.
Objectives
1) Transgenic suspension cells lacking AOX (AS) were grown under P limitation to
determine if rates of ROS generation would be increased.
2) Some morphological differences between WT and AS suspension ceils were qmtified
through measurement of cellular dimensions and photography.
6.2.1 Hydrogen Peroxide Production
Washed suspension cells were incubated in moâified g~owtb medium with the
fluorescent probe DCFH-DA as described in section 2.8 on day 3 after subculture. As
shown in Fig. 6.1, AS C cells tended to have higher fluorescence (indicating higher rates
of H202 production) than WT C cells, although the dflerence was not significant (P=
0.2). Addition of FCCP, an uncoupler of oxidative phosphorylation, greatly reduced the
rate of H202 generation by both the WT C and AS C cells.
A difference in H202 production rate between WT and AS cells was very apparent
under P limitation (Fig. 6.2). AS LowP cells had a significantly higher rate of H202
production than WT LowP cells (P= 0.001). This Merence was abolished by addition of
FCCP through reduced H202 production by the AS LowP cells while WT LowP cells
were unaffected by FCCP addition.
6.2.2 Citalase Aaaiysis
Soluble protein extraction fiom suspension ceils on day 5 aller subculture was
perfonned as described in section 2.6.5 and anaiyzed as described in section 2.5. A
monoclonal antibody raised against catdase (3B6) detected protein in suspension cells
grown in complete medium (Fig. 6.3). It was observed that AS C cultures consistentiy
contained higher levels of catalase than WT C cultures.
6.2.3 Cellular Dimensions
Cellular dimensions were determined as described in section 2.6.5 on days 3 and
5 d e r subcuiture. Scatter plots of WT C, WT LowP, AS C and AS LowP cellular
dimensions are show Ui Fig. 6.4 (&y 3) and Fig. 6.5 (&y 5 ) (see Appendk D for
statistical tests). Wid type and antisense cells have an immediately obvious morphology
difference. WT cells had significantly increased widths and signifïcautiy decrwised
lengths
Figure 6.1: Hydrogen peroxide production of WT and AS suspension ceUs grom in
complete medium for 3 days. Control experiments and +FCCP (1 pM) experiments are
shown. Units of fluorescence are based on hydrogen peroxide production in suspension
ce11 culnues of 4 mg DW * mL" density. In the case of control experiments (-FCCP),
the data are the average (f se) h m 5 independent experiments. The +FCCP &ta are the
average (f se) fiom 3 independent experirnents done aiongside three of the control
expehents.
+ WT C control 4 AS C control
WT C +FCCP
5 10 15 20 25
time af'ter probe addition (minutes)
Figure 6.2: Hydrogen peroxide production of WT and AS suspension cells grown in low
P medium for 3 days. Control experiments and +FCCP (1 @A) experiments are shown.
Units of fluorescence are based on hydrogen peroxide production in suspension ce11
cultures of 4 mg DW * mL" density. in the case of control experiments (-FCCP), the
data are the average (f se) fiom 5 independent experiments. The +FCCP data are the
average (k se) fiom 3 independent experiments done dongside three of the control
experiments.
+ WT LowP control + AS LowP control + WT LowP +FCCP *-V-, AS LowP +FCCP
5 10 15 20 25
time after probe addition (minutes)
Figure 6.3: Immunoblot analysis of catalase in WT and AS suspension cells p w n in
complete medium for 3 days. Analysis was performed on total soluble protein extracts
using a monoclonal antibody (3B6) to catalase.
Figure 6.4: Cellular dimensions of WT and AS suspension ceiis grown in complete or
low P medium for 3 days. Average values (* se) fiom 3 independent experiments (totai
n=lOO) are shown.
width 37.8 +/- 0.9 length 44.9 +/- 2%
WT LowP width 38.0 +/- 0.8 length 47.3 +/- 1.8
O O
AS C width 22.0 +ln 2.6
OO O length 55.0 +/- 0.4 O 0 0
O 0 O
O AS LowP
O O
width 22.5 +/- 0.5 O length 74.6 +/- 3 .O
width (microns)
Figure 6.5: Cellular dimensions of WT and AS suspension cells gmwn in complete or
low P medium for 5 days. Average vaiues (.f; se) fiom 3 independent experiments (total
n= t 00) are shown.
