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.

References

Aien AC, Fluhr R 1997. Two distinct sources of elicited reactive oxygen species in

tobacco epidemal cells. Plant Celi. 9: 1559- 1572.

Amcs BN. 1966. Assays of phosphate and phosphatases. In: Methods in Enzymology.

edited by Neufeld EF, Ginsberg V. Academic Press. New York. Vm: 1 15-1 17.

Barniex AJ, Breteler H, van de Geijn SC. 1984. Gas and ion exchanges in wheat roots

after nitrogen supply. Physiol. Plant. 6 1 : 3 57-362.

Becker WM, Deamer DW, editors. 1991. The world of the cell. Benjamin/Cumrnins

Publishing Company Inc. Don Mills, Ont. 640-670.

Bieleski RL, Ferguson IB. 1989. Physiology and metabolism of phosphate and its

compounds. in: Inorganic Plant Nutrition. editor Bieleski RL. 422-449.

Bingham IJ, Famr R. 1988. Regulation of respiration in roots of barley. Physiol.

Plant. 73: 278-285.

Bosse D, Kock M. 1998. Mluence of phosphate starvation on phosphohydrolases during

development of tomato seedlings. Plant Cell Environ. 2 1 : 325-332.

Boum M, Faber AM, Charbonnier M, Briquet M. 1984. Microanalysis of plant

mitoc hondrial protein synthesis products. Plant Md Biol. 3 : 445-452.

Calhoun MW, Thomas JW, Gennis RB. 1994. The cytochrome oxidase superfamily of

redox-driven proton pumps. T m . 19: 325-3 30.

Carswell MC, Grant BR, Plarton WC. 1997. Disruption o f the phosphate-starvation

response of oilseed tape suspension cells by the fuagicide phosphonate. Plonta. 543: 1-7.

Chandel NW, Maltepe E, Goldwasser E, Mathewu CE, Sion MC, Schumacker PT.

1998. Mitochondrial reactive oxygen species trigger hypoxia induced transcription. Cell

Biol. 95: 11715-1 1720.

Cbaph FS, Bieleski RL. 1982. Mild phosphorus stress in barley and a related low

phosphorus adapted barley grass: phosphorus fractions and phosphate absorption in

relation to growth. Physiol. Plant. 54: 309-3 17.

Dalton CC, Stnet HE. 1977. The influence of applied carbohydrates on the growth and

greening of cultured spinach (Spinacea oleracea L.) cells. Plunt Physiol. 56: 752-756.

Dalton CC. 1983. Chlorophyll production in fed batch cultures of Ocimum basilicum

(sweet basil). Plant Sci. Lett. 32: 263-240.

Dancer J, Veitb R, Fei1 R, Komor E, Stitt M. 1990. Independent changes of inorganic

pyrophosphate and the ATPIADP or UTPRIDP ratios in plant ce11 suspension cultures.

Plant Sei. 66: 59-63.

Degousee N, Triantaphylides C, Montille! JL. 1994. Involvement of oxidative

processes in the signahg mechanisms leading to the activation of glyceollin synthesis in

soybean (Glycine mm). Plant Physiol. 104: 945-952.

Delhaize E, Randall PJ. 1995. Characterization o f a phosphate-accumulator mutant of

Arabidopsis thaliana. Plant Physiol. 1 07(1): 207-2 1 3.

Dennis DT, Huang Y, Negm FB. 1997. Glycoiysis, the pentose phosphate pathway and

anaerobic respiration. In: Plant Metabolism. editors Dermis DT, Turpin DH, Lefebvre

DD, Layzell DB. Longman Publishers. England. pp. 106- 123.

Dong B, Reagel 2, Delbaize E. 1998. Uptake and translocation of phosphate bypho2

mutant and wild-type seedlings of Arabidopsis thaliana. PZW 205: 25 1 -256.

Eltbon TE, Nickels RL. McIntosh L. 1989a. Monoclonal antibodies to the alternative

oxidase of higher plant mitochondria. Plant Physiol. 89: 1 3 1 1 - 13 17.

Goldstein AH, Mayfield SP, Danon A, Tibbot BK. 1989. Phosphate starvation

inducible metabolism in Lycopersicon esculentum. Plant Physiol. 9 1 : 1 75-1 82.

