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Interaction between respiratory carbon flow and photosynthetic light harvesting in the green alga, selenastrum… Holmes, Jody J. 1993

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We a^this thesis as conformingequired standardINTERACTION BETWEEN RESPIRATORY CARBON FLOW ANDPHOTOSYNTHETIC LIGHT HARVESTING IN THE GREEN ALGA,SELENASTR UM MINUTUMbyJODY J. HOLMESB.Sc.H., Queen's University, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Botany)THE UNIVERSITY OF BRITISH COLUMBIASeptember 1993© Jody J. Holmes, 1993(SignatureIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of /OkaniThe University of British ColumbiaVancouver, CanadaDate SeiptoAxA/ Zel ) I Rct 3DE-6 (2/88)llABSTRACTAlthough the regulation of respiration by photosynthesis has been extensively studied,very little is known about the regulation of photosynthesis by respiration. In this thesis, it wasproposed that changes in respiratory carbon flow would affect the ratio of reduced/oxidizedpyridine nucleotides and affect reduction of the PQ(cyt b6f) pool via a thylakoid-boundNAD(P)H-PQ oxidoreductase. In turn, reduction of the PQ(cyt b6f) pool would result in astate 1 to 2 transition which could poise the photosynthetic electron transport chain for adecrease in NADPH production. The purpose of the present study was to rigorously test thehypothesis that increased respiratory carbon flow, which increased reduced/oxidized pyridinenucleotide ratios, would increase PQ reduction and result in a state transition. The corollary ofthis hypothesis was that increased respiratory carbon flow, which did not affect the ratio ofreduced/oxidized pyridine nucleotides, would not affect PQ(cyt b6f) reduction or result in astate transition. In the green alga, Selenastrum minutum, five treatments were shown toincrease dark respiratory carbon flow as measured by CO2 release and/or starch degradation.These treatments were further subdivided on the basis of their ability to cause a state transition.Class 1 treatments included NH4+ assimilation by N-limited cells, anaerobic treatment, anduncoupling with CCCP and all resulted in large perturbations in room temperature and 77Kfluorescence emission indicative of a state 1 to 2 transition. These changes were correlated withreduction of the PQ pool as measured by changes in the kinetics of time-resolved fluorescencedecay and induction. Class 2 treatments included NO3 - assimilation by N-limited cells and Piassimilation by Pi-limited cells. Both of these treatments resulted in only small changes influorescence emission suggesting that a state 1 to 2 transition had not occurred. Both NO3 -and Pi treatment had only minor effects on PQ(cyt b6f) reduction as measured by fluorescencedecay kinetics. However, in cells treated with NO3 - , measurement of PQ reduction made usingfluorescence induction kinetics was not consistent with the original hypothesis. Thisinconsistency was proposed to result from actinic effects of signal averaging. Increases in theiuNADH/NAD but not NADPH/NADP ratio were correlated with class 1 treatments while class 2treatments resulted in small (Pi) or intermediate (NO3 -) changes in NADH/NAD ratios. Keyrespiratory metabolites were examined for each of the five treatments. An examination of thecombined mass action ratio for TP to PGA conversion indicated that carbon flow, via NAD-GAPDH and PGA kinase, was significantly enhanced by class 1 treatments while no significantchange was noted for class 2 treatments. NAD-GAPDH has been shown to be 86% localized inthe chloroplast in Chlamydomonas reinhardtii (Klein, 1986 Planta 167:81). It was proposedthat chloroplastic NAD-GAPDH activity was responsible for an increase in chloroplasticNADH/NAD ratios, reduction of PQ, and a state transition. The results of the present studysuggest that respiratory carbon flow can regulate the poising of the photosynthetic electrontransport chain for the NADPH/ATP production ratio via the state transition. This may haveramifications for interactions between respiration and photosynthesis in both the dark and thelight. The physiological significance of this interaction was discussed.TABLE OF CONTENTSABSTRACT^ iiTABLE OF CONTENTS^ ivLIST OF FIGURES viiLIST OF TABLES^ ixLIST OF SYMBOLS AND ABBREVIATIONS^ xACKNOWLEDGMENTS^ xiiPROLOGUE^ xiiiCHAPTER 1: THEORETICAL CONSIDERATIONSGENERAL INTRODUCTION AND LITERATURE REVIEW^ 1Photosynthesis^ 1Respiration 6Interaction between photosynthesis and respiration^ 8A mechanism to regulate the ratio of linear to cyclic electron transport^ 14Can respiratory carbon flow influence the redox state of the PQ(cyt b6f) pooland poise the ratio of linear to cyclic electron flow?:^ 20The hypothesis to be tested:^ 31CHAPTER 2: EXPERIMENTAL RATIONALE: A FRAMEWORK TO TESTTHE HYPOTHESISINTRODUCTION^ 33Rationale for the hypothesis to be tested^ 33Methods which can be used to measure state transitions^ 34Rationale for working with Selenastrum minutum 37MATERIALS AND METHODS^ 38Cell culture (chemostats) 38Treatments^ 39Experimental 40Fluorescence measurements^ 40Other measurements 41RESULTS^ 42Effect of treatments on CO2 efflux and starch degradation^ 42Effect of treatments on fluorescence emission^ 42DISCUSSION^ 47Effect of treatments on respiratory carbon flow 47Effect of treatments on fluorescence emission ^ 51ivSUMMARY^ 55CHAPTER 3: TESTING THE MODEL: THE EFFECTS OF CLASS 1 andCLASS 2 TREATMENTS ON REDUCTION OF THE PQ(CYT b6f) POOLINTRODUCTION^ 57Methods which can be used to infer the redox state of the PQ pool^ 58MATERIALS AND METHODS^ 60Experimental conditions 60Fluorescence^ 60RESULTS^ 63Time-resolved fluorescence decays^ 63Time-resolved fluorescence inductions 65DISCUSSION^ 69DCMU and Class 1 treatments:^ 69Class 2 treatments (Pi and NO3 - assimilation)^ 71SUMMARY^ 73CHAPTER 4: THE EFFECTS OF CLASS 1 AND CLASS 2 TREATMENTSON THE REDOX STATE OF THE PYRIDINE NUCLEOTIDE POOLINTRODUCTION^ 75Techniques to measure pyridine nucleotides^ 77MATERIALS AND METHODS^ 78Experimental^ 78Pyridine nucleotide determinations^ 78Fluorescence 80RESULTS^ 80NADP, NADPH, and the NADPH/NADP ratio^ 81NAD, NADH, and the NADH/NAD ratio 83Fluorescence^ 87DISCUSSION 90NADP, NADPH, and the NADPH/NADP ratio^ 90NAD, NADH, and the NADH/NAD ratio 92SUMMARY^ 96CHAPTER 5: INTERACTION BETWEEN RESPIRATORY CARBON FLOWAND THE STATE TRANSITION AFTER UNCOUPLING WITH CCCPINTRODUCTION^ 98MATERIALS AND METHODS^ 99Experimental .^ 99Starch degradation 100Metabolites 100Gas exchange^ 100Fluorescence 101RESULTS^ 101The effect of CCCP on key respiratory metabolites ^  101viDISCUSSION^ 111Metabolic changes observed after uncoupling with CCCP^ 111SUMMARY 122CHAPTER 6: DEVELOPMENT OF A COMPREHENSIVE MODEL FORTHE INTERACTION BETWEEN RESPIRATORY CARBON FLOW ANDPOISING OF THE STATE TRANSITIONINTRODUCTION^ 124RESULTS^ 126Kinetics of changes in ADP, Pyr/PEP and FBP/F6P^ 126PGA/TP, NADH/NAD, ATP/ADP and F ([PGA][NADH][ATP] /[TP] [NAD] [ADP])^ 129DISCUSSION^ 131Activation of respiratory carbon flow : the effect of class 1 and 2 treatments onADP, Pyr/PEP, FBP/F6P and PGA/TP^ 131The effect of class 1 and 2 treatments on the combined mass action ratio forNAD-GAPDH and PGA kinase 132The physiological significance of interaction between respiratory carbon flowand photosynthesis via the CRETC in vivo^ 136CHAPTER 7: GENERAL SUMMARY AND CONCLUSIONS^ 139LITERATURE CITED^ 143APPENDIX 1: APPROXIMATION OF qNp AND qp^ 163APPENDIX 2: CORRECTION OF TIME-RESOLVED FLUORESCENCEDECAYS FOR THE ACTINIC EFFECTS OF THE MEASURING BEAMINTRODUCTION^ 166MATERIALS AND METHODS^ 167RESULTS^ 167DISCUSSION 173APPENDIX 3: METABOLITE ASSAYS^ 177CURRICULUM VITAE^ 179LIST OF FIGURESFigure 1: A diagrammatic representation of the photosynthetic electron transportchain and carbon fixation in the chloroplast^ 2Figure 2: Pathways of respiratory carbon flow and mitochondrial respiratory electrontransport in photosynthetic organisms^ 7Figure 3: The phosphorylation mobile antennae model for the state transition^ 16Figure 4: Thylakoid electron transport pathways which share plastoquinone in greenalgae^ 23Figure 5: The effects of treatments which increase respiratory carbon flow on steady-state saturation pulse fluorescence analysis^ 44Figure 6: The effect of class 1 treatments on absolute fluorescence emission at 77K ^ 48Figure 7: The effect of class 1, class 2 and DCMU treatments on corrected time-resolved fluorescence decay kinetics^ 64Figure 8: The effect of class 1, class 2 and DCMU treatments on time-resolvedfluorescence induction kinetics^ 67Figure 9: The effect of class 1 treatments on NADP, NADPH and theNADPH/NADP ratio^ 82Figure 10: The effect of class 2 treatments on NADP, NADPH and theNADPH/NADP ratio^ 84Figure 11: The effect of class 1 treatments on NAD, NADH and the NADH/NADratio^ 85Figure 12: The effect of class 2 treatments on NAD, NADH and the NADH/NADratio^ 88Figure 13: The effect of oxygen re-addition on room temperature fluorescence fromcells which were anaerobically adapted for 20 minutes^ 89Figure 14: The effect of CCCP treatment on the long term rate of starch degradation ^ 103Figure 15: The effect of CCCP treatment on the cellular concentration of adenylates ^ 104viiFigure 16: The effect of CCCP treatment on the cellular concentration of pyruvateand phospho enol pyruvate ^  105Figure 17: The effect of CCCP treatment on cellular levels of fructose bisphosphate,fructose-6-phosphate and triose-phosphate^ 106Figure 18: The effect of CCCP treatment on cellular levels of glucose-6-phosphateand glucose-l-phosphate^ 108Figure 19: The effect of CCCP treatment on the rate of dark CO2 efflux as measuredby an open gas exchange IRGA system^ 110Figure 20: The effect of CCCP treatment on the rate of dark 02 consumption^ 112Figure 21: The effect of CCCP treatment on room temperature and 77K fluorescenceparameters^  113Figure 22: The proposed mechanism for interaction between respiratory carbon flowand poising of the PETC by the state transition after uncoupling with CCCP^ 115Figure 23: The effect of light intensity on the measurement of room temperaturefluorescence parameters^ 165Figure 24: The effect of DCMU and class 1 and 2 treatments on uncorrected time-resolved fluorescence decay kinetics^  169Figure 25: The effect of DCMU and class 1 and 2 treatments on the level offluorescence induced by the 100 kHz measuring beam^ 172viiiLIST OF TABLESTable 1: The effect of a variety of treatments on the rate of respiratory carbon flow inS. minuturn as measured by the rate of CO2 efflux and starch degradation^ 43Table 2: A summary of the effect of treatments which increased respiratory carbonflow on room temperature and 77K fluorescence parameters in S. minuturn ^ 46Table 3: The effect of DCMU and class 1 and 2 treatments on the amplitude and half-times of the fast and medium components of time-resolved fluorescence decays whichwere corrected for the actinic effect of the 100 kHz measuring beam 66Table 4: The effect of DCMU and class 1 and 2 treatments on^the area abovetime-resolved fluorescence induction curves^ 68Table 5: The effect of CCCP on the PEP/TP, ATP/ADP, NADH/NAD and combinedmass action ratio for NAD-GAPDH and PGA kinase compared to the dark aerobiccontrol^ 109Table 6: A comparison of the effects of class 1 and class 2 treatments on changes inkey respiratory intermediates and their ratios^  127Table 7: The effect of illumination and treatments which increase the rate ofrespiratory carbon flow on the PGA/TP, ATP/ADP, NADH/NAD and combined massaction ratio compared to the dark aerobic control^ 130Table 8: The effect of DCMU and treatments which increase respiratory carbon flowon the amplitude and half times of the fast and medium components of time-resolvedfluorescence decays which were not corrected for the actinic effect of the 100 kHzmeasuring beam^ 170ixLIST OF SYMBOLS AND ABBREVIATIONS: amplitude of the ith component of a time-resolved fluorescence decayadenosine diphosphateAccelerator of the Deactivation Reaction of Y, the H2O oxidizing complex ofPS2area between FM and the time-resolved fluorescence induction curveadenosine monophosphateadenosine triphosphatecarbonyl cyanide-m-chlorophenyl-hydrazonechlorophylltreatments which increase respiratory carbon flow and cause a state transitiontreatments which increase respiratory carbon flow but do not cause a statetransitionchloroplastic respiratory electron transport chaincytochrome3-(3,4-dichloropheny1)-1,1 methyl ureadibromothymoquinoneelectronthe level of fluorescence induced by the measuring beam after treatmentflavin adenine dinucleotidefructose 1,6, bisphosphatefructose 1,6 bisphosphataseminimal level of fluorescence induced by the "non-actinic" measuring beam indark-adapted cellsquantum yield of PS2 upon illuminationferredoxinmaximal level of fluorescence induced by a saturating flashferredoxin NADP reductasefructose 6-phosphateglyceraldehyde 3-phosphate dehydrogenaseglucose-6-phosphateglucose-6-phosphate dehydrogenaseglucose 6-phosphateglucose 1-phosphatehydrogenasepotassium cyanidemajor light harvesting complex of PS2 in higher plants and green algaemajor light harvesting complex of PS 1 in higher plants and green algaemitochondria electron transport chainnicotinamide adenosine dinucleotidenicotinamide adenosine dinucleotide phosphateammoniumnitrate/nitriteociADPADRYAmaxAMPATPCCCPchlclass 1class 2CRETCcytDCMUDBMIBe-FFADH2FBPFBPaseFocpFdFMFNRF6PGAPDHG6PG6PDHG6PGIPH2aseKCNLHC2LHC1METCNADHNADPHNH4+NO3 -/NO2 -OPPP680 , P700PCPEPPETCPFKPGAPi6PG6PGDHPKPRKPQ/PQH2PS2PS1PyrQAQBqEclIqNpclPq(t)(ITRUBISCORuBPSHAMTCATiTPoxidative pentose phosphate (pathway)reactive chlorophylls of photosystem 2 and 1, respectivelyplastocyaninphospho enol pyruvatephotosynthetic electron transport chainphosphofructokinasephosphoglycerateinorganic phosphate6-phosphogluconate6-phosphogluconate dehydrogenasepyruvate kinasephosphoribulokinaseplastoquinone/plastoquinolphotosystem 2photosystem 1pyruvateprimary quinone acceptor of PS2secondary quinone acceptor of PS2energized quenching of fluorescence due to ApHquenching of fluorescence due to photoinhibitionnon photochemical quenchingphotochemical quenchingfraction of reduced PS2 reaction centres at time, tquenching of fluorescence due to a state transitionribulose bisphosphate carboxylase/oxygenaseribulose bisphosphatesalicylhydroxamic