width 34.4 +/- 0.6 length 50.8 +/- 2.3
WT LowP width 38.4 +/- 0.9 length 58.9 +/- 3.5
--
AS C width 22.6+1- 2.1 length 59.8 +/- 2.4
AS LowP O O
O width 23.7 +/- 1.2
O 0 0 ~ 0 length 116.2 +/- 4.8
70 0) 10 20
width (microns)
compared to AS cells grown in complete or low P medium. AS C ceils were thus more
rod-like in appearance in addition to their tendency to chah in culture. T'he low
phosphorus treatments had diflerent eEects on the morphology of WT and AS cells. WT
C and WT LowP cells did not ciiffer significantiy in length or width on day 3 although by
day 5 the WT LowP ceils were slightly Uicreased in width and length compand to WT C
cells. In contrast, AS LowP cells quickly had a greatly increased length compared to AS
C cells (P= 0.000 on day 3 afler subculhire) while maintainhg the same width. The
difference Ui length became even more dramatic by day 5.
On days 3 and 5, WT cells tended to be spherical to slightly oblong whether
grown in complete or Low P medium (Fig. 6.6 and Fig. 6.7). WT cells tended to exist as
either single cells or as small clumps of a few cells, although on occasion they would also
exist as short chahs of cells. AS cells were very different. In cornparison to WT cells,
these cells were much more rod-like in appearance as a result of being both longer and
nanower than WT cells. Also, these cells were much more likely to exist in longer chahs
of cells. These morphologicai differences between the WT and AS cells became more
pronounced during growth in low P medium and with time (compare day 3 to day 5).
6.3 Discussion
High hydrogen peroxide generation in AS cells indicated that alternative pathway
respiration is Unportant in prevention of ROS production, especially during P limitation.
This concurs with suggestions by Purvis and Shewfeft (1993) that AOX acts as a
rnediator of tesistance to stress. In diis study, AS LowP cells had significantly higher
H202 generation rates compared to WT LowP cells. In conrrast, while AS C cells tended
to have higher H202 generation rates than WT C cells, this dflerence was not significant
indicating that the importance of alternative paîhway respiration in prevention of ROS
production hcreases during P limitation. Furthemore, addition of FCCP to low P grown
cells resulted in significantly decreased Hz02 generation in AS LowP cells, but not in WT
Figure 6.6: Cell morphology of WT and AS suspension ceils grown in complete or low P
medium for 3 days.
Figure 6.7: Ce11 morphology of WT and AS suspension ceils p w n in complete or low P
medium for 5 days.
LowP cells whereas both WT C and AS C celis had significautly decreased H202
generation by FCCP addition. In other words, only WT LowP cells that had high AOX
capacity and protein were d e c t e d by FCCP alleviation of respiratory restriction. This
implies that high alternative pathway respiration successfully prevents ROS production in
WT LowP ceils by allowing continued respiratory electron flow.
Increased H202 production in AS C cells led to detection of increased catalase
protein content compared to WT C cells. This concurs with the literature that
accumulation of H202 may induce antioxidant enzymes such as catalase, which
subsequently could provide protection against enhanced, damaging H202 production
(Willekens et al., 1994). Catalase was not detected in WT LowP or AS LowP cells
possibly due to the slightly lower rates of H202 production in low P cells compared to
cornplete medium grown cells (Fig. 6.1 and Fig. 6.2) or oxygen scavenging enzymes
other than catalase may be employed during P limitation. For example, photosynthetic
electron transport regulation of peroxidase during excess light stress (Karpinski et al.,
1997) or differential regulation of superoxide dismutases during environmental stress
(Tsang et al., 199 1).
Some inconclusive results fiom other H202 production experiments are presented
in Appendices D-1 and D-2. I hypothesized that restriction of respiratory electron flow
by inhibition of the cytochrome pathway would increase H202, particularly in AS cells.
Two inhibitors were used: antimycin A and myxothiazoi. Antimycin A and myxothiazol
inhibit at the same site of the electron transport chah (cytochrome c in complex III) (Fig.
1m2), however, their inhibition has different effects on ROS generation. Chandel et al.