Guan L, Scandalios JC. 1995. Developmentally related responses of rnaize catalase

gews to salicylic acid. Proc.Nut1. Acud. Sci. 92: 5930-5934.

Gutsehick VP, Kay LE. 1995. Nutrient limited growth rates: quantitative benefits of

stress responses and some aspects of regulation. J. Erp. Bot. 46(289): 995-1 009.

Guy RD, Berry JA, Fogel ML, Boering TC. 1989. Differentiai hctionation of oxygen

isotopes by cyanide-resistant and cyanide-sensitive respiration in plants. Plantu. 177:

483-491.

Hashimoto T, Yamada Y. 1991. Organ culture and manipulation. In: Methods in Plant

Biochemistry. Academic Press. Toronto. 6: 323-350.

Heath MC. 1998. Involvement of reactive oxygen species in the response of resistant

(hypersensitive) or susceptible cowpeas to the cowpea rust fungus. New PhytoZ. 138: 251-

263.

Heldt HW, editor. 1997. In: Plant Biochemistry and Molecular Biology. Oxford

University Press, Oxford. P. 12 1-146.

Henry LT, Raper CDJr. 1991. Soluble carbohydrate allocation to roots, photosynthetic

rate of leaves and nitrate assimilation as affected by nitrogen mess and irraâience. Bot.

Ga. 152(1): 23-33.

Hilal M, Castagnaro A, Moreno H, Massa EM. 1997. Specific localization of the

respiratory alternative oxidase in meristematic and xylematic tissues from developing

soybean roots and hypocotyls. Plant Physiol. 1 15: 1499-1 503.

Hill SA. 1997. Carbon metabolism in mitochondria. in: Plant Metabolism. edited by

Derinis DT, Turpin DH, Lefebvre DD, Layzell DB. Longman Publishers. England. pp.

181-199.

Hoefnagel MHN, van I n n F, Libbenga KR. 1993. In suspension cultures of

Catharanthus roseus the cyanide resistant pathway is engaged in respiration by excess

sugar in combination with phosphate or nitrogen starvation. Physiol. Plant. 87: 297-304.

Hoefnagel MHN, Millar AH, Wiskicb JT, Day DA. 1995. Cytochrome and alternative

respiratory pathways compte for electrons in the presence of pynivate in soybean

mitochondria. Arch. Biochem. Biophys. 3 18: 394400.

Ingestad T. 1979. Nitrogen stress in birch seedlings. Physiol. Plant. 45: 149-1 57.

Johnson JF, Vance CP, Allan DL. 1996. Phosphorus deficiency in Lupinus albus. Plant

Physiol. 1 12: 3 1-41.

Karpinski S, Escobar C, Karpinska B, Creissen G, MuMilieaux PM. 1997.

Photosynthetic electron transport regulates the expression of cytosolic ascorbate

peroxidase genes in Arabidopsis during excess light stress. Plant Cell. 9: 627-640.

Kearns A, Whelan J, Young S, Elthon TE, Day DA. 1992. Tissue-specific expression

of the alternative oxidase in soybean. Plant Physiol. 99: 7 12-7 17.

Korsbunov SS, Skulachev VP, Starkov AA. 1997. High proton potentid actuates a

mechanism of production of reactive oxygen species on mitochondria. FEBS. 4 16: 15-1 8.

Kumar AM, Soll D. 1992. Arabidopsis alternative oxidase sustains Escherichia coli

respiration. Proc. Natl. Acad. Sci. USA 89: 10842- 1 0846.

Lambers H. 1982. Cyanide-resistant respiration: a non-phosphorylating electron

transport pathway acting as an energy ovefflow. Physiol. Plant. 55: 478-485.

Lamben H. 1985. Respiration in intact plants and tissues: its regulation and dependence

on environmental factors, metabolism and invaded organisms. In: Encyclopedia of Plant

Physiology, New Series, Vol 18: Higher Plant Ce11 Respiration. editors Douce R, Day

DA. Springer-Verlag, New York, pp 41 8-473.

Lambers H. 1997a. Respiration and the Alternative Oxidase In: A Molecular Approach

to Primary Metabolism in Higher Plants. editors Foyer, Quick. P. 295-309.

Lambers H. 1997b. Oxidation of mitochondnal NADH and the synthesis of ATP. in:

Plant Metabolism. editors Dennis DT, Turpin DH, Lefebvre DD, Layzell DB. Longman

Publishers. England. P. 200-220.