acidtricarboxylic acid (cycle)the half-time of the ith component of a time-resolved fluorescence decaytriose phosphate (refers to dihydroxyacetone phosphate andglyceraldehyde-3-phosphate)xixiiACKNOWLEDGMENTS: First and foremost, I must acknowledge the contribution of Dr. David H. Turpin whohas always encouraged his students to look at the "big picture". Not only has he helped me seethe forest for the trees, but he has also ensured that each tree was acknowledged on the way.Secondly, I want to thank all of the members of the traveling Turpin road show whoprovided some continuity in an otherwise tumultuous world of musical labs. A big thank you toDaggo for advice on metabolite assays, Troyboy for great hair days, general lab merriment andtube ticklin', Stormin' Norman for late night company and introducing me to some great folkguitarists, Annaramalamadingdong for an eye into the young and the restless, Dericko for hisOK black T-shirts, Fartee for tube ticklin', retaining a semblance of order in chaos and incitingTroyboy to new heights and Feather for some great non-scientific advice given along the way.In addition, thank you to the members of my committee, Dr. Edith Camm and Dr.Beverly Green for thoughtful advice. Also, a very big thank you to Dr. Robert Guy who helpedme through the mathematical complexities of Sigma Plot and statistics when time was at apremium.Last, but not least, a HUGE thank you to Nicholas Beatty who has selflessly providedsustenance and support throughout and who taught me that fear is all in the mind.PROLOGUEThis thesis is an integrative work and covers topics ranging in diversity from physiologyto biophysics. The challenge in engaging these different disciplines is to introduce each conceptin context and to provide enough theory for the uninitiated reader. To this end, I have writtenan extremely general literature review to introduce the larger concepts on which theexperimental rationale for this thesis is based. Each subsequent chapter deals with a subset ofthe larger, integrative hypothesis developed in the first chapter. A more specific review of thepertinent theory and techniques utilized is provided in the introductory section of each chapter.For added clarity, each chapter is briefly set into the context of conclusions from the precedingchapters.ICHAPTER 1: THEORETICAL CONSIDERATIONSGENERAL INTRODUCTION AND LITERATURE REVIEWFrom an anthropocentric viewpoint, human survival depends upon the use of energyinitially fixed by photosynthetic organisms. Photosynthesis is an anabolic process whichconverts light energy into chemical energy in the form of Al? and NADPH which are used asthe energetic currency of the living world. In the plant cell, ATP and NADPH equivalents arestored in reduced carbon compounds via photosynthetic carbon fixation. In turn, the energystored in these compounds can be released for cellular metabolism by respiration which iscatabolic in nature. Since cellular metabolism, including respiration, is not completely efficient,some of the energy stored in reduced carbon compounds is lost as heat. Without photosynthesisto continually replace energy depleted by the respiratory and metabolic needs of heterotrophicand photosynthetic organisms, the f ee energy level of the biosphere would rapidly decrease.Despite the importance of photosynthesis to life on this planet, relatively little is known about itsregulation or interaction with other metabolic processes in the photosynthetic cell. Even less isknown about the interaction between photosynthesis and respiration.The following discussion will review the pertinent concepts of photosynthesis andrespiration and move on to explore what is known about interactions between photosynthesisand respiration. Then, a theoretical mechanism will be developed whereby respiration mayregulate photosynthetic light harvesting to alter the poising of ATP and NADPH production bythe light reactions of photosynthesis. This will provide an explanation of the experimentalrationale for this research.Photosynthesis:Photosynthesis consists of three distinct but intimately related processes which includelight harvesting, the photosynthetic electron transport chain (PETC, see list of abbreviations)and the production of reduced carbon compounds (Calvin cycle) (Figure 1).thylakoidmembranePS2P680H2O 1/2 02+ 2 H+lumen2 H+ PETCATPCalvin CycleATP (1)^Pi ■ ^-44 )(D 101Ru5P^sedoheptuloseribose5-P erythrose F-4P^BP1,3 bis PGA NADPHCHLOROPLASTRuBP sedoheptulose -1,7 bis P^DHAP -41:1-310" GAPCO2 3PGAC)— export(cytosol)starch[W]NADPH^4 ATPCDXyulose5Pstroma-7 PFigure 1: A diagrammatic representation of the photosynthetic electron transport and carbonfixation in the chloroplast. For an explanation see text. Enzymes: 1, Ribulose bisphosphatecarboxylase/oxygenase; 2, phosphoglycerate kinase; 3, glyceraldehyde phosphate dehydrogenase;4, Triose-phosphate isomerase; 5,8, aldolase; 6, Fructose bisphosphatase; 7,10,12, transketolase;9, sedoheptulose bisphosphatase; 11, Ribose-5 phosphate isomerase; 13, Ribulose-5 phosphate 3-epimerase; 14, phosphoribulokinase. Abbreviations: Ru5P, ribulose 5-phosphate; RuBP, ribulosebisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; TPP,thiamine pyrophosphate; C2, 2 carbon compound; CF0/CF1, coupling factor or chloroplastic ATPsynthase; hv, light. For rest of abbreviations, see list of abbreviations.3Photosynthetic light harvesting and electron transport:^Light harvesting by accessoryantenna pigments is required to provide excitation to the reaction center chlorophylls ofphotosystem 2 (PS2) and photosystem 1 (PS 1), P680 and P700, respectively. The efficiency ofelectron transport, and hence carbon fixation, is dependent on the light harvesting capacity ofthe two photosystems. In a process known as primary charge separation excitation of thereaction center chlorophyll allows reduction and oxidation of the primary electron (e -) acceptorand donor of each photosystem . With the help of a series of electron transfer components suchas plastoquinone (PQ), cytochrome (cyt) b6f, and plastocyanin (PC), electrons are transferred ina coordinated light dependent fashion from water to ferredoxin via PS2 and PS 1 in a mannerwhich has been formalized in the Z scheme (Hill and Bendal, 1960). The components of thelight reactions of photosynthesis, known as the photosynthetic electron transport chain (PETC),are located in the thylakoid membrane such that electrons are transported from the thylakoidlumen to the thylakoid exterior and protons are transported in the opposite direction; the resultis the production of a transthylakoid electrochemical gradient. Dissipation of this gradient isaccomplished by the ATP synthase which converts the free energy of the transthylakoidelectrochemical gradient into ATPDuring linear electron transport, electrons from Fd red are utilized to produce NADPHvia Fd/NADP reductase (FNR). Although the regulation of electron flow pathways is not wellunderstood, electrons from Fdred can also be utilized to reduce 02 (pseudocyclic e transportor Mehler reaction), PQ(cyt b6f) (cyclic electron transport) or NO3 -/NO2 - . The relative ratioof these types of electron transport can affect the stoichiometry of the ATP/NADPH productionratio via the light reactions. For more detailed reviews see Lawlor (1987), Murphy (1986) andNelson and Prezelin (1990).Thylakoid lateral heterogeneity:^In green algae and higher plants, improvements inthylakoid sub-fractionation techniques and immunocytochemical electron microscopy haveshown that intrinsic thylakoid protein complexes exhibit heterogeneous lateral distribution4(Anderson, 1992, 1989; Vallon et al., 1986; Olive et al., 1986). The two photosystems areobserved to be unequally distributed; PS2 is found almost exclusively in the stacked granalthylakoids while PS1 is found in the unstacked stromal thylakoids (Allen, 1992). Some PS1 isfound in the margins of the grana thylakoids and is thought to contribute to linear electrontransport. In addition, PS213, a photochemically inactive form of PS2, is found in the stromalregions (Albertsson et al., 1990; Anderson, 1989, 1992). Immunogold labeling shows that cytb6f complexes are distributed laterally between the grana, stroma and grana margins (Olive etal., 1986) although distribution is dynamic and appears to be under redox control (Vallon et al.,1991). Similarly, PQ and PC are distributed throughout the three thylakoid domains. Stericconsiderations appear to govern the distribution of ATP synthase which has a bulky head group.ATP synthase is found in both the stromal thylakoids and the granal margins but not the stackedgranal thylakoids (Svensson et al., 1991). 84% of FNR is located in the stroma and granalmargins while 16% is located in the grana (Vallon et al., 1986).Lateral heterogeneity has several implications. First, mobile electron carriers arerequired for transfer of electrons from PS2 to PS 1. Although PQ was initially favoured as themobile carrier, recent work by Joliot and coworkers has suggested that rapid diffusion of PQ islimited to small domains of less than 8 connected PS2 centres (Joliot et al., 1992; Joliot andJoliot, 1992; Lavergne et al., 1992). On the other hand, immunolocalization of PC has showndramatic light to dark differences in the location of PC indicating that PC may well be capableof facilitating rapid long range e - transfer between PS2 and PS 1 (Haehnel et al., 1989). Anotherimportant consequence of lateral heterogeneity of PETC components is that it may provideseparate domains for cyclic and linear electron transport (Svensson et al., 1991). At high lightintensity, it is unlikely that long range transport of e - would occur between the grana and thestroma. Since PS213 is not photochemically functional, Anderson (1992) suggests that underthese circumstances linear e - flow would be restricted to the grana and the grana margins whilePS1 in the stroma would be restricted to cyclic photophosphorylation. Although in vivoevidence for this theory is scarce, Anderson (1989) reported that, in in vitro preparations, the5rate of linear photophosphorylation in prestacked thylakoids was 2 fold higher than in artificiallyunstacked thylakoids at saturating light while the opposite was true for the rate of cyclic e -flow.Photosynthetic carbon fixation: ATP and NADPH produced by the light reactions ofphotosynthesis are utilized in the chloroplast stroma for carbon fixation and reduction in aprocess known as the Calvin cycle (or the reductive pentose phosphate pathway) (see Figure 1).CO2 is fixed via ribulose bisphosphate carboxylase/oxygenase (RUBISCO) which carboxylatesRuBP to form 2 molecules of PGA. ATP and NADPH are used to phosphorylate and reducePGA to triose phosphate (DHAP and GAP) in reactions catalyzed by PGA kinase and GAPDH.Triose phosphate can be utilized to regenerate RuBP, produce starch, or exported from theplastid to form sucrose. The ratios of these three processes depends on the tissue type and therelative source and sink demands outside the tissue (Macdonald and Buchanan, 1990).Although tight coupling exists between the light reactions and CO2 fixation, reductantand ATP produced by the light reactions can also be utilized for assimilation of inorganic N, Por S and for biosynthetic reactions in the stroma such as amino acid and protein biosynthesis(Lawlor, 1987; Turpin and Weger, 1990). Fixation of CO2 via the reductive pentose phosphatepathway in C3 plants requires approximately 1.5 ATP per NADPH (Heber and Walker, 1992).Assuming that the H -Ele-ratio of linear electron transport is 2, however, the ATP/NADPHproduction ratio of linear electron transport is 1.3. This implies that some other form ofelectron transport is necessary to supplement ATP production (Heber and Walker, 1992).Moreover, metabolism which had a different ATP/NADPH requirement ratio occurringsimultaneously with, or instead of CO2 fixation, could also result in ATP requirements in excessof those provided by linear electron transport alone. It has been proposed that increased cellularATP requirements in the light are met either by pseudocyclic or cyclic electron transport(Horton, 1985; Heber and Walker, 1992; Lawlor, 1987).6Respiration:Respiration involves 4 major related pathways including glycolysis, the oxidative pentosephosphate (OPP) pathway, the tricarboxylic acid (TCA) cycle and the mitochondria' electrontransport chain (METC). Respiratory carbon flow (OPP pathway, glycolysis and TCA cycle) iscompartmentalized in the stroma of the chloroplast, the cytosol and in the matrix of themitochondrion (Figure 2). Starch is initially degraded to glucose-6-P (G6P) via starchphosphorylase and amylase and further oxidized to triose phosphates (TP) and/orphosphoglycerate (PGA) in the chloroplast. Oxidation of G6P to TP and PGA can occur eithervia glycolysis or via the oxidative pentose phosphate pathway (Stitt, 1990). The ratio of flowthrough these two pathways usually indicates the metabolic status of a tissue with high OPPpathway activity generally indicative of high levels of biosynthesis (Miernyk, 1990). TP or PGAis then transported from the chloroplast to the cytosol via a TP(PGA)/Pi translocator where it isfurther oxidized to pyruvate via the "lower half' of glycolysis. Pyruvate is transported into themitochondria where it is oxidized and decarboxylated by the tricarboxylic acid (TCA) cycle.Oxidation of carbon via respiratory carbon flow is accompanied by the production of reductant(NADH, FADH2). Reductant can be oxidized either by the METC with concomitantchemiosmotic production of ATP and consumption of 02 or by other processes such as NO3 -/NO2 - reduction (Turpin and Weger, 1990).The components of the METC are membrane-bound and exhibit remarkable similarity tothe components of the PETC. Both electron transport systems contain mobile quinone carriers(ubiquinone in the METC, plastoquinone in the PETC) and a membrane-boundquinone/cytochrome be-type oxidoreductase (Scherer et al., 1990). Electron transport iscoupled to proton transport across the membrane and, in both cases, a membrane-specific ATPsynthase (chloroplastic: CF0/CF1, mitochondria': Fo/F 1) converts the free energy of theresultant electrochemical gradient into ATP. The major difference between the two is that theMETC