(1998) reported that unlike myxothiazol, antimycin A augments increases in DCFH
fluorescence during hypoxia of Hep3B cells. This appeared to be due to different abilities
of the MO inhibitors to alter the lifetime of ubisemiquinone and, therefore, ROS
generation. However, my findings do not agree with those of Chandei et al. (1998).
Antimycin A inhibition of cytochrome pathway respiration in the tobacco suspension
cells resulted in slight increases in ROS compared to large increases during myxothiazol
inhibition of cytochrome pathway respiration. Further experimentation would by
necessary to clarify these resdts.
hcreased H202 production in AS cells could lead to oxidative damage. H a 2
contributes to structural reinforcement of plant ce11 w d s (Yamada et al., 1998) which
may effect the morphology of cells. AS cells were rod-like in appemce compared to
sphencal WT cells. As well, AS ceils have a tendency to fom chahs in culture and, in
particdar, AS LowP ce11 cultures showed discolouration @y day 5 a f k subculture) that
was similar to discolouration associated with aging of cultures n o d l y not seen until
much later &er subculture. A recent study using tobacco suspension cells (Winicur et ai.
1998)' removal of the homione auxin h m growth medium changed ce11 and organelle
morphology and the tendency of cells to chain in culture. Measurement of width and
lengths of WT and AS cells grown in complete or low P medium indicated that AS cells'
growthidimensions were affected by P limitation sooner than WT cells. AS LowP cells
had a significantly increased length compared to AS C cells by day 3 after subculture
whereas WT LowP cells were not significantly different fiom WT C cells until day 5 aller
subculture. This suggests that P limitation has a more severe effect on AS cells than WT
cells.
Chapter 7- General Discussion: The role of alternative ~athwav
res~iration dunng growth under P limitation
Induction of AOX during P limitation was observed in both WT tobacco plants
and suspension cells cultures. In contrast, AOX induction during P limitation was not
observed in either plants or suspension ce11 cultures of the AS tobacco line. Other
differences in mponse to P limitation were observed between WT and AS ceils through
cornparisons of respiratory characteristics, growth and morphology. Furthemore,
examination of metabolism h o u & fiee amino acid level and hydrogen peroxide
production indicated that AOX may have a role in respiratory metabolism duting P
limitation.
Increased respiratory restriction in AS LowP cells compared to WT LowP cells
resulted in altered growth and metabolism. For example, while there was a large decrease
in dry weight growth of WT LowP cells compared to WT C cells, AS LowP cells did not
display a large decrease in comparison to AS C cells. Furthemore, WT LowP and AS
LowP cells significantly differed in cellular composition (proteinlg DW) while WT C and
AS C cells had a similar composition.
The absence of AOX respiration in the AS line contributed to a tendency towards
increased H202 production in AS C cells compared to WT C cells. P limitation of the WT
and AS lines amplified these differences resulting in significantly higher rates of H202
production in AS LowP cells compared to WT LowP cells. Thus, AOX appears to have a
role during P limitation in prevention of respiratory restriction that may leaâ to increased
ROS production.
Altered metabolism was evidenced by differences in free amino acid levels in the
WT versus AS cells. WT C cells accumulated -16 fold higher levels of the fixe amino
acid alanine compared to AS C cells. This rnay indicate that even in complete medium,
AS cells experience respiratory restriction due to their lack of AOX. P limitation of the
WT and AS cells amplified differnces in tke amino acid composition. For example, AS
LowP cells accumulated amino acids derived fiom "upsûeam" respiratory intemediates
(serine, tyrosine) compared to WT LowP cells which tended to accumulate amino acids
derived h m c4downstteam~' respiratory intemediates (glutamine).
Finally, WT and AS suspension cells had obvious morphological differences
suggesting that differences in metabolism existed even before introduction of P
limitation. P limitation amplifies these morphological differences between WT and AS
cells in a similar manner to the P limitation amplification of metabolic differences. Thus,
1 have developed a good system for study of the d e of AOX in prevention of respiratory
restriction through P limitation of WT and AS plant cells.