LaRosa PC, Haseywa PM, Bnssan RA. 1984. Photoautotrophic potato cells:

transition fkom heterotrophic to autotrophic growth. Physiol. Plant. 61: 279-286.

Lanon E, Howlett B, Jagendorf A. 1986. Artificial reductant enhancement of the

Lowry method for protein determination. Anal. BBiochem. 155: 243-248.

Lauer MJ, Blevins DG, Sienputowska-Gria 8. 1988. "P-nuclear magnetic

resonance determination of phosphate compiirtmentation in leaves of reproductive

soybeans as affected by phosphate starvation. Plant Physiol. 93: 504-5 1 1 .

Leifert C, Murphy KP, Lumsden PJ. 1995. Mineral and carbohydrate nutrition of plant

ce11 and tissue cultures. Crit. Views Plant Sci 14(2): 83-109.

Li XN, Ashihan H. 1989. Effects of inorganic phosphate on the utilization of sucrose by

suspension cultured Catharanthus roseus cells. Anals Bot. 64: 33-36.

Li XN, Asbihan 8. 1990. Effects of inorganic phosphate on sugar catabolisrn by

suspension cultured Catharanthus roseus. Phytochem. 29(2): 497-500.

Li Q, Ritzel RG, McLean LLT, McIntosb L, Ko T, Bertrand H, Nargang FE. 1996.

Cloning and analysis of the alternative oxidase gene of Neurospora crassa. Genetics.

142: 129-140.

Linsmaier EM, Skoog F. 1965. Organic growth factor requirement of tobacco tissue

cultures. P hysiol. Plant. 1 8 : 1 00- 1 27.

Lynch JP, Beebe SE. 1995. Adaptation of bans ( Phaseolus vulgaris L.) to low

phosphom availabiliîy. Hort-Sci. 306(6): 1 1 65- 1 1 7 1.

Makino A, Osmond B. 1991. Effects of nitrogen nutrition on nitrogen partitionhg

between chloroplasts and mitochondria in pea and wheat. Plant Physiol. 96: 355-362.

McHugb SC, Knowles VL, Blakeley SD, Sangwan RS, Miki BL, Demis DT, Pluton

WC. 1995. Differentiai expression of cytosolic and plastid pynrvate kinase isozymes in

tobacco. Physiol. Plant. 95: 507-514.

Mclntosh L. 1994. Molecular biology of the alternative oxidase. Plont Physiol. 105: 78 1 - 786.

Meeuse BJD. 1975. Themiogenic respiration in aroids. Anm. Rev. Plant Physiol. 26:

117-126.

Mikubka M, Bomsel JL, Rychter AM. 1998. The influence of phosphate deficiency on

photosynthesis, respiration and adenine nucleotide pool in bean leaves. Photosynthetica.

35(1): 79-88.

Millar AH, Day DA. 1997. Alternative solutions to radical problems. Trends in Plant

Sciences. 2(8): 289-299.

Millar AH, Atkin OK, Meaz RI, Henry B, Farquhar G, Day DA. 1998. Analysis of

respiratory chah regulation in roots of soybean seedlings. Plant Physiol. 1 17: 1083- 1093.

Mishn NP, Fatma T, Singhal GS. 1995. Development of antioxidative defense system

of wheat seedlings in response to high light. Physiol. Plant. 95: 77-82.

Moller IM, Berczi A, van der Plas L W , Lambers H. 1988. Measurement of the

activity and capacity of the altemative pathway in intact plant tissues: identification of

pro blerns and possible solutions. Physiol. Plant. 72: 642-649.

Moore AL, Siedow JN. 1991. The regulation and nature of the cyanide-tesistant

aitemative oxidase of plant mitochondria. Biochim. Biophys. Acta 1059: 12 1 - 140.

Murasbige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with

tobacco tissue cultures. Physiol. Plant. 1 5: 473497.

Murley VR, Theodorou ME, Pluton WC. 1998. Phosphate starvation-inducible

pyrophosphate-dependent phosphofructokinase occurs in plants whose roots do not form

symbiotic associations with myorrhizal fun@. Physiol. Plant. 1 03 : 405-4 1 4.