oxidizes NADH to produce ATP whereas the PETC utilizes light energy to reduceNADP+ and produce ATP.starchstarch^pphosphorylaseir glucose-1 PPGMglucose-6PNADPHOPP pathway^ 6P-gluconateHPIATP^Fructose-6PPFKFructose-1,6 bisPATPA ) so, 3PGA4111PGA kinase■^AoAr3PGA PiPGA kinaseNADHPi Triose-P uk--^Db. 1,3 bisPGAGAPDHAIDATPI^ NADHTriose-P 1^110.GAPDH1,3 bisPGAcytosol CO2PEPc'asePGA mutaseenolaseacetyl CoA tollfmPDHC  Pyr ^Cm ito^YirNADH^OAAPEP^TPPyrTCAcycle^isocitratecitrateisocitrateMDHmalateNADHsuccinate^ DH^NADHDH2-oxoglutaratesuccinate^2-OGDHFADH2NADHATPNADH^METC H+intermembranespaceH+H+[W]H+chloroplastNADPHRibulose-5P6PGDHG6PDHFigure 2: Pathways of respiratory carbon flow and mitochondrial respiratory electron transportin photosynthetic organisms. See text for discussion. Abbreviations: PGM,phosphoglucomutase; HPI, hexose phosphate isomerase; PDHC, pyruvate dehydrogenasecomplex; DH, dehydrogenase; 2OGDH, 2-oxoglutarate dehydrogenase; G6PDH, glucose-6phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; cyt bc1, cytochromebc1; CoA, coenzyme A; UQ, ubiquinone; Fo/F 1, mitochondrial ATP synthase. See list ofabbreviations for further information.8Interaction between photosynthesis and respiration:In photosynthetic organisms, the interaction between respiration and photosynthesis isnot well understood. Despite reductionist attempts to separate photosynthesis from respiration,we are beginning to understand that these two processes must interact at a fundamental level. Itis important to keep in mind that photosynthesis and respiration are opposing processes.Photosynthesis converts light energy into ATP, reductant, and ultimately into reducedcarbohydrate while CO2 is consumed and 02 produced. Respiration, on the other hand,involves the oxidation of reduced carbon from photosynthesis to produce reductant which canthen be used in the production of ATP with concomitant consumption of 02 and production ofCO2. To prevent futile cycling, it is extremely important that these two processes becoordinately regulated.The most likely mediators of interaction between photosynthesis and respiration are theadenine nucleotides (ATP, ADP, AMP) and the pyridine nucleotides (NADH, NAD, NADPH,NADP), effectors which have been shown to regulate many key regulatory enzymes of carbonflow (Graham, 1980). Respiration and photosynthesis involve processes which consume andproduce ATP and NAD(P)H in a dynamic fashion and with potentially different stoichiometriesrequiring an intimate co-regulation between the individual components of both photosynthesisand respiration. Our understanding of the interaction between photosynthesis and respiration isstill frustratingly limited and much of the information available at present is conflicting. Muchof the present understanding of this interaction has been derived from biochemical studies of keyregulatory enzymes and studies of the effects of the induction of photosynthetic carbon fixationon respiration. Although the effects of light-induced changes in pyridine and adeninenucleotides on mitochondrial respiration has been studied in some depth, little is known aboutthe effect respiration has on photosynthesis. This is particularly important as respiration inplants has a major biosynthetic function.9Direct regulation of respiration by photosynthetic activity in the light:Much confusion about the interaction between photosynthesis and respiration has arisenfrom measurements of gas exchange. This is due to the fact that measurement, in the light, ofrespiration in photosynthetic cells is hampered by the superimposition of other gas exchangeprocesses such as photosynthesis, photorespiration and the Mehler reaction (Turpin and Weger,1990). Measurement of respiratory 02 consumption in the light has resulted in conflictingobservations of the effect of photosynthesis on respiration. For example, some authors reportcomplete inhibition of respiration upon illumination (Mehler, 1951; Radmer and Kok, 1976;Shiraiwa et al., 1988; Avelange and Rebeille, 1991) while others observe little or no effect(Gerbaud and Andre, 1980; Peltier and Thibault, 1985; Weger et al., 1988). Similarly,measurements of respiratory CO2 efflux in the light have shown values ranging from 20 to200% of dark CO2 release rates (Piesker and Apel, 1980; Sharp et al., 1984; Azcon-Bieto andOsmond, 1983; Brooks and Farqhuar, 1985; Avelange and Rebeille, 1991; Weger et al., 1989).Because no biochemical measurements have accompanied gas exchange measurements in mostcases, it is difficult to resolve these discrepancies.Classic work on the effect of illumination on pyridine and adenine nucleotides was doneby Heber and Santarius who showed that, in the first few minutes of a dark-to-light transition,energy charge (Heber and Santarius, 1965) and the ratio of reduced/oxidized pyridinenucleotides both increased (Santarius and Heber, 1965). Levels of cofactors were measured inthe chloroplastic and cytosolic compartments by means of non-aqueous fractionation; thesecofactor levels showed that pyridine nucleotides were strictly compartmentalized sincechloroplastic NADP was observed to become rapidly reduced while cytosolic NADP was muchless affected (Heber, 1974). Longer-term experiments with chloroplasts confirmed increases inthe redox state of the chloroplast upon illumination (Harvey and Brown, 1969). In contrast,these same experiments suggested that the ATP/ADP ratio returned to levels close to darklevels in the light after a few minutes (Hampp et al., 1982; Stitt et al., 1982).10It is generally accepted that the METC is under adenylate control (Lambers, 1990; Dayet al., 1987; Wiskich, 1980). This control may function directly at the level of ADP-limitationof the ATP synthase or by ADP-limitation of respiratory carbon flow via pyruvate kinase orPGA kinase which would, in turn, limit reductant availability for METC oxidation (Lambers,1990). It has been proposed that the high ATP/ADP ratio observed during illumination in thechloroplast stroma is transferred to the cytosol via a TP/PGA shuttle and that the consequenttransient increase in the cytosolic ATP/ADP ratio causes ADP-limitation of glycolysis and therate of METC activity (Douce, 1985; Santarius and Heber, 1965). Importantly, the increase inthe cytosolic ATP/ADP ratio is transient and returns to dark levels within approximately oneminute; this makes it unlikely that ADP-limitation, via photosynthesis, is a large factor undernormal photosynthetic conditions (Hampp et al., 1982; Stitt et al., 1982). The recovery of thecytosolic ATP/ADP ratio during illumination may result from the induction of photosyntheticCO2 fixation and sucrose synthesis which would decrease stromal ATP/ADP ratio (Hanson,1992).Photosynthetic control of respiration could also function at the level of high reducingpower generated by the light reactions since transfer of ATP to the cytosol via a TP/PGAshuttle would also involve transfer of reductant. It is likely that at least some of the decrease inrespiratory CO2 efflux observed upon illumination is a result of inhibition of substratedecarboxylation via the OPP pathway since isolated G6P dehydrogenase is inhibited byFd/thioredoxinred and by high NADPH/NADP ratios (Lendzian and Bassham, 1975). Thedehydrogenase reactions of the TCA cycle, in particular malate and isocitrate dehydrogenase,are also thought to be sensitive to increases in the NADH/NAD ratio (Wiskich, 1980; Graham,1980) which might occur if reductant were shuttled from the chloroplast to the cytosol.In a RUBISCO-deficient Chlamydomonas reinhardtii mutant, illumination resulted in a65% inhibition of CO2 efflux corresponding to a large increase in the NADPH/NADP ratiowhile 02 consumption was stimulated. ADP levels were extremely low and unaffected by alight to dark transition. Light-induced inhibition of CO2 efflux and stimulation of 0211consumption could be reversed by DCMU leading Gans and Rebeille (1985) to propose that theobserved 02 consumption was due to the Mehler reaction and that respiratory 02 consumptionwas inhibited. Uncoupling with CCCP could reverse inhibition of CO2 efflux suggesting, in thiscase, that at least a portion of the inhibition was due to competition between the chloroplastsand mitochondria for ADP (Gans and Rebeille, 1985). It is also possible that some 02consumption was due to reductant shuttling to the mitochondria resulting in the inhibition ofCO2 efflux by means of an increase in mitochondrial NADH/NAD ratios (Gans and Rebeille,1988). In contrast, illumination of wild-type Chlamydomonas, which is capable of CO2fixation, results in DCMU-insensitive 02 consumption (Peltier and Thibault, 1985). The factthat the Calvin cycle provides an efficient sink for both ATP and reductant suggests that thedegree of inhibition of respiration in the light depends on a subtle balance between Calvin cycleactivity and the PETC. This may explain why a greater inhibition of CO2 efflux is observedwhen cells are illuminated at the CO2 compensation point or during water stress (Azcon-Bietoand Osmond, 1983; Canvin et al., 1980; Stuhlfauth et al., 1991) and why only a transientinhibition of respiration is seen upon illumination under high CO2 conditions (Hampp et al.,1982). Illumination also inhibits both respiratory CO2 efflux and 02 consumption inphotoautotrophic higher plant cell suspensions (Avelange and Rebeille, 1991; Avelange et al.,1991). It appears, then, that changes in photosynthetic the ATP/NADPH production ratio,resulting from changes in coupling between photosynthetic electron transport and carbonfixation, can regulate both respiratory carbon flow and METC activity.Indirect regulation of respiration by photosynthesis:Photosynthesis in the preceding light period has been observed to affect dark respiratoryactivity in the light and the subsequent dark period (Weger et al., 1989; Stokes et al., 1990). Inspinach leaf discs after prolonged illumination, respiratory 02 consumption in the ensuing darkperiod was transiently enhanced by up to 3-fold over steady-state dark levels and this increasewas referred to as light enhanced dark respiration (LEDR) (Stokes et al., 1990; Reddy et al.,121991). Comparison of LEDR under high and low CO2 conditions, sensitivity to light intensity,and DCMU all suggested that the substrate for LEDR was photosynthetically generated (Stokeset al., 1990). Although it has been suggested that LEDR is due to increased METCconsumption of reductant generated by glycolytic metabolism of enhanced levels ofphotosynthetic metabolites (Azcon-Bieto and Osmond, 1983), the lack of CO2 effluxenhancement makes this unlikely (Stokes et al., 1990). Stokes et al. (1990) have suggested thatLEDR results either from alternative pathway activity or chloroplastic respiratory electrontransport activity (Stokes et al., 1900). On the other hand, measurements of gross gas exchangein a marine diatom have led Weger et al. (1989) to suggest that LEDR is, in fact, a misnomer.These authors suggest that respiratory 02 consumption increases upon illumination as aconsequence of increased anabolic biosynthesis during photosynthesis. In turn, the enhancedrespiratory rate occurring during the light period can be observed for a brief period afterexposure to the dark. In either case, photosynthesis appears to affect the rate of respiratory e -transport and carbon flow whether directly in the light or indirectly in the subsequent darkperiod.Regulation of photosynthesis by respiratory activity:Even if ATP production via METC activity is superfluous in light-saturated conditions,respiratory carbon flow and/or METC activity is necessary because of the requirement for ketoacids from the TCA cycle for light-dependent biosynthesis of porphyrins and amino acids(Graham, 1980; Turpin and Weger, 1990). In general, very little is known about the directregulation of photosynthesis by respiratory activity.Limited evidence suggests that changes in the relative rates of respiratory carbon flowand/or METC affects the rate of photosynthetic electron transport and/or carbon fixation bychanges in ATP/NADPH production and utilization ratios. In barley protoplasts and leaves,selective inhibition of mitochondrial oxidative phosphorylation by oligomycin results in a 40 to60% inhibition of photosynthetic carbon fixation (Kromer et al., 1988; Kromer and Heldt,131991). This inhibition suggests that excess reductant from the light reactions could be shuttledto the METC via a malate/oxaloacetate shuttle. This, in turn, would prevent over-reduction ofthe PETC and could potentially result in up to a 3-fold increase in quantum efficiency for ATPproduction compared to that provided by linear electron transport (Ebbighausen et al., 1987).Consistent with this role is the fact that oligomycin can greatly increase photoinhibitory damageof the PETC at saturating light intensities (Saradadevi and Raghavendra, 1992). It is alsopossible that METC activity in the light may provide the ATP required for cytosolic sucrosesynthesis (Kromer and Heldt, 1991; Hanson, 1992) or, in extreme cases, to supportphotosynthetic carbon fixation (Lemaire et al., 1988).A potential interaction between respiratory carbon flow and poising of photosyntheticelectron transport (for ATP and NADPH production)Although relatively little research has investigated the question of the interactionbetween respiratory carbon flow and poising of photosynthetic electron transport, anexamination of what is known about regulation of photosynthetic light harvesting and electrontransport can help in proposing a potential mechanism for regulatory interaction betweenrespiration and photosynthetic electron transport.When high levels of TCA cycle activity are required for amino acid biosynthesis, it hasbeen shown that carbon flow from glycolysis is required to replenish TCA cycle intermediatesused in biosynthesis (Turpin, 1991). This, in turn, could affect the rate of respiratory reductantproduction in both the chloroplast and the mitochondria. In order to maintain high rates ofrespiratory carbon flow required for biosynthesis it would be necessary for the reduced pyridinenucleotides to be recycled In addition, if the chloroplastic pyridine nucleotide pool were tobecome reduced, this might limit linear electron transport and decrease the efficiency of