Future directions in which this system could be used include:
1. Measurement of H202 pduction rates in WT and AS suspension cells could
be M e r studied under several different experimental conditions to understand the
nature of mitochondrial H202 production. Cornparison of the effects of inhibitors of the
mitochondrial electron transport chain on H202 production rates may increase knowledge
of mitochondrial ROS production in plants. For example, prelhinary experiments with
the inhibitors antimycin A and myxothiazol that each have unique inhibitory properties
(Chandel et al., 1998 as discussed in chapter 6) could be continued. Alternatively,
inhibition of catalase activity (e.g. by use of 3-aminotriazole, an inhibitor of catalase,
Willekens et al, 1994) might indicate whether H202 is an Unportant signal in induction of
AOX during P limitation. For example, inhibition of catalase in WT C cells might lead to
accumulation of H202 and induction of AOX. Further observations of P limitation
enhancement of H202 production in cornparisons between WT and AS cells may also be
usefiil. For example, high H202 production rates in AS LowP cells might be reversed by
supplementation of the growth medium with additional P. Finally, replication of al1 or
some of these experiments in WT and AS whole plants during P limitation would be
useful in understanding the nature of Hz@ production in plants compared to plant
suspension cells.
2. Further examination of the different composition and morphology of WT and
AS suspension cells. For example, fatty acid or carbohydrate (in addition to starch)
composition may heip explain rny obsemation of different dry weight growth between
WT and AS cells as well as contributing to a better understanding of metabolism in the
two cell types.
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Appendix A-l : Dry weight and protein growth of WT and AS suspension cells. Average values (+/- se) fiom 6 independent experiments on days 3 and 5 after subculture are shown. See Fig. 5 .2 for graph of day 5 data and Appendix A-3 for graph of day 3 data. P-values for comparisons of suspension cell growth are presented in Appendix A-2.
g DW * L-'
T!re!!lent !&!Y 3
W C 6.7 * 0.4
AS C 6.6 * 0.6
WT LowP 4.7 * 0.7
AS LowP 4.7 * 0.3
WT LowN 5.2 A 0.2
AS LowN 3.7 0.3
mg prote!' * L-'
looy 5 0 9 ~ 3 OOY _5
15.4 * 0.4 599 * 39 1423 * 136
13.2 * 1.7 717* 163 1353 * 166
5.2 * 0.3 676 i 125 829 * 59
8.9 * 0.8 542* I l l 1103 199
8.1 * 1.1 367 ft 59 443 * 62
6.2 * 0.9 434 98 410* 51
*. a"'
WT LowN
T
AS LowN
Appendix A-3: Dry weight and protein growth o f WT and AS suspension ce11 cultures grown in either complete, low P or low N medium on day 3 af?er subculture. Average values (+/- standard error) fiom 6 independent experiments are shown.
Appendix A-4: Proteidg DW of WT and AS suspension cells. Average values (+/- se) fiom 6 independent experiments on days 3 and 5 are shown. See Fig. 5.3 for graph of day5 data and Appendix A- 6 for graph of day 3 data. P-values for comparison of proteidg DW are presented in Appendix A-8.
Treatment
W C
AS C
WT LowP
AS LowP
WT LowN
AS LowN
Appendix A-5: unpaired t-test P-values for cornparisons of data presented in Appendix A-4 (proteidg DW of WT and AS suspension cells).
'mshnent
w'r C vs WT LowP
WT C vs WT LowN
AS C vs AS LowP
AS C vs AS LowN
WTCvsASC
WT LowP vs AS LowP
WT LowN vs AS LowN
Appendix A-6: Proteidg DW of WT and AS suspension ceIl cultures grown in complete, low P or low N medium for 3 days. Average values (+/- standard error) of 6 independent experiments are shown.
Appendix A-7: Starch content of WT and AS suspension cells (mg glucose * g -'DW). Average values (+ln se) fiom 3 independent experiments on days 3 and 5 are shown. See Fig. 5.4 for graph of day 5 data and Appendix A-9 for graph of day 3 data. P-values for cornparisons of starch content are presented in Appendix A-8.
Treatments
W C
WT LowP 39.1 f 10.4 41.5 + 6.5
AS LowP 18.3 + 2.6 21.4+, 8.3
WT LowN 25.2 i 7.7 19.3 f 0.2
AS LowN 43.2 & 12.0 24.2 k 10.2
Appendix A-8: unpaired t-test P-values the data presented in Appendix A-7 (starch content of WT and AS suspension cells). P-values of less than 0.05 denote a significant difference between the suspension ce11 cultures.
mg starch * g-' DW
Trcniment D ~ Y 3
WT C vs WT LowP 0.29
WT C vs WT LowN 0.95
AS C vs AS LowP 0.44
AS C vs AS LowN 0.27
WTCvsASC 0.94
WT LowP vs AS LowP 0.13
WT LowN vs AS LowN 0.27
WT LowP
AS LowP
WT LowN
t
AS LowN
Appendix A-9: Starch content of WT and AS suspension ceil cultures grown in complete, low P or lowN medium for 3 days. Average values (+/- standard error) fiom 3 independent experiments are show.
WT LowP
AS LowP
Appendir B-3: Respiration of WT and AS suspension ceii cultures grown in cornplete, low P or low N medium for 3 days. Respiration is expressed on a DW (A) or protein (B) basis. Average values (+/- standard error) for 6 independent experirnents are shown.
Appendix B-4: Respiratory capacity of WT and AS suspension cells. Respiratory capacity was detemined in the presence of FCCP. Average values (+/O se) fiom 6 independent experiments are shown. See Fig. 5.6 for graph of day 5 data and Appendix B-6 for a graph of day 3 data. P-values for cornparisons of respiratory capacity are presented in Appendix B-5.
WT LowP 18.8 k 2.4 17.0 f 1.6 154.7 -t 37.6 107.2 + 11.6
AS LowP 16.2 f 1.9 7.5 & 2.1 157.9 k 23.9 66.6 -t 19.8
WT LowN 12.7 k 0.9 10.4 $: 2.0 215.3 + 50.1 127.7 + 28.6
AS LowN 12.1 + 1.7 6.0 + 0.3 162.6 it 42.1 89.8 k 5.8
Appeadix B-5: unpaired t-test P-values for data presented in Appendix B-4. P-values of less than 0.05 denote a significant difference between the suspension ceIl cultures.
'hotmests
WT C vs WT LowP
WT C vs WT LowN
AS C vs AS LowP
AS C vs AS LowN
WTCvsASC
WT LowP vs AS LowP
WT LowN vs AS LowN
WT LowP
AS LowP
WT LowN
3-
AS LowN +
Appendix Bd: Respiratory capacity of WT and AS suspension ce11 cultures grown in complete, low P or Iow N medium for 3 days. Respiratory capacity was determineci in the presence of FCCP and is expressed on a DW (A) and protein (B) basis. Average values (+/O standard error) h m 6 independent experiments are shown.
L I ' 8 1
Appendix B-8: unpaired t-test P-values for data presented in B-7 (alternative oxidase capacity of WT and AS suspension cells). P-values of less than 0.05 denote a significant difference between the suspension cell cultures.
T r e r r m l
WT C vs WT LowP
WT C vs WT LowN
AS C vs AS LowP
AS C vs AS LowN
WTCvsASC
WT LowP vs AS LowP
WT LowN vs AS LowN
nmol O2 * m i i ' * mg' DW
DBY 3 D ~ Y 5
0.005 0.000 1
0.12 0.04
0.88 0.4 1
0.2 1 1 .O1
O. 19 0.72
0.003 0.000 1
0.55 0.009
nmol O2 * min"' * mg-' --- - protein
Day 3 !MY 5
0.02 0.0006
0.1 1 0.03
0.64 0.80
0.56 0.24
0.12 0.33
0.0 1 0.0004
0.07 0.03
WT LowP
t
Appendis 8-9: Alternative oxidase capacity of WT and AS suspension ce11 cultures grown in complete, low P or low N medium for 3 &YS. Alternative oxidase capacity was detemhed by successive addition of 1 mM KCN then 2 mM SHAM and is expressed on a DW (A) and protein (B) basis. Average values (+/- standard ewr) fiom 3 independent experhents are show.
Appendix B-10: Alternative oxidase capacity of WT and AS suspension cells. Alternative oxidase capacity was determined by successive addition of sodium azide then SHAM. Average values (+/- se) fiom 3 independent experiments are show. See Appendix B-12 for graph of day 3 data and Appendix B- 13 for graph of day 5 data. P-values for cornparisons of alternative oxidase capacity are presented in Appendix B- 1 1.
Trea!meat
WTC
AS C
WT LowP
AS LowP
WT LowN
AS LowN
nmol 02* min-' * mg-'DW
D ~ Y 3 D ~ Y 5
0.2 + 0.1 0.3 2 0.1
0.2 0.1 0.1 k 0.0
3.4 0.5 2.3 0.2
0.7 0.1 0.4 + 0.0
0.7 -t 0.1 0.2 k 0.0
0.3 k 0.1 0.2 f 0.1
nmol O2 * min " * mg O' protein
D ~ Y 3 D.Y 5
2.1 k 1.0 3.9 f 1.7
3.4 1.2 0.4 & 0.4
25.7 k 4.5 14.0 & 0.9
9.1 k 1.7 5.6 I 1.0
12.9 I 2.3 3.3 $: 1.0
4.6 k 1.7 2.9 I 1.0
Appendix B-Il : unpaired t-test P-values for data presented in Appendix B-10 (alternative oxidase capacity of WT and AS suspension cells). P-values less than 0.05 denote a significant difference between the suspension ce11 cultures.
Trcatmea!
WT C vs WT LowP
WT C vs WT LowN
AS C vs AS LowP
AS C vs AS LowN
WTCvsASC
WT LowP vs AS LowP
WT LowN vs AS LowN
nmol O2 * min" * mg-' DW
D ~ Y 3 D w 5
0.01 0.003
0.02 0.27
0.03 0.00 1
0.65 0.07
0.68 0.06
0.02 0.004
0.15 0.64
nmol O2 * min-' * mg' p r m n
DW 3 DayS
0.03 0.01
0.007 0.84
0.1 1 0.002
0.7 1 0.03
0.50 0.16
0.08 0.003
0.06 0.80
AS WT LowP LowN
Appendix B-12: Alternative oxidase capacity of WT and AS suspension ce11 cultures grown in complete, low P or low N medium for 3 days. Alternative oxidase capacity was detennined by successive addition of 10 rnM NaN3 then 2 mM SHAM and is expressed on a DW (A) and protein (B) bais. Average values (+/- standard erm) fiom 3 independent experiments are shown.
amiao acids ala
asn
as!' =Ys gin glu B ~ Y his ile leu lys met
phe Pro ser thr
trp tyr val
total
Appendir C-1: Free amino acid composition of WT and AS suspension cells. Average values (+/- standard emr) from 3 independent experiments on day 5 after subculture are show except for low nitrogen grown suspension cells for which 2 experiments were performed.
A ~ ~ e n d ù C-2: unpaired t-test P-values for cornparison ofWT and AS suspension ceii amino acid content from 3 independent experiments on day 5 after subculture. P-values of less than 0.05 denote a sigmfïcant difference between the suspension ceU cultures.
ser ( P W
total
Treatment
WTCvsASC WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
WTCvsASC WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
WTCvsASC 'WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
WTCvsASC WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
WTCvsASC WT LowP vs AS LowP RT C vs WT LowP AS C vs AS LowP
W C v s A S C 'WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
WTCvsASC WT LowP vs AS LowP WT C vs WT LowP AS C vs AS LowP
160
-e- WTC I 4 O - 4 ASC
tirne after probe addition (minutes)
Appendix D-1: Hydrogen peroxide production of WT and AS suspension celi cultures grown in complete or low P medium for 3 days. Hydrogen peroxide production was detennined in the presence of antimycin A and is based on cultures of 4 mg DW * ml-1 density. Average values (+/- se) for 5 independent experiments are shown.
WTC + ASC -v- WT LowP -i~- AS LowP
5 10 15 20 25 tirne d e r probe addition (minutes)
Appendu D-2: Hydrogen peroxide production of WT and AS suspension ceIl cultures grown in complete or low P medium for 3 days. Hydrogen peroxide production was determined in the presence of myxothiazol and is based on cultures of 4 mg DW * mCI density. Average values (+/- se) for 2 independent experiments are shown.
Appendix D-4: unpaired t-test P-values for data presented in Appendix D-3 (cell dimensions of WT and AS suspension cells). P-values of less than 0.05 denote a significant difference between the suspension ce11 cultures.
DW 3
Tiatments width
WTCvsASC 0.0000
WT LowP vs AS LowP 0.0000
WT C vs WT LowP 0.92
AS C vs AS LowP 0.44