Newcomb W. 1997. Mitochondnal structure. In: Plant Metabolism. editors Dennis DT,

Turpin DH, Lefebvre DD, Layzell DB. Longman Publishen. England. P. 163-165.

Nielsen TH, Krapp A, Roper-Schwan U, Stitt M. 1998. The sugar-mediated

regulation of genes encoding the small subunit of rubisco and the regulatory subunit of

ADP glucose pyrophosphorylase is modified by phosphate and nitrogen. Plant Ce11

Environ. 2 1 : 443-454.

Oparka KJ, Reid ND. 1994. The use of fluorescent probes for studies of living plant

cells. In: Ce11 Biology: A practical approach. 26-50.

Palet A, Ribas-Carbo M, Argiles JM, Azcon-Bieto J. 1991. Short-term effects of

carbon dioxide on carnation callus ce11 respiration. Plant Physiol. 96: 467472.

Pamplin EJ, Chapman TM. 1975. Sucrose suppression of chlorophyll synthesis on the

activity of the enzymes of the chlorophyll synthetic pathway. J. Exp. But. 26: 2 12-220.

Pascal B, Douce R. 1993. Effect of uon deficiency on the respiration of sycamore (Acer

pseudoplutanus L.) cells. Plant Physiol. 1 30(4): 1 329- 1 3 3 8.

Paul MJ, Stitt M. 1993. Effects of nitrogen and phosphorus deficiencies on levels of

carbohycùates respiratory enzymes and metabolites in seeàlings of tobacco and their

response to exogenous sucrose. Plant Cell Environ. 16: 10474057.

Peel E. 1982. Photoautotrophic growth of suspension cultures of Asperagus oflcinaliis L.

cells in turbidostats. Plant Sci. Lett. 24: 147-1 55.

Popov VN, Sirnonian RA, Skulachev VP, Starkov AA. 1997. Inhibition of the

alternative oxidase stimulates H202 production on plant mitochonària. FEBS. 41 5: 87-90.

Purvis AC, Shewfelt RL. 1993. Does the alternative pathway arneliorate chilling injury

in sensitive plant tissues? Physiol. Plant. 88: 7 12-7 18.

Purvis AC, Shewfelt RL, Gegogeine JW. 1995. Superoxide production by mitochondria

isolated fiom green bel1 pepper fruit. Physiol. Plant. 94: 743-749.

Purvis AC. 1997. Role of the alternative oxidase in lirniting superoxide production by

plant mitochondria. Physiol. Plant. 100: 165- 170.

Rebeüle F. 1988. Photosynthesis and respiration in air-grown and COz-grown

photoautotrophic ce11 suspension cultures of carnation. Plant Sci. 54: 1 1-2 1.

Rhoads DM, McIntosh L. 1991. Isolation and characterization of a cDNA clone

encoding an alternative oxidase protein of Sauromatum guttatum (Schott) . Proc. Natl.

Acad. Sci. 88: 312202126.

Rhoads DM, McIntosh L. 1993. Cytochrome and alternative pathway respiration in

tobacco. Effects of salicylic acid Plant Physiol. 103: 877-883.

Royal JA, Ischiropoulos H. 1993. Evaluation of 2', 7-dichlorofIuorescin and

dihydrorhodamine 123 as fluorescent probes for intraceilular H202 in ~Ultured endotheliai

cells. Arch Biochem. Biophys. 302(2): 348-355.

Rufty TWJr, Huber SC, Voik RJ. 1988. Alterations in leaf carbuhydrate metabolism in

response to nitrogen stress. Plant Physiol. 88: 725-730.

Rychter AM, Mikuiska M. 1990. The relationship between phosphate status and

cyanide resistant respiration in bean mots. Physiol. Plant. 79(4): 663-667.

Sakajo S, Minagawa N, Komiyama T, Yoshimoto A. 1991. Molecular cioning of

cDNA for antimycin A-inducible mRNA and its role in cyanide-cesistant respiration in

Hansenula anomala. Biochim. Biophys. Acta. 1 090: 1 02- 1 08.

Salisbury FB, Ross CW, editors. 1978. PIant Physiology 2nd Edition. Wadsworth

Publishing Company, Inc. Belmont, California. pp. 174-1 84.

Shigenaga MK, Hagen TM, Ames BN. 1994. Oxidative damage and mitochondrial

decay in aging. Proc. Natl. Acad Sei. 9 1 : 1077 1 - 1 0778.

Siedow JN, Umbach AL. 1995. Plant mitochondrial electron transfer and molecular

biology. Plant Cell. 7: 821-83 1.

Siedow JN, Umbach AL, Moore AL. 1995. Hypothesis: The active site of the cyanide-

resistant oxidase from plant mitochondria contains a binuclear iron centre. FEBS. 362:

10-14.

Taiz L, Zeiger E, editors. 1991. Respiration In: Plant Physiology (Redwood City CA:

Benjdcummings Publishing). pp. 265-29 1.

Theodorou ME, Elrifi IR, Turpin DH, Pluton WC. 1991. Effects o f phosphorus

limitation on respiratory metabolism in the green alga Selenasmm minutum. Plant

Physiol. 95: 1089-1095.

Theodorou ME, Plutoa WC. 1993. Metaboiic adaptation of plant respiration to

nutritional phosphate deprivation. Plant Physiol. 101 : 339-344.

Theodorou ME, Pluton WC. 1994. Induction of PPi-dependent phosphofnctokinase

by phosphate starvation in seedlings of Brassica nigru. Plant Cell Environ. 17(3): 287-

294.

Thordal-Christensen A, Zbang 2, Wei Y, Collinge DB. 1997. Subcellular localization

of HzOz in plants. H202 accumulation in papillae and hypersensitive response during the

barley powdery mildew interaction. Plant J. 1 l(6): 1 1 87- 1 194.

Thontciosson B, Tillkrg JE. 1990. Changes in photosynthesis/respiration ratio and

levels of few carbohydrates in leaves of nutrient depleted barley and pas. J Plant

Physiol. 136: 532-537.

Tillberg JE, Rowley JR. 1989. Physiological and structural effects of phosphorus

starvation on the unicellular green alga Scenedesmus. Physiol. Plant. 75: 3 15-324.

Tsang EWT, Bowler C, Herouart D, Van Camp W, Vülarroel R, Genetello C, Van

Montap M, Ime D. 1991. Differential regdation of superoxide dismutases in plants

exposed to environmental stress. Phnt Cell. 3: 783-792.

Umbach AL, Siedow JN. 1993. Covalent and noncovalent dimers of the cyanide-

resistant alternative oxidase protein in higher plant rnitochondria and their relationship to

enzyme activity. Plant Physiol. 103: 845-854.

Umbach AL, Wiskich JT, Siedow JN. 1994. Regdation of alternative oxidase kinetics

by pynivate and intennolecuiar disulfide bond redox status in soybean seedling

mitochondria. FEBS. 348: 18 1-1 84.

Usuda H, Simogawarr K. 1992. Phosphate deficiency in maize. Changes in enzyme

activities during the course of phosphate deprivation. P h Physiof. 99: 1680- 1685.

Van Camp W, Capiau D, Van Mantagu M, Ime 4 Slooten L. 1996. Enhancement of

oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide

dismutase in chloroplasts. Plant Physiol. 1 1 2 1 703- 17 14.

Vance CP. 1997. Oxidation of mitochondnal NADH and the synthesis of ATP. In: Plant

Metabolism. editors Demis DT, Turpin DHy Lefebvre DDy Layzell DB. Longman

Publishers. England. pp. 200-220.

van Emmerik WAM, Wagner AM, vin der Plas L W . 1992. A quantitative

comparison of respiration in cells and isolated mitochondria fiom Petunia hybrida

suspension cultures: A high yield isolation procedure. J . Plant Physiol. 139(4): 390-396.

vanden Hoek TL, Becker LB, Shao 2, Li C, Schumacker PT. 1998. Reactive oxygen

species released fiom mitochondria during bief hypoxia induce preconditioning in

cardiomycetes J. Biot. Chem. 273 : 1 8092- 1 8098.

Vanlerbergbe CC, Schuller KA, Smith RG, Fei1 F, Pluton WC, Turpin DA. 1990.

Relationship between assimilation rate and in vivo phosphoenolpynivate

carboxylase activity. Plant Physiol. 94: 284-290.

Vanlerberghe CC, McIntosh L. 1992. Lower growth temperature increases alternative

pathway capacity and alternative oxidase protein in tobacco. Plant Physiol. 100: 1 15-1 19.

Vanlerberghe CC, McIntosh L. 1994. Mitochondrial electron transport regdation of

nuclear gene expression: studies with the alternative oxidase gene of tobacco. Plant

Physiol. 105: 867-874

Vanlerôerghe GC, Vanlerberghe AE, McIntosh L. 1994. Molecular genetic alteration

of plant respiration: silencing and ovetexpression of alternative oxidase in transgenic

tobacco. Plant Physiol. 106: 1503-1 5 10.

Vanlerberghe CC, Day DA, Wiskich JT, Vanlerberghe AE, McIntosh L. 1995.

Alternative oxidase activity in tobacco leaf mitochondria: dependence on tricarboxylic

acid cycle-mediated redox regulation and pynivate activation. Plant Physiol. 109: 353-

361.

Vanlerberghe GC, McIntosh L. 1996. Signals regulating the expression of the nuclear

gene encoding alternative oxidase of plant mitochondria. P h i Physiol. 1 1 1 : 589-595.

Vanlerberghe GC, Vaiilerberghe AE, Mclntosh L. 1997. Molecular genetic evidence

of the ability of alternative oxidase to support respiratory carbon metabolism. Plant

Physiol. 1 1 3 :65 7-66 1.

Vanlerberghe GC, McIntosh L. 1997. Alternative Oxidase: From Gene to Function.

Annu. Rev. Plant Physiol. Plant M d Biol. 48 :703-34.

Vanlerberghe CC, McIntosh L, Yip JYH. 1998. Molecular localization of a redox-

modulated process regulating plant mitochondrial electron transport. Plant Cell. 10: 1-1 1.

Veitb R, Komor E. 1993. Regulation of growth, sucrose storage and ion content in

sugarcane cells, measund with suspension cells in continuous culture grown under

nitrogen, phosphorus or carbon limitation. J. Plant Physid. 142: 41 4-424.

Visser R de, Lamben 8. 1983. Growth and the efficiency of mot respiration of Pisum

sarivum as dependent on the source of nitrogen. Physiol. Plant. 58: 533-543.

Wagner AM, Moore AL. 1997. Structure and hction of the plant alternative oxidase:

its putative role in the oxygen defence mechanism. Biosci. Rep. 17(3): 3 19-333.

Wagner AM, van den Bergen CWM, Wincencjusz 8. 1995. Stimulation of alternative

pathway respiration by succinate and malate. Plant Physiol. 1 O8(3): 1 O3 5- 1042.

Wagner AM, Knb K. 1995. The altemative respiration pathway in plants: role and

regdation. Physid. Plant. 95: 3 18-325

Weger HG. 1996. interactions between respiration and inorganic phosphate uptake in

phosphate-limited cells of Chiumydomonas reinhurdlii. Physiol. Plant.. 97: 635-642.

Wbelaa J, Hugosson M, Glaser E, Day DA. 199%. Studies on the import and

processing of the alternative oxidase precursor by isoiated soybean mitochondria. Plant

Mol. Biol. 27: 769-778

Whelan J, Smith MK, Meijer M, Yu JW, Badger MR, Price GD, Day DA. 1995b.

Cloning of an additional cDNA for the alternative oxidase in tobacco. Plant Physiol. 107:

146% 1470.

Whelan J, Mclntosb L, Day DA. 1996a. Sequencing of a soybean alternative oxidase

cDNA clone. Plant Physiol. 1 02: 1 48 1.

Whelan J, Mülar AH, Day DA. 1996b. nie alternative oxidase is encoded in a

multigene famil y in soybean. P lantu. 1 98: 1 97-20 1.

Wekens H, Villarroel R, Van Montagu M, Iaze D, Van Camp W. 1994. Molecular

identification of catalases h m Nicotiuna plumbaginifolia (L.). FEBS. 352: 79-83.

Wiaicur ZM, Zhang GF, Sbehelin LA. 1998. Auxin deprivation induces synchronous

golgi differentiation in suspension-cultured tobacco BY-2 ceils. Plunt Physiol. 1 17: 501-

513.

Yamada T, Imaishi H, Obkawa A. 1998. Molecular cloning and sequence analysis of

catalase cDNA fiom green pepper seedlings elicited with arachidonic acid Biosci.

Biotechnol. Biochem. 62 (4): 753-758.

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