carbonfixation. Although the METC can recycle reductant in the mitochondria, it is also possible thatdown-regulation of reductant production by the PETC would make more oxidized pyridinenucleotide available for respiratory carbon flow. Down-regulation of reductant production14could be accomplished by changing the ratio of linear to cyclic electron flow. Althoughregulation of the ratio of linear to cyclic electron transport is poorly understood, one mechanismwhich has been proposed to regulate this ratio is the state transition.A mechanism to regulate the ratio of linear to cyclic electron transport:Molecular mechanism of the state transition:Until 1969, scientists were puzzled by what was referred to as the "quantum yieldanomaly". The steady state quantum yield of photosynthesis (CO2 fixed or 02 evolved perquantum of absorbed light) remains the same over a broad spectrum of light wavelengthsalthough the two photosystems have quite different absorption spectra (Myers, 1971; Barber,1986). The existence of a regulatory mechanism to explain the quantum yield anomaly was firstdemonstrated in the green alga Chlorella pyrenoidosa (Bonaventura and Myers, 1969) and thered alga, Porphyridium cruentum (Murata, 1969) by examining the effects of differentialexcitation of the two photosystems using monochromatic light and was termed the "light" statetransition. Subsequent research has suggested that a number of methods, in addition todifferential excitation of the two photosystems, can result in a state transition as defined below.A model for the molecular basis for state transitions in green algae and higher plants hasdeveloped from the discovery of a) reversible phosphorylation of the LHC2 in vitro (Bennett,1979), b) correlation between phosphorylation of LHC2 and fluorescence changes associatedwith the state transition (Bennett et al., 1980; Horton and Black, 1981; Chow et al., 1981;Steinback et al., 1982; Black et al., 1984; Farchaus et al., 1985) c) evidence for lateralheterogeneity and localization of PS2 and PS 1 in appressed and non-appressed thylakoidsrespectively (Anderson and Andersson, 1982) d) evidence for lateral movement of a mobilesubpopulation of phospho-LHC2 from appressed to non-appressed thylakoids (Chow et al.,1981; Staehelin et al., 1982; Anderson et al., 1982; Kyle et al., 1983; Bassi et al., 1988; Larssonet al., 1987), and evidence for redox sensitivity of the LHC2 kinase (Allen and Horton, 1981;Bennett, 1984). Despite its discovery and descriptive phenomenology over 30 years ago, the15actual mechanism and physiological significance of state transitions is still controversial today.The subsequent discussion will focus on the most recently reviewed model for the statetransition in green algae and higher plants (Allen, 1992) and will incorporate some additionalrecent developments (Anderson, 1992).Phosphorylation and the mobile antenna/cyt b6f model:In green algae and higher plants, the mechanism proposed to explain the state transitionin vivo is the phosphorylation mobile antenna model (see Figure 3). If the PQ pool and quinolbinding sites on the cyt b6f complex become reduced a protein kinase is activated whichphosphorylates LHC2 and/or cyt b6f (Gal et al., 1992). Although it was originally proposedthat phosphorylation caused an increase in the negative charge on phospho-LHC2 and thesubsequent electrostatic repulsion provided the driving force for movement of the phospho-LHC2 from the appressed to non-appressed regions of the grana (Barber, 1986), it has recentlybeen proposed that conformational changes in docking sites on LHC2 and cyt b6f due tophosphorylation are responsible for disassociation of these two proteins with PS2 and diffusionto the non-appressed thylakoids where the conformational changes may, in fact, favourassociation of phospho-LHC2 and cyt b6f with PS1 (Allen, 1992; Anderson, 1992). This state,where the light harvesting antennae complement (and consequently fluorescence) of PS2 isdecreased is termed state 2. Transition back to state 1 (PS2 has full antennae complement andhigh fluorescence) occurs if PQ(cyt b6f) is oxidized which deactivates the kinase. Phosphatasescleave the phosphate groups from LHC2 and cyt b6f and they diffuse back to the grana toreassociate with PS2. Transitions between these two conditions are known as state transitions.Function of the state transition:Although it is generally agreed that the state transition involves reversiblephosphorylation and migration of LHC2 from appressed to non-appressed thylakoids, the exactfunction of the state transition is still disputed. On the basis of differential light excitation of the2.H 2O 02state 1 - state 2 transitionI state 2 - state 1 transition I state 2 4.16Figure 3: The Phosphorylation Mobile Antennae Model: A putative model for the mechanism ofthe state transition (modified from Allen, 1992 and Anderson, 1992). In state 1, LHC2 and cytb6f associate with PS2 in the appressed thylakoids (1). This configuration favours linear electrontransport from PS2 to PS1. When PS2 is overexcited relative to PS 1, the PQ(cyt b6f) poolbecomes reduced and activates a kinase. This kinase phosphorylates LHC2, cyt b6f and itself (2).Phosphorylation of LHC2 and cyt b6f results in conformational changes and affects theirassociation with PS2 leaving them free to diffuse to the non-appressed membranes where theyreassociate with PS 1. This association favours cyclic electron transport around PS 1 and is knownas state 2 (3). Over excitation of PS1 relative to PS2 oxidizes the PQ(cyt b6f) pool and results indeactivation of the kinase. Phosphatases dephosphorylate LHC2 and cyt b6f which then diffuseback to the appressed thylakoids and reassociate with PS2 (4).17two photosystems it has been proposed that the light state transition functions to maximize thequantum yield of linear electron transport by minimizing non-photochemical de-excitation(Bonaventura and Myers, 1969). However, reversible phosphorylation and migration ofphospho-LHC2 has also been observed under conditions of constant light quality and/orintensity. Under these conditions, it has been proposed that the state transition may function tooptimize the relative rates of ATP synthesis and NADPH reduction in response to metabolicdemands (Allen et al, 1981; Allen and Bennett, 1981; Allen and Horton, 1981; Fernyhough et al,1984; Turpin and Bruce, 1990). Thus, it is envisaged that, in state 1, LHC2 and cyt b6f arelargely associated with PS2 which favours linear electron transport and maintains a high ratio oflinear to cyclic electron transport (Figure 3, Anderson, 1992). However, upon phosphorylationand migration of LHC2 and cyt b6f to the non-appressed grana, association with PS 1 isfavoured and this, in turn, enhances cyclic electron flow around PS1. At constant non-saturating light intensity and quality, the overall effect of a state 1 to 2 transition is, therefore, todecrease the ratio of linear to cyclic flow. This would have the effect of down-regulatingNADPH production and decreasing the NADPH/ATP production ratio.Evidence for a structural basis for changes in the ratio of linear to cyclic e - flow:Evidence for a structural basis for changes in the ratio of linear to cyclic electron flowafter a state 1 to 2 transition has come from a variety of sources including studies of a) thelateral distribution of cyt b6f complexes (Wollman and Bulte, 1990; Vallon et al., 1991), b)super-complex formation (Wollman and Bulte, 1990), c) phosphorylation of FNR (Hodges etal., 1987), and d) the functional absorption cross section of PS1 (Allen, 1984, 1992).On the basis of highly accurate membrane fractionation techniques andimmunocytochemical microscopy, Vallon et al. (1991) demonstrated a marked change in thelateral distribution of cyt b6f complex during state transitions induced both in vivo and in vitroin maize and Chlamydomonas reinhardtii. When cells were adapted to state 2, the proportionof cyt b6f located in the stromal thylakoids was significantly greater than after a transition to18state 1. This marked change in cyt b6f distribution was accompanied in all cases by theredistribution of phospho-LHC2 although the proportion of cyt b6f redistributed wassignificantly larger (30-40 %) than that of LHC2 (10-20%). Similarly, changes in lateraldistribution of cyt b6f or phospho-LHC2 in vitro required addition of exogenous ATP,suggesting that the distribution of cyt b6f depended on LHC2 kinase activity. Vallon et al.(1991) postulated that changes in lateral distribution of cyt b6f during state transitions provide amechanism for the modulation of linear and cyclic flow. Cytochrome b6f is less abundant thaneither PS 1 or PS2 in thylakoids isolated from plants grown at a variety of light intensities(Anderson, 1992). Thus, increased cyt b6f in the stroma during state 2 would increase theamount of cyt b6f available for interaction with PQ and PS1 for cyclic flow.Studies on super-complex formation have also suggested a physical basis for changes inthe ratio of linear to cyclic electron transport. Precedence for super-complex formation comesfrom kinetic studies in photosynthetic bacteria which have suggested super-complex formationbetween cyt bci, the cytochrome oxidase, and the photochemical reaction center (Joliot et al.,1989). In green algae, the evidence for super-complex formation comes from several sources.First, cyt b6f co-migrated with PS 1 in sucrose density gradients following centrifugation ofsolubulized Chlamydomonas membranes isolated from cells in state 2 (Wollman and Bulte,1990). Second, in procedures used to purify cyt b6f, both the LHC2 kinase and FNR wereshown to co-purify with cyt b6f, indicating some level of association between these threecomplexes (Clark et al., 1984; Gal et al., 1990a). Third, the rate of cyt f oxidation by PS1 aftera single turnover flash was increased under conditions very similar to those used to induce astate 2 transition while state 1 conditions decreased the rate of cyt f oxidation (Delosme, 1991).Delosme (1991) suggested that the faster rate of cyt f oxidation is due to the closer associationof cyt b6f with PS 1 during state 2. Finally, the size and distribution of freeze-fracture particlessuggested that some cyt b6f complexes were associated in the same intermembrane particleswith PS2a in the stacked membranes while others were associated in the same inter-membraneparticles with PS113 in the unstacked regions (Olive et al., 1986). Vallon et al. (1991) proposed19that, in state 1, cyt b6f may be able to migrate freely between the two compartments but thatstate 1 to 2 transitions would favor super-complex formation between cyt b6f and PS1, thus,enhancing the amount of cyt b6f found in the stroma.Studies on the effects of phosphorylation on FNR activity have suggested anothermechanism whereby the state transition may affect the ratio of linear to cyclic electron transport.Hodges et al. (1987) report an increased recovery of phosphorylated FNR in the soluble fractionafter incubation of pea thylakoids with ATP in the light or dark. FNR is weakly bound to thestromal side of the thylakoid where it forms a cation-sensitive complex with Fd. Itsmodification might be expected to alter binding to both Fd and the membrane and affect e -transfer to NADP. The kinase which phosphorylates FNR is redox-controlled and hasproperties similar to those of the LHC2 kinase. Hodges et al. (1987) reported that PS1 inhibitsreduction of NADP and that this inhibition is ATP-dependent but independent of effects ofphosphorylation of LHC2. The inhibition of NADP reduction was reversed by antimycin Awhich also inhibits cyclic electron transport. This led them to suggest a role for FNRphosphorylation in the regulation of the relative rates of cyclic and non-cyclicphotophosphorylation.The final factor which could provide a structural basis for an increase in the ratio ofcyclic to linear electron transport after a state 1 to state 2 transition is the complementarychange in light harvesting allocation which is thought to accompany the state transition. It isgenerally agreed that a mobile sub-population of the phospho-LHC2 dissociates from PS2 andmoves from the grana to the PS 1 rich stroma. However, determination of the functionalassociation of phospho-LHC2 with PS1 has been far more controversial (for a review see Allen,1992; see also Wendler and Holzwarth, 1987; Allen and Melis, 1988). It has been suggestedthat discrepancies in functional phospho-LHC2 association with PS 1 may arise from variabilityinduced by temperature (Havaux, 1988; Timmerhaus and Weis, 1990) and zeaxanthin formationassociated with the development of thylakoid ApH (Horton, 1989). In a recent review, Allen(1992) proposes that mechanisms which can switch between excitation-transfer to PS1 and20dissipation as heat, depending upon light intensity and temperature, would determine whetherthe state 2 transition functions to increase or decrease the quantum yield of PS 1, that is, toconserve or dissipate excitation energy.Phosphorylation of LHC2 could affect either the efficiency of cyclic electron flow or therelative extent of cyclic vs. linear flow dependent on whether phospho-LHC2 was functionallyassociated with PS 1. As was discussed previously, under normal conditions of CO2 fixationlinear electron transport would not be able to provide the ATP/NADPH ratio required for CO2fixation suggesting that cyclic or pseudocyclic electron transport would be required to providethe excess ATP requirements for efficient CO2 fixation (Heber and Walker, 1992). At constantsub-saturating light intensity and quality, the quantum yield of PS 1 cyclic flow would increaseonly if phospho-LHC2 redirected its excitation energy to PS 1. The effect of dissociation ofphospho-LHC2 from PS2 under these conditions, however, would be to decrease the quantumyield of linear electron transport. Assuming that some cyclic electron transport occurred in bothstate 1 and state 2, a decrease in the quantum yield of linear electron transport would stillfunction to decrease the ratio of linear to cyclic transport although it might not increase theabsolute quantum yield of cyclic electron transport.Can respiratory carbon flow influence the redox state of the PQ(cyt b6f) pool and poisethe ratio of linear to cyclic electron flow?:It is likely that respiratory carbon flow could interact directly with photosyntheticelectron transport by changing production of ATP and/or NAD(P)H. The pertinent questionthen becomes: can changes in the production of ATP or NAD(P)H, as a result of respiratorycarbon flow, affect the redox state of the PQ(cyt b6f) pool and result in a state 1 to 2transition? In the light, there are two potential mechanisms whereby increases in theNAD(P)H/ATP production ratio by respiratory carbon flow might affect the redox state of thePQ(cyt b6f) pool. Linear electron transport produces approximately 0.77 NADPH per ATP(Nelson and Prezelin, 1990). Hence, if the ratio of NADPH/ATP in the chloroplast were to21increase significantly, due to an increase in the NAD(P)H/ATP production ratio fromrespiratory carbon flow, this could potentially affect the poising of the state 1 to 2 transition inat least two ways. The first involves direct substrate (NADP) limitation of electron transportand the second involves direct reduction of the PQ(cyt b6f) pool by NAD(P)H.Substrate (NADP) limitation of the PETC:If respiratory carbon flow were to increase in the light, an increase in NADPH or ATP inthe chloroplast could potentially affect the reduction of the PQ(cyt b6f) pool. An increase in theATP/ADP ratio would lead to substrate limitation of the chloroplast ATP synthase and limitdissipation of the proton gradient. Since electron flow from PQ to cyt b6f is coupled to protontranslocation, the rate of electron flow is decreased by an increase in thylakoid ApH (Lawlor,1987). However, it is quite unlikely that an increase in respiratory carbon flow would beaccompanied by an increase in the ATP/ADP ratio since this would tend to limit rather thanenhance respiratory carbon flow (Day et al., 1987). It is much more likely that increases inrespiratory carbon flow would lead to an increase in the ratio of reduced/oxidized reductant(Lambers, 1990). An increase in reduced/oxidized pyridine nucleotide ratios in the chloroplastcould potentially affect the redox state of the PQ(cyt b6f) pool by substrate-limiting linearelectron transport due to a lack of NADP. Testing this hypothesis would involve examiningchanges in respiratory carbon flow which occur in the light during photosynthetic electrontransport. Such tests would be extremely difficult to perform because of the difficulty inresolving the effect of changes in the NAD(P)H/NAD(P) ratio directly attributable torespiration.Direct interaction between reductant and the PQ(cyt b6f) pool:Respiratory carbon flow could also affect the reduction of the PQ(cyt b6f) pool bydirect reduction of PQ with NAD(P)H. Evidence for direct interaction between pyridinenucleotides and the photosynthetic electron transport chain has come from two independent22lines of research. One line of investigation has focused on photoevolution of H2 in green algaewhile a second has developed from studies of respiratory 02 uptake in the chloroplast in thedark. The electron transport pathways involved in both of these processes and their interactionwith the PETC are outlined in Figure 4.112 photoevolution:^In hydrogenase-inducible green algae under anaerobic conditions,sustained H2 production occurs in the absence of CO2 or when CO2 fixation is inhibited(Gaffron and Rubin, 1942; Stuart and Gaffron, 1972; Kaltwasser et al., 1969; Graves et al.,1989). This has led Graves et al. (1989) to suggest that H2 photoevolution competes with CO2fixation for reductant. Kaltwasser et al. (1969) were the first to propose that in vivo PS1-dependent H2 photoevolution depended upon degradation of organic substrate in Scenedesmusobliquus (see Figure 4). H2 photoevolution was inhibited by starvation and stimulated byexogenous substrates such as glucose, acetate and ethanol (Wiessner and Gaffron, 1964; Tanneret al., 1965; Kaltwasser et al., 1969; Bishop et al., 1977; Senger and Bishop, 1979). Inaddition, H2 photoevolution was accompanied by CO2 release and was inhibited bymonofluoroacetic acid (a TCA cycle inhibitor) suggesting that oxidative carbon metabolism wasrequired (Healey, 1970b; Stuart and Gaffron, 1971). These observations were consistent withthe existence of P51-dependent e - flow from reductant, produced by oxidative carbonmetabolism, to a high potential redox carrier capable of reducing H+ (Healey, 1970b).Although the pathway for H2 photoevolution appears to be species-specific, in Chlamydomonassp., H2 photoevolution was shown to be independent of PS2 on the basis of work with PS2mutants and lack of inhibition by DCMU (Healey, 1970a). Stimulation of H2 photoevolution byreduced pyridine nucleotides was first observed in vitro by Abeles (1964) in thylakoidpreparations of Chlarnydomonas eugmetos. NADH-dependent H2 photoevolution inChlamydornonas reinhardtii thylakoid preparations (Ben-Amotz and Gibbs, 1975) and whole23H2 O 0.5 02FNRtNAD(P)HC substratenOPP orglycolysisNO2 -or Z2Fd H2aseNADPH / PQoxidoreductaseDCMUcyt b6cyt bc (putative)terminaloxidasemyxothiazolFigure 4: Thylakoid electron transport pathways which share PQ in green algae (modified fromMaione and Gibbs, 1986b). Black arrows, electron transport which occurs only in the dark; greyarrows, electron transport which occurs only in the light; white arrows, electron transport whichcan occur in both the dark or the light. Double lines indicate sites of inhibitor action. Electronflow from NAD(P)H to 02 via the NAD(P)H-PQ oxidoreductase is defined as the chloroplastrespiratory electron transport chain (CRETC). Flow from H2O to Fd via PS2 and PS1 is definedas the photosynthetic electron transport chain (PETC). Electrons from Fd can be used to a)reduce NADP (linear electron transport), b) reduce 02 (pseudocyclic electron transport), c)produce H2 (H2 photoevolution), d) reduce PQ (PS1 cyclic electron transport). Depending uponillumination, PQ can accept electrons from PS2 and/or NAD(P)H and can donate electrons toPS1 or the terminal oxidase of the CRETC. See text for further discussion. Abbreviations are asin the list of abbreviations.24cells (Gfeller and Gibbs, 1985) was observed to be sensitive to DBMIB but not DCMU,consistent with e - transfer from NADH to PQ.A thylakoid-bound protein component responsible for the transfer of e- from NAD(P)Hto PQ was partially purified by Godde and Trebst (1980) and further characterized by Godde(1982). Photosynthetically active chloroplast particles from C. reinhardtii were observed toevolve H2 in the light at the expense of intermediates of glycolysis which produced NAD(P)H(G6P, FBP, lactate). Both exogenous NADPH and NADH were observed to act as substratesfor this activity although NADPH interacted with 2-fold less activity (Godde and Trebst, 1980).The H2 photoevolution activity was inhibited by rotenone and thenoyl trifluoroacetate, both ofwhich act to inhibit iron sulfur centres in mitochondrial NADH dehydrogenase (complex 1) andsuccinate dehydrogenase, respectively (Figure 4). The activity was also shown to utilize PQwith much higher efficiency than ubiquinone (UQ) making mitochondrial contamination unlikely(Godde, 1982). The involvement of PQ led to the conclusion that this protein component actedas an NAD(P)H-PQ oxidoreductase.Succinate dehydrogenase (SDH) may also act as an e - donor to PQ. 25% of the cellularSDH activity is observed in isolated Chlangdomonas chloroplasts and exogenous succinate hasbeen observed to support PS1 activity in thylakoid preparations (Willeford et al., 1989).Respiratory 02 consumption in the chloroplast: It has been proposed that in the dark,electrons from NAD(P)H are transferred to 02 via NAD(P)H-PQ oxidoreductase and the PQpool (Bennoun, 1982; Diner and Mauzeral, 1973; Peltier et al., 1987) (see Figure 4). The termchlororespiration was first coined by Bennoun (1982) to describe the pathway of electrontransport from NAD(P)H to 02 and to distinguish it from the pathway in the mitochondria.However, the term "chlororespiration" has since become ambiguous because respiratory carbonflow (Singh et al., 1992; Gibbs et al., 1990), electron transport from NAD(P)H to 02 via PQ(Bennoun, 1982), and electron transport from NAD(P)H to 02 via FNR (Kow et al., 1982) areall thought to occur in the chloroplast. For the purposes of this work, chloroplastic respiratory25e transport chain (CRETC) activity involving PS1- and PS2- independent electron transportfrom NAD(P)H to 02 via PQ will be distinguished from both chloroplastic respiratory carbonflow and PS1/FNR-associated 02 consumption.Goedheer (1963) was the first to propose the existence of a putative thylakoidrespiratory electron transport chain which consumed 02 and shared components with thePETC. Evidence consistent with dark e - flow from components of the PETC to 02 wasprovided by observations that the redox state of both the PQ pool (Diner and Mauzeral, 1973;Diner, 1977) and cyt b563 (Hiyama et al., 1969) was affected by anaerobiosis. On the basis of02 flash yield measurements and photosynthetic light intensity curves in anaerobically treatedChlorella, Diner and Mauzeral (1973) hypothesized that PQ was shared between the PETC anda chloroplastic respiratory e - transport chain which accepted electrons from reduced pyridinenucleotides and donated them to 02 (Figure 4). It was subsequently shown that PQ could bereduced by the products of anaerobic starch metabolism in Chlamydomonas reinhardtii (Gfellerand Gibbs, 1985). At the extinction point (the lowest p02 at which anaerobic fermentation iseliminated), DBMIB could be shown to increase fermentative ethanol production which, in turn,resulted in a further increase in the extinction point from 2 to 5% 02. This led Gfeller andGibbs (1985) to propose that oxidation of pyridine nucleotides occurs via PQ in the presence of02 or via ethanol formation in the absence of electron transfer to 02 (anaerobiosis or DBMIBinhibition).Oxidation of reductant (via the CRETC) was shown to involve the NAD(P)H-PQoxidoreductase because a) respiratory CO2 efflux associated with CRETC activity in isolated C.reinhardtii chloroplasts was inhibited by rotenone, an inhibitor of the NAD(P)H-PQoxidoreductase (Singh et al., 1992) and b) exogenous NAD(P)H increased the reduction of PQas measured by fluorescence induction in open cell preparations of a PS1 mutant of C.reinhardtii (Bennoun, 1982).Although the NAD(P)H-PQ oxidoreductase has been isolated and partially characterized(Godde and Trebst, 1980), very little is known about the identity of the components that accept26e- from the PQ pool and transfer them to 02. Using C. reinhardtii mutants, Bennoun (1983)demonstrated that the Rieske iron sulfur protein, cyt f, PC, PS1, and the ATP synthase were notnecessary for CRETC activity, suggesting that interaction between the CRETC and PETCoccurred only at the level of PQ. An amperometric signal associated with CRETC flow to 02(Peltier et al., 1987) was inhibited by myxothiazol and 5 [tM antimycin A, inhibitors whichspecifically affect cytochrome bc 1 but not b6f complexes. This fact led Ravenel and Peltier(1991) to suggest that a distinct cyt bc type complex was involved in the CRETC. Furthermore,two novel cytochromes, cyt h1 and h2 were identified (Lemaire et al., 1986; Rolfe et al., 1987)and were shown to increase under conditions of N-limitation, which enhances CRETC activityin C. reinhardtii (Peltier and Schmidt, 1991). Ravenel and Peltier (1991) suggested that thesetwo cytochromes might be components of the putative myxothiazol- and antimycin A-inhibitedcyt bc complex.The existence of a terminal oxidase was suggested by inhibitor studies. InChlamydomonas, dark oxidation of the PQ pool was inhibited by CO, NO, KCN, and sodiumazide, indicating the involvement of a cytochrome-type oxidase in the oxidation of PQ. InChlorella, however, SHAM (but not KCN) inhibited PQ oxidation suggesting that the terminaloxidase may act more like an alternative oxidase in this organism (Bennoun, 1982).Competition between 02 and PS1 as terminal electron acceptors for the CRETC / Theeffects of illumination:Kok effect: Kok (1949) first observed a non-linearity, at low light intensities, of thephotosynthetic light saturation curve in the green alga Chlorella. On the basis of DCMUinsensitivity, it was proposed that the Kok effect resulted from inhibition of mitochondrialrespiration, in the light, by an increase in the ATP/ADP ratio from cyclic photophosphorylation(Kok 1949; Hoch et al., 1963). However, since the Kok effect was also insensitive to CCCPand was enhanced in far red light, Healey and Myers (1971) suggested that inhibition of 02consumption at low light intensities was a result of the diversion of reductant from respiratory27electron transport to PS 1. Similarities in the Kok effect observed in Anacystis nidulans, acyanobacterium in which the respiratory and photosynthetic electron transport chains occur inthe same membrane and share components, led Jones and Myers (1963) to suggest that arespiratory activity was directly inhibited by competition with PS 1 for reductant (Jones andMyers, 1963).In C. reinhardtii, mass spectrometric measurements of gross gas exchange allowedPeltier and Sarrey (1988) to distinguish between two respiratory components. The firstcomponent was inhibited by low concentrations of antimycin A and SHAM, was light-independent and was thought to result from METC activity. A second component was inhibitedby light but insensitive to low concentrations of antimycin A and SHAM, suggesting that itresulted from CRETC and not METC activity. Consistent with these observations is theobservation that, in Chlorella vulgaris, the Kok effect was enhanced by anaerobiosis (whichwould prevent oxidation of PQ) and abolished by benzoquinone (which rapidly oxidizes the PQpool) (Diner and Mauzeral, 1973).Flash inhibition of CRETC: Using amperometric and mass spectrometric measurements in C.reinhardtii, Peltier et al. (1987) observed a respiratory 02 consumption component which wasinhibited by short (2 [isec) saturating flashes. This component was related to 1802 uptake, wasinsensitive to DCMU, and was stimulated by acetate and high p02. It was suggested that theflash-inhibited component resulted from inhibition of 02 consumption via the CRETC because ita) had a higher km for 02 than the METC, b) was insensitive to concentrations of antimycin Aand SHAM which completely inhibited respiration and c) was inhibited by concentrations ofKCN which have no effect on the METC. In addition, in mutants which were missing eitherPS1 or cyt b6f, this respiratory component was enhanced after illumination (Ravenel and Peltier,1992; Peltier and Thibault, 1988). De-convolution of the flash-induced amperometric signal inwild-type cells indicated that flash-induced stimulation also occurred but that this stimulationdeveloped more slowly than flash-induced inhibition by PS1 (Ravenel and Peltier, 1992). All28these data support the idea that light inhibition of CRETC activity is due to competition,between PS1 and 02, for e - from PQ. Stimulation of the respiratory component by illuminationsuggests that 02 may accept e - from PS2 as well as from reductant and highlights PQ as across-over point between oxidizing and reducing reactions in the thylakoid. Similaritiesbetween this flash-inhibited component and that seen in photosynthetic prokaryotes, where thePETC and respiratory electron transport chain share components, lends further support to theidea that PS1 and 02 compete for e - from PQ (Vermeglio and Carrier, 1984; Lavorel et al.,1989; Baccarini et al., 1978; Vermeglio and Joliot, 1984; Richaud et al., 1986).Evidence for the existence of a CRETC in higher plants:Although strong evidence exists for the occurrence of a CRETC in chl b-containing(Bennoun, 1982) and chl c-containing (Wilhelm and Duval, 1990; Buchel and Wilhelm, 1990;Ting and Owens, 1993) unicellular algae, the existence of a CRETC in higher plant cells is stilldebated. Chloroplast genome analysis in liverwort, tobacco, sugar beet, rice, and broad beanhas revealed the presence of open reading frames which have a high homology with themitochondrial NADH dehydrogenase (complex 1) (Umesono and Ozeki, 1987; Oyhama et al.,1988; Meng et al., 1986; Shinozaki et al., 1986). A recent study has also shown that thiscomplex is expressed in mono- and dicotyledonous plants and is localized in the stromallamellae of the thylakoid membrane (Berger et al., 1993). H2 photoevolution, correlated withNAD(P)H-PQ oxidoreductase activity, was not detected in spinach thylakoids provided withexogenous hydrogenase (Ben-Amotz and Gibbs, 1975; Godde and Trebst, 1980). However,NADPH reduced cyt b560 (Cramer and Butler, 1967) and PQ (Mills et al., 1979) in spinachchloroplasts. Flash-induced inhibition of respiratory 02 uptake was not observed inphotoautotrophic cell cultures or protoplasts of Euphorbia (Avelange and Rebeille, 1991;Ravenel and Peltier, 1992). However, Garab et al. (1989) demonstrated in tobacco and sugarbeet that both KCN and SHAM inhibited the oxidation kinetics of PQ in vitro and in vivo. Inaddition, oxidation of exogenous glucose in isolated spinach chloroplasts was inhibited by29rotenone, amytal, antimycin A, propyl galate, sodium azide (Singh et al., 1992), andanaerobiosis (Ahluwalia et al., 1989), all of which have themselves been demonstrated to inhibitCRETC activity in isolated Chlamydomonas reinhardtii chloroplasts (Singh et al., 1992). Inspinach leaf discs, reduction of the PQ pool resulted from anaerobiosis and the PQ pool was re-oxidized by either 02 or far red light, suggesting that electron transport from PQ to a terminaloxidase occurs in higher plants (Harris and Heber, 1993).Evidence to suggest that changes in the cellular NAD(P)H/ATP ratio can regulate thestate 1 to 2 transition:In the light: Treatments thought to cause an increase in the NADPH/ATP ratio have beenshown to induce a state 1 to 2 transition in maize (Horton and Lee, 1986; Horton, 1987,1989;Horton et al., 1989; Turpin and Bruce, 1989). In maize mesophyll chloroplasts, theNADPH/ATP ratio can be manipulated by providing endogenous carbon with differingNAD(P)H/ATP requirement ratios (Fernyhough et al., 1984; Horton et al., 1989). OAA (1NADPH required per OAA assimilated to malate via NADP-malate dehydrogenase), whichwould decrease the cellular NADPH/ATP ratio, was observed to strongly inhibit LHC2phosphorylation. Pyruvate (2 ATP required per pyruvate reacted to PEP via pyruvate PPidikinase), which would increase the cellular NADPH/ATP ratio, increased LHC2phosphorylation (Fernyhough et al., 1984; Fernyhough et al., 1989). Similarly, in the light in N-limited green algae, it was shown that assimilation of NH4+, which requires 0.2 NADPH perATP, resulted in a state 1 to 2 transition (as measured by absolute 77K fluorescence), whileNO3 - and CO2 assimilation which require 0.83 and 0.67 NADPH per ATP, respectively, didnot cause a state 1 to 2 transition (Turpin and Bruce, 1990). A transition fromphotoautotrophic to photoheterotrophic acetate metabolism, which has a low NAD(P)H/ATPrequirement ratio, has been observed to increase the absorption cross section of PS1 asmeasured by the quantum yield of H2 photoevolution in Chlamydobotris stellata (Boichenko etal., 1992). An extreme state 1 to 2 transition was also observed in high CO2-grown Chlorella30vulgaris upon transfer to low CO2 conditions leading Demidov and Elfimov (1992) to suggestthat inhibition of the Calvin cycle (due to CO2-limitation) and increased ATP consumption,necessary to induce the CO2-concentrating mechanism, decreased the cellular NADPH/ATPrequirement ratio. In all of the above cases, it is likely that a NADPH/ATP utilization ratiolower than the linear electron transport NADPH/ATP production ratio of 0.77 would tend toincrease the cellular NADPH/NADP ratio. This, in turn, could influence the redox state of thePQ(cyt b6f) pool either by substrate-limitation or by reduction of PQ via the NAD(P)H-PQoxidoreductase. It is, however, very difficult to differentiate between these two possibilities inthe light.In the dark: State 1 to 2 transitions have also be shown to occur in the dark, leading severalauthors to suggest that an increase in the dark NAD(P)H/NAD(P) ratio might increase theredox state of the PQ(cyt b6f) pool and result in a state 1 to 2 transition (Rebeille and Gans,1988; Gans and Rebeille, 1990; Bulte et al., 1990; Mohanty et al., 1990). In Chlamydomonasreinhardtii, treatment of darkened cells with uncouplers or antimycin A and SHAM increasedcellular NADPH/NADP ratios and resulted in a state 1 to state 2 transition (Bulte et al., 1990;Gans and Rebeille, 1990). Similarly, assimilation of NH4+ by N-limited Selenastrum minutumresulted in an increase in the cellular NADPH/NADP ratio (Vanlerberghe et al., 1992) and astate 1 to 2 transition (Mohanty et al., 1990). In the absence of illumination, substrate-limitationof the PETC would not occur and it is likely that the above treatments increased the reductionof the PQ(cyt b6f) pool as a result of NAD(P)H-PQ oxidoreductase activity.The rationale for working in the dark:In most studies to date, the direct measurement of either pyridine or adenine nucleotidelevels has been limited and a correlation between a decreased NADPH/ATP requirement ratioand a state 1 to 2 transition has been implied but not observed. It is extremely difficult todetermine the mechanism responsible for the reduction of PQ in the light because of the variety31of processes which may contribute to regulation of the PETC. In the light, NAD(P)H can beconsumed by carbon fixation, NO3 - assimilation, and amino acid biosynthesis. At the sametime, NAD(P)H is produced by the PETC and respiratory carbon flow. In the presence ofmultiple sources and sinks for NAD(P)H, it is extremely difficult to isolate the effects ofreductant production from respiratory carbon flow on the PETC in vivo during illumination. Inaddition, the redox status of the PQ(cyt b6f) pool can be affected by its rate of reduction by PS2or reductant (Figure 4, see also Ravenel and Peltier, 1992) and by its rate of oxidation by PS1,which, in turn, can be affected by light-induced trans-thylakoid ApH. Measurement of a statetransition using fluorescence quenching techniques would be complicated because at least 2other processes are thought to quench fluorescence in the light; these are energized quenching(due to thylakoid ApH) and photoinhibitory quenching (Krause and Weis, 1991). All of thesefactors make it extremely difficult to clearly demonstrate the regulation of the photosyntheticNADPH/ATP production ratio by respiratory carbon flow in the light.It is equally possible, however, that respiratory activity in the dark might affect the redoxstate of the PQ(cyt b6f) pool and poise the PETC for a decrease in the NADPH/ATPproduction ratio upon illumination. Testing this hypothesis would be considerably easier. Themultiple sources and sinks for NAD(P)H in the dark would be absent largely because productionand consumption of NAD(P)H, via photosynthetic processes, would not occur. In addition, theredox state of PQ would not be affected by PS 1 and PS2 activity making it possible to isolatethe effects of NAD(P)H-PQ oxidoreductase activity on the PQ redox state. Under theseconditions, it is possible to isolate and test the effects of increases in the NADPH/NADP ratioon the redox state of the PQ(cyt b61) pool by direct electron transfer from the NAD(P)H-PQoxidoreductase (Godde and Trebst, 1980).The hypothesis to be tested:The theoretical relationship between respiratory carbon flow and the PETC viaNAD(P)H-PQ oxidoreductase activity in the dark can be formalized as a testable hypothesis.32Essentially, I propose that increases in respiratory carbon flow which cause an overall increasein the NAD(P)H/NAD(P) ratio will increase the reduction of the PQ(cyt b6f) pool andconsequently poise the PETC to decrease the ratio of linear/cyclic electron transport via a state1 to 2 transition. The corollary to this hypothesis is that increases in respiratory carbon flow,which do not result in an overall increase in the NAD(P)H/NAD(P) ratio, will not increase thereduction of the PQ pool and will not affect the poising of the PETC for cyclic vs. linearelectron transport. The purpose of this thesis will be to test this hypothesis and its corollary invivo, in the green alga Selenastrum minutum, as stringently as possible, to develop a model forthe interaction between respiratory carbon flow and photosynthetic light harvesting.It has been suggested that the role of the CRETC is to recycle reductant for respiratorycarbon flow in the chloroplast in much the same manner that the METC functions inmitochondria (Peltier et al., 1987; Peltier and Schmidt, 1991; Singh et al., 1992). If the abovehypothesis is supported, however, it will have major implications for our understanding of therole of the CRETC in metabolism. Essentially, the CRETC would allow respiration to directlymodulate photosynthesis. First, the CRETC could allow respiration to communicate directlywith the PETC and decrease NADPH production via the state 1 to 2 transition in the light. Asecond implication is that respiratory activity in the dark could affect the poising of thephotosynthetic NADPH/ATP production ratio upon illumination. Increases in NAD(P)H in thechloroplast due to respiratory carbon flow in either the dark or light could, therefore, becommunicated to the PETC and cause a modulation of the NADPH/ATP production ratio tocompensate.33CHAPTER 2: EXPERIMENTAL RATIONALE: A FRAMEWORK TO TEST THEHYPOTHESIS INTRODUCTIONRationale for the hypothesis to be tested:Considerable evidence exists to suggest that the activity of both the PETC andphotosynthetic carbon fixation can affect respiration through changes in the ratio of pyridineand/or adenine nucleotides (Graham, 1980; Dry and Wiskich, 1987; Turpin and Weger, 1990).Considerably less information is known, however, about whether respiration regulatesphotosynthesis. Since both the PETC and respiratory carbon flow produce NAD(P)H and ATPbut with potentially different stoichiometries, it is possible that changes in respiratory carbonflow could affect NAD(P)H/ATP ratios in the cell, making it necessary for the PETC to down-regulate the NADPH/ATP production ratio to prevent over-reduction of the cell.The previous chapter examined the theoretical possibilities of an interaction betweenrespiratory carbon flow in some detail. It was proposed that the state 1 to 2 transition mightfunction to down-regulate the ratio of NADPH/ATP production by decreasing the ratio of linearto cyclic e - transport (Allen and Horton, 1981; Horton et al., 1989). To support thiscontention, state 1 to 2 transitions were observed during treatments which are thought toincrease the cellular NADPH/ATP ratio in the light (Fernyhough et al., 1984; Horton and Lee,1986; Horton et al., 1989; Turpin and Bruce, 1989; Boichenko et al., 1992; Demidov et al.,1992). During these treatments, it was proposed that the increase in the cellular NADPH/ATPratio resulted in reduction of the PQ pool, either by NADP -Llimitation of linear e - transport orby NAD(P)H-PQ oxidoreductase activity, and initiated a state 1 to 2 transition (Turpin andBruce, 1989; Horton et al., 1989). It is, however, extremely difficult to distinguish between thetwo mechanisms which might contribute to PQ reduction in the light.It is particularly difficult to isolate the interaction between respiratory carbon flow andthe PETC in the light. Physiological measurements of gas exchange are hampered by thesuperimposition of a variety of processes (Turpin and Weger, 1990) and biochemical34measurements are difficult because cofactors (ATP and NAD(P)H) potentially responsible forthe interaction between respiratory carbon flow and the PETC can be affected by several sourceand sink reactions at once. In the dark, however, it is considerably easier to isolate the effectsof changes in the NADPH/ATP ratio on the redox state of the PQ(cyt b6f) because neitherphotosynthetic carbon fixation or electron transport occur. Under these conditions, it should bepossible to isolate and test the effects of increases in NAD(P)H/NAD(P) ratios on the state 1 to2 transition resulting from increased reduction of the PQ(cyt b61) pool by direct electrontransfer from the NAD(P)H-PQ oxidoreductase.Methods which can be used to measure state transitions:In order to examine the interaction between respiratory carbon flow and the statetransition in the dark, accurate measurements of the state transition were required. The statetransition has traditionally been measured by changes in fluorescence emission or LHC2phosphorylation. LHC2 phosphorylation measurements have proven extremely useful for themeasurement of state transitions in vitro but are intrusive and do not provide any informationabout the functional association of phospho-LHC2. On the other hand, chlorophyll afluorescence provides a sensitive, non-intrusive probe of the photosynthetic apparatus and,when several methods are used, allows for resolution and rapid measurement of state transitionkinetics.Light emission (or fluorescence) arises from chl a if excitation energy is not dissipatedby photochemistry or radiationless dissipation. At room temperature, most fluorescence isemitted by chl a molecules in the light harvesting antennae of PS2 (Krause and Weis, 1991).Fluorescence yield is rarely maximal due to the existence of photochemical and non-photochemical processes which can "quench" fluorescence. Photochemical quenching (qp) isrelated to the redox state of QA, and occurs when excitation energy of the reaction centrechlorophyll is used for photochemistry. If the primary quinone acceptor associated with the PS2reaction centre (QA) is reduced, the reaction centre is "closed" and cannot contribute to35photochemical quenching of fluorescence. On the other hand, an "open" or oxidized reactioncentre is capable of quenching fluorescence photochemically (Bradbury and Baker, 1981;Krause and Weis, 1984; Sivak and Walker, 1985).Quenching of fluorescence may also result from non-photochemical processes whichinclude energization of the thylakoid membrane, the state transition, and photoinhibition(Krause and Weis, 1991; Horton and Bowyer, 1991). A major component of non-photochemical quenching (qNp) is energized quenching, or qE, which is associated with thebuildup of the thylakoid proton gradient. The molecular mechanism of qE is still not wellunderstood (Ruban and Horton, 1992) but it is thought that qE results in energy release in theform of heat rather than fluorescence (Krause et al., 1982; Horton, 1982). A xanthophyll cycleis one mechanism that has been proposed to account for qE. The xanthophyll cycle wouldinvolve dissipation of excess excitation energy by zeaxanthin, which is formed by de-epoxidationof violoxanthin when the thylakoid lumen pH is low (Demmig-Adams, 1990). Horton andcoworkers have recently proposed that qE involves a protonation-induced aggregation ofLHC2, which serves to decrease fluorescence yields and may be enhanced by zeaxanthin (Rubanand Horton, 1992; Ruban et al., 1992; Horton et al., 1991). It is important to note, though, thatqE cannot be attributed solely to the formation of thylakoid ApH. In higher plants, antimycin Ahas been demonstrated to inhibit qE without affecting thylakoid ApH, suggesting that qE may beregulated by both the thylakoid ApH and the redox state of an electron transport chaincomponent (Oxborough and Horton, 1987).The Dl protein of PS2 appears to be highly susceptible to photodamage and this can beaccelerated by prolonged illumination at high light intensities or at extreme temperatures.Photodamage of PS2 can convert PS2 from high fluorescent, photochemically active centres tolow fluorescent, photochemically inactive centres. The quenching of fluorescence resultingfrom this process is referred to as photoinhibitory quenching, or qi (Krause and Weis, 1991).Room temperature fluorescence can also be affected by the occurrence of statetransitions. In state 1, the amount of LHC2 functionally associated with PS2 is greater than in36state 2 (Allen, 1992). In the absence of other non-photochemical quenching mechanisms, theintensity of fluorescence emitted from antennae pigments is dependent upon the absorptioncross section or amount of antennae pigments associated with the reaction centre (Krause andWeis, 1991). Thus, a decrease in the absorption cross section of PS2 due to a state 1 to state 2transition can be measured as a decrease in fluorescence emitted from PS2 at room temperature.This quenching of fluorescence is referred to as state transition quenching, or qT.It is possible to distinguish between photochemical and non-photochemical quenchingwith room temperature fluorescence measurements using the light doubling technique ofBradbury and Baker (1981). A non-actinic measuring beam (does not cause photochemistry) isused to measure the minimal level of fluorescence of the antennae pigments, Fo , where qp = 1and qNp = 0. Application of a short, saturating flash of light results in full reduction of QA andprovides a measure of the maximal fluorescence possible when qp is 0. The level offluorescence excited by a saturated flash, under conditions where qNp is negligible, is defined asFM . Differences between FM and FM ' (the saturated flash level of fluorescence when qNp > 0)allow a determination of the degree to which qNp contributes to fluorescence quenching.Resolution of the separate components of qNp has been made possible by examining thekinetics of fluorescence relaxation in the dark in the presence of DCMU, sodium fluoride (aphosphatase inhibitor which inhibits a state 2 to 1 transition), and chloramphenicol. A fastphase of relaxation (t112=1 min) is attributed to qE, the second phase (t112=8 min) is attributedto qT, and a slow phase (t112=40 min) is attributed to qi (Horton and Hague, 1988).Resolution of these components, however, can be complicated in green algae and should beperformed with caution (Lee et al., 1990). Quenching of PS2 fluorescence at room temperaturewhich persists after uncoupling of the proton gradient is thought to be due to qT (if the lightintensity is non-photoinhibitory). This, however, should be confirmed by alternativemeasurements (Lee et al., 1990).Absolute 77K fluorescence measurements have traditionally been used to confirm theoccurrence of a state transition (Krause and Behrend, 1983; Saito et al, 1983; Catt et al, 1984;37Rebeille and Gans, 1988). At room temperature, most fluorescence emission arises from PS2.However, upon cooling to liquid nitrogen temperatures (77K), fluorescence emission peaksassociated with both PS2 and PS1 are observed. Peaks associated with the CP43 and CP47proteins of PS2 are observed at 685 and 695 nm, respectively (Krause and Weis, 1991). Longerwavelength peaks with maxima ranging from 715 to 735 nm are thought to arise from the PS1core ("PS1-65") and LHC1 (Krause and Weis, 1991). When fluorescence emission spectra arenormalized to an external fluorescence standard (e.g. fluorescein), it is possible to determine the"absolute" fluorescence emission associated with PS2 and PS1. A complementary decrease inthe absolute fluorescence emission arising from PS2 (F686, F695) and an increase in that arisingfrom PS 1 (F715- F735) have been strongly correlated with the occurrence of LHC2phosphorylation and a state 1 to 2 transition both in vitro (Krause and Behrend, 1983) and invivo (Saito et al, 1983) in higher plants and green algae. Furthermore, Krause et al (1983) haveshown that the absolute fluorescence arising from PS2 is greatly affected by 4H-dependentquenching (qE) while the absolute fluorescence arising from PS 1 is not. Since both qE and qiare thought to have negligible effect on PS1 fluorescence, absolute 77K fluorescence provides auseful tool to resolve quenching mechanisms and determine the contribution of the statetransition to qNp at room temperature (Krause and Weis, 1991).Rationale for working with Selenastrum minutum:In this study, the green alga Selenastrum minutum was chosen to test the interactionbetween respiratory carbon flow and the state transition. Regulation of respiratory carbon flowhas been well characterized physiologically and biochemically in this alga (for a review seeTurpin, 1991). Furthermore, S. minutum is easily grown at steady-state under nutrientlimitation in chemostats. Nutrient limitation of this alga results in large accumulation of starchwhich can support high rates of respiratory carbon flow in the dark (Elrifi and Turpin, 1985). Inaddition, it has been shown that activity of the CRETC, in particular the NAD(P)H-PQoxidoreductase, is enhanced under N-limitation in green algae (Peltier and Schmidt, 1991).38This, in turn, suggested that the effects of respiratory carbon flow on PETC poising might bemore significant and better observed in nutrient-limited cells.This chapter will focus on the question of whether respiratory carbon flow in the darkcan affect the poise of the PETC for the ratio of NADPH/ATP production via a state transition.In order to answer this question, respiratory carbon flow was enhanced by a variety oftreatments and fluorescence characteristics were examined to determine whether a state 1 to 2transition had occurred. Two classes of treatments were distinguished. Both classes resulted inincreases in respiratory carbon flow but the first class resulted in complementary changes in PS2and PS 1 fluorescence emission indicative of a state 1 to 2 transition. The second class oftreatments did not.MATERIALS AND METHODSCell culture (chemostats)The green alga Selenastrum minutum (Naeg.) Collins (UTEX 2459) was culturedaxenically under conditions of NO3 - or Pi-limitation in temperature-regulated (20 °C)chemostats as described previously (Elrifi and Turpin, 1985). Cells were aerated with 5% CO2and magnetically stirred. Continuous illumination was provided by Sylvania cool whitefluorescence VHO tubes with an average PFD of 150 4Ei.m -2 -s-1 . Cells were grown on asubstantially modified Hughes medium buffered with 25 mM Hepes/KOH at pH 8.0. NO3 -limited medium contained 1 mM NaNO3 and 200 uM K2PO4 while Pi limited mediumcontained 12 mM NaNO3 and 30 uM K2PO4. Because the chemostat has a fixed volume, eachdrop of fresh medium forces an equal volume out of the chemostat. Consequently, when cellsare provided with medium which is limited by a particular nutrient, the steady state growth rateof cells is equal to the dilution rate of the culture. NO3 - and Pi-limited cells were grown under39steady-state conditions at 0.3 and 0.6 d -1 , respectively. This represents 18 and 36% of theirmaximal growth rate. For an overview of chemostat theory see Turpin et al. (1985).Treatments:1) NH4:^Re-supply of NH4+ to dark, aerobic N-limited S. minutum results in rates of Nassimilation into amino acids of up to 180 .tmol N•mg -1 Chl•h-1 (Weger and Turpin, 1989).To ensure that cells acclimated during N-assimilation, cells for these experiments were providedwith 180 [tmol NH4Ch1•mg -1 CM which required approximately 1 hour of assimilation.2) Anaerobiosis:^Initiation of strict anaerobiosis was achieved by incubation of cells with aglucose/glucose oxidase 02 scavenging system (Vanlerberghe et al., 1990), consisting of 5 mMglucose, 40 p.g mL -1 catalase, 400 p.g mL -1 glucose oxidase, and replacement of air bubblingwith N2. It has been shown previously that these cells are unable to utilize exogenous glucose(Vanlerberghe et al., 1989).3) CCCP: CCCP was provided to a final concentration of 3.3 wnol CCCP mg -1 Chl. Thisconcentration was shown to uncouple both mitochondrial and chloroplastic electron transportchains.4) NO3- :^NO3- was provided at a concentration of 55 [tmol NaNO3 mg -1 Chl whichallowed cells to fully acclimate to N-assimilation and required approximately 1 hour forassimilation (Weger and Turpin, 1989).5) Phosphate:^KPi ( K2HPO4/KH2PO4, pH 8.0) was re-supplied to Pi-limited cells at aconcentration of -- 80 wnol mg -1 Chl which was shown to require approximately 1 hour forassimilation (Gauthier and Turpin, 1993).40Experimental:Cells were concentrated by centrifugation (5000 rpm, 5 min) and resuspension insupernatant at the concentrations noted in each technique. All experiments were performed in adarkened, temperature-regulated (20 °C) cuvette bubbled with 5% CO2 in air and magneticallystirred unless otherwise stated. Cells were dark-adapted for 20 minutes before treatment.Treatments 1 to 4 were applied to N-limited cells while treatment 5 was applied to Pi-limitedcells. In all cases, cells had fully acclimated to treatment within 20 minutes.Fluorescence measurements:Room temperature steady state fluorescence:All room temperature fluorescence was measured using a PAM fluorometer (HeinzWalz, Effeltrich, FRG) as previously described (Schreiber et al., 1986). Dilute cells (3 -5 ptgChl mL-1 ) were dark adapted for 20 minutes in a temperature-regulated, magnetically-stirred,aerated, 3.0 mL disposable acrylic cuvette. The fibre optic cable was placed at the surface ofthe cuvette. Fluorescence parameters were essentially the same as those designated by vanKooten and Snel (1990) although some measurements were approximations rather than truemeasurements (see Appendix 1). For the purposes of these measurements, Fo was designatedas the level of fluorescence induced in dark aerobic control cells by the 1.6 kHz measuring beam(0.3 [tEi•m-2 •s-1 , < 680 tun). Increases in the fluorescence induced by the measuring beam aftertreatments were designated as F. Maximal fluorescence (F M) was induced by a saturating (10,000 1.tEi•m-2 •s-1 ) 50 msec multiple turnover xenon flash provided by the PAM XMT 103 unit.(The 50 msec pulse results in induction to the 12 level which reflects full suppression ofphotochemical quenching at PS2 (Schreiber et al., 1989)). The 50 msec pulses were used tominimize the actinic effects of the saturating pulses in dark-adapted cells. Changes in FMresulting from treatments were designated as F M '. Because changes in photochemical and non-photochemical quenching (qp and qNp, respectively) and not absolute amounts of quenching41coefficients were of interest, qp and qNp were estimated relative to the control with controlvalues arbitrarily set to qp = 1 and qNp = 0 (see Appendix 1). Calculation of the potentialcorrected quantum yield of linear electron transport for each treatment was performed asdescribed in Holmes et al. (1989): Op = J/I = qp(0.4777 - 0.3282 qNp), where Op is thecorrected quantum yield, J is gross linear photosynthetic electron flow and I is the incident lightintensity.Absolute 77K Fluorescence:For measurement of absolute 77K fluorescence, cells (200 4, 3-5 tg Chl inI: 1 ) wereinjected into uniform NMR tubes and rapidly frozen in liquid N2. Absolute emission spectrawere measured on a custom-built spectrofluorometer fitted with a low temperature quartzdewar filled with liquid N2 (Bruce et al., 1989). Fluorescence was induced by chlorophyll aexcitation (435 nm, 10 nm bandwidth). NMR tubes were mounted in a spinning sample holderdriven by compressed N2. Fluorescence from the whole tube surface was averaged by spinningthe sample at approximately 1000 rpm, during the 10 second measuring period. Repeatedfluorescence yield determinations from identical samples were within 4%. Higher accuracy wasachieved via the spinning method than with the internal fluorescence standard fluorescein.Other measurements:All measurements of metabolites and gas exchange were standardized to chlorophyll.Chlorophyll was extracted for 1 to 4 hours in 100% methanol at -20°C and samples werecentrifuged to remove particulate matter before reading absorbance (Elrifi and Turpin, 1985).Chlorophyll concentration (gg mL -1 ) was calculated as 25.5*(A650)1965).+ 4.0 (A*,--665) (Holden,42RESULTSEffect of treatments on CO2 efflux and starch degradation:1) NH4+: Assimilation of NH4+ resulted in an increase in respiratory carbon flow which wasobserved as a 2.5-fold increase in respiratory CO2 efflux or a 6-fold increase in the rate ofstarch breakdown (Table 1).2) Anaerobiosis:^Anaerobiosis in N-limited S. minutum resulted in a "Pasteur effect", whichwas observed as a 2-fold increase in the rate of starch breakdown (Table 1). The rate ofrespiratory CO2 efflux declined 4.7-fold during anaerobiosis (Table 1).3) CCCP: Uncoupling of N-limited cells with CCCP resulted in a 2.1-fold increase in starchdegradation and a 1.8-fold increase in respiratory CO2 efflux (Table 1; see also Figures 16 and21, Chapter 5).4) NO3-: Treatment of N-limited cells with NO3 - resulted in a 3.5-fold increase in the rate ofCO2 efflux and an 8-fold increase in the rate of starch breakdown (Table 1).5) Pi:^In cells grown under phosphate limitation, re-supply of Pi was correlated with a 2.5-fold increase in both respiratory CO2 efflux and the rate of starch breakdown (Table 1).Effect of treatments on fluorescence emission:Figure 5 shows the effects of the 5 treatments which caused increased respiratory carbonflow on steady state room temperature fluorescence emission from S. minutum. These effectswere tentatively grouped into two classes on the basis of their effects on room temperature and77K fluorescence emission (Table 2).Class 1 treatments (NH4,+ anaerobiosis and CCCP):Perturbation in both FM and Fo occurred after treatment of N-limited cells with NH4+,anaerobiosis or CCCP. F, the level of fluorescence induced by the measuring beam, increased43Table 1: The effect of a variety of treatments on the rate of respiratory carbon flow in S.minutum measured either as the rate of CO2 efflux or starch degradation. Values arenormalized to the respective dark, aerobic control. Pi treatment was made to Pi-limited cells, allother treatments were made to N-limited cells.Treatment rate of CO2 efflux rate of starch breakdowndark, aerobic control (N or Pi-limited) 1.0 * 1.0 **+ NH4+ t 2.5 6.0+ anaerobiosis^ft 0.2 2.0+ CCCP ttt 1.9 2.1+ NO3 - t 3.5 8.0+ N # 2.5 2.5* Actual values were 88.5 (± 32.6) j_tmol CO2 mg -1 Chl h-1 and 724 nmol CO2 mg-1 Chl h-1for N- and Pi-limited cells, respectively.** Actual values were 12.7 (± 0.7) wriol gluc equiv mg -1 Chl h-1 and 10.71..tmol gluc equivmg 1 Chl h-1 for N- and Pi- limited cells, respectively.t from Weger and Turpin, 1989.ft from Vanlerberghe et al., 1989, 1990.ttt see Figures 14 and 19, Chapter 5.t from Weger and Turpin, 1989; Turpin, 1992.tt from Gauthier and Turpin, 1993.F0 homomowsoossolowoossowirmookftopows+piFFm 'FFMFMmi4+0,00,00010#40400011,110+ anaerobiosis1+ CCCPFMF0B.^NOFMF0FNIFM0+ NO3 -FMFD.Fm'E.Fm '10 minutesFigure 5: The effect of treatments which increase respiratory carbon flow on steady-statesaturation pulse analysis as measured with a pulse amplitude modulated (PAM) fluorometer.Control cells (3-5 .tg Chl mL-1 ) were dark, aerobically adapted for 20 minutes before treatmentwith A. 750 p.M NH4C1; B. anaerobiosis; C. 10 pM CCCP; D. 10 uM DCMU; E. 200 p.MNaNO3; F. 400 1.1,M KPi. Treatments A-E and F were made to N-limited and Pi-limited cells,respectively.45approximately 24% within 1 minute of NH4+ treatment and then recovered gradually to controllevels within 20 minutes (Fig 5A). Similarly, F increased a maximum of 38% within 4 minutesof the onset of anaerobiosis (Fig 5B). Maximal change in F did not occur until approximately10 minutes after uncoupling with CCCP by which time it had increased by 50% (Fig 5C). Themaximal level of fluorescence, F M, decreased 29 and 20%, respectively, after 20 minutes ofNH4+ assimilation or anaerobiosis in N-limited cells. Uncoupling with CCCP initially caused a6% increase in FM and then resulted in a 14% decrease in F M within 20 minutes (Figure 5C).This resulted in a 20% overall decrease in fluorescence after treatment with CCCP.Fo is defined as the minimal level of fluorescence when all the reaction centers are open(QA is oxidized). The fluorescence resulting from the low intensity measuring beam in the"dark" was not the true Fo because it increased upon the addition of 10 .tM DCMU (Figure5D). This problem is inherent in the fluorometer because, at the lowest possible measuringbeam light intensity, 10 1.tM DCMU still resulted in an increase in fluorescence induced by themeasuring beam (data not shown). This increase was presumably due to photochemistry causedby the measuring beam (i.e. the measuring beam has an actinic effect) in the presence of DCMU.Thus the fluorescence level measured under dark aerobic control conditions would moreaccurately be defined as variable fluorescence, Fv, but, following convention, has been termedFo .The assumptions used to approximate the quenching parameters (qp and qNp) from thefluorescence transients shown in Fig 5 are provided in Appendix 1. In all cases, the amount ofnon-photochemical quenching (qNp) induced by treatment with NH4+, anaerobiosis or CCCPincreased greater than 0.23 in comparison to dark aerobic control cells within 20 minutes oftreatment (Table 2). Treatment with NH4+, CCCP, and anaerobiosis decreased the amount ofphotochemical quenching (qp) by 6, 27, and 31%, respectively, relative to dark control cells(Table 2). Approximation of Op, the potential quantum yield of linear electron transport(quantum yield which would be observed immediately upon illumination or that observed understeady-state illumination assuming qNp and qp values were the same as those calculated in the46Table 2: A summary of the effect of treatments which increased respiratory carbon flow onroom temperature and 77K fluorescence parameters in S. minutum. qNp and qp wereapproximated relative to the dark aerobic control as described in Appendix 1. The potentialquantum yield of linear electron transport, Op, was calculated as in Holmes et 0.(1989).F686/F717 were calculated from absolute fluorescence spectra.^All values indicatemeasurements taken after 20 minutes adaptation to a treatment.limited cells, all other treatments were made to N-limited cells.Pi treatment was made to Pi-Treatment CiNP (IP CDp F686/F717Dark aerobic controls:N-limited cells 0.0 1.0 0.478 2.33Pi-limited cells 0.0 1.0 0.478 2.82Class 1 treatments:+ NH4+ 0.32 0.94 0.344 1.95+ anaerobiosis 0.27 0.73 0.260 1.90+ CCCP 0.23 0.69 0.254 1.73Class 2 treatments:+ NO3 - 0.08 0.92 0.439 2.25+ Pi 0.03 0.98 0.458 2.8347dark) was made for each treatment (Table 2). Relative to the dark control, Op decreased 85%after 20 minutes of anaerobiosis or CCCP uncoupling and 40% during NH4+ assimilation.Absolute 77K fluorescence emission spectra from cells which were treated with NH4+,CCCP, or anaerobiosis are shown in figure 6. The fluorescence at 686 nm (F686) arises fromPS2 (CP43) while the fluorescence at 717 nm arises from the peripheral antennae of PS1(Krause and Weis, 1991). The effect of NH4+, anaerobiosis, or CCCP was to decrease F686 by10, 9 and 14 % respectively and increase F717 by 21, 13, or 27%, respectively. The absolutedecrease in F686 after treatment with NH4+, anaerobiosis or CCCP was 1.3-, 1.6- and 1.2-foldgreater, respectively, than the absolute increase in F717 (Figure 6). NH4+, anaerobiosis andCCCP treatment decrease the ratio of F686/F717 fluorescence by 20% (Table 2).Class 2 treatments (NO3 -and Pi):In contrast to class 1 treatments, NO3 - assimilation by N-limited cells or Pi assimilationby Pi-limited cells resulted in minimal perturbation in room temperature fluorescence (Figure 5)and caused much smaller changes in qNp or qp relative to dark control cells (Table 2). NO3 -treatment resulted in a 0.08 increase in qNp and a 8% decrease in qp. Pi treatment increasedqNp by 0.03 and decreased qp by 2%. In addition, NO3 - and Pi treatments decreased thepotential quantum yield of linear electron transport by only 9 and 4% respectively (Table 2).The effect of NO3 - or Pi assimilation on 77K emission spectra was also minimal (data notshown) and the F686/F717 ratio decreased by < 5% after treatment with NO3 - or Pi (Table 2).DISCUSSIONEffect of treatments on respiratory carbon flow:In green algae, starch is degraded to hexose phosphates in the chloroplast (Levi andGibbs, 1984). Hexose phosphate can then be oxidized via the oxidative pentose phosphatepathway or the "upper half" of glycolysis to the level of phosphoglyceric acid (PGA) in the800060004000200048e650^675^700^725^750Wavelength (nm)Figure 6: The effect of class 1 treatments (NH4+, anaerobiosis or CCCP) on absolutefluorescence emission at 77K. Control cells (3-5 .tg Chl inL- 1 ) were dark, aerobically adaptedfor 20 minutes (—) before treatment with 750 ptM NH4C1 (----), anaerobiosis (••••) or 10 .tMCCCP All treatments were made to N-limited cells.49chloroplast before being exported to the cytosol (Klein, 1986). PGA is converted to pyruvate(PYR) in the cytosol and imported into the mitochondria where it is converted to acetyl CoAand enters the TCA cycle (Lambers, 1990). CO2 efflux arises from decarboxylation steps in theOPP pathway, the pyruvate dehydrogenase complex, and the tricarboxylic acid cycle (TCAcycle). Increases in respiratory carbon flow can, therefore, be measured directly as increases inthe rate of starch degradation or respiratory CO2 efflux. On the basis of these measurements,all 5 treatments increased dark respiratory carbon flow in the green alga S. minuturn (Table 1).1) The effects of NH4+ assimilation: Treatment of darkened, N-limited cells with NH4+resulted in a 3-fold enhancement of respiratory CO2 efflux and a 2-fold increase in the rate ofstarch degradation (Table 1). In N-limited cells, re-supply of NH4+ results in rapid assimilationinto amino acids (Turpin et al., 1990; Weger and Turpin, 1989). In contrast to assimilation of Nin N-sufficient cells, assimilation of N by N-limited cells is independent of recent photosynthateand can occur in both the dark and the light (Amory et al., 1991). An increase in TCA cycleactivity is necessary to supply carbon skeletons required for amino acid biosynthesis. This hasthe further effect of "drawing down" carbon from starch via glycolysis to replenish carbon in theTCA cycle.2) The effects of anaerobiosis: Anaerobiosis resulted in a 2-fold increase in the rate ofstarch breakdown in darkened N-limited cells (Table 1). Anaerobiosis prevents oxidation ofreduced pyridine nucleotide via the mitochondrial electron transport chain. This treatmentresults in a "Pasteur effect" in green algae (Gfeller and Gibbs, 1984; Peavey et al., 1983;Vanlerberghe et al., 1990) and higher plants (Barker et al., 1967; Faiz-ur-Rahman et al., 1974;Givan, 1968; Kobr and Beevers, 1971). In S. minutum, the Pasteur effect involves a stimulationof glycolytic starch degradation (Table 1) concurrent with an increase in the production offermentative end products such as lactate, ethanol, and succinate (Vanlerberghe et al., 1990).The 5-fold decrease in CO2 efflux, measured during anaerobiosis (Table 1), is consistent with50the observation that only partial oxidative TCA cycle activity occurs during anaerobiosis.Although CO2 is released from ethanol formation (Vanlerberghe et al., 1989), only 1 CO2 isreleased per pyruvate for ethanol formation compared to 3 CO2 released per pyruvate for fullTCA cycle decarboxylation. The occurrence of other fermentative pathways which do notinvolve CO2 efflux would further decrease the rate of respiratory CO2 efflux (Vanlerberghe etal., 1989).3) The effect of uncoupling with CCCP:^Uncoupling of cells with CCCP resulted in anapproximately 2-fold increase in respiratory carbon flow as measured by CO2 efflux or starchdegradation (Table 1). This increase is thought to be a response to decreased levels ofchemiosmotically-generated ATP which serves to activate glycolysis. Specifically, pyruvatekinase (which catalyzes the conversion of PYR to PEP) is thought to be ADP-limited underphysiological conditions and PGA kinase has been proposed to be controlled by adenylateenergy charge (Turner and Turner, 1980; Lambers, 1990). Increases in ADP during uncouplingare thought to increase the activation of PK and increase the rate of respiratory carbon flow viaglycolysis and the TCA cycle (Turpin et al., 1990; Vanlerberghe et al., 1990a; Bulte et al.,1990).4) The effects of NO3 - assimilation:^Treatment of N-limited cells with NO3 - resulted in a3.5-fold increase in the rate of CO2 efflux and an 8-fold enhancement of starch degradation inthe dark (Table 1). Although assimilation of NO3 - into amino acids occurs at one third the rateof NH4+ assimilation in the dark, (Weger and Turpin, 1989) it requires 1.5-fold morerespiratory carbon flow due to the reductant requirements for NO3 - reduction to NH4+(Turpin, 1991).5) The effects of Pi assimilation: Treatment of Pi-limited cells with Pi resulted in a 2.5-foldincrease in both the rate of CO2 efflux and the rate of starch degradation (Table 1). Re-supply51of Pi to Pi-limited cells was observed to result in rapid rates of uptake and assimilation and it isthought that assimilation of Pi results in a rapid increase in respiratory carbon flow (Table 1)due to the high ATP requirements associated with Pi uptake (Gauthier and Turpin, 1993).Effect of treatments on fluorescence emission:Although all 5 treatments resulted in an increase in respiratory carbon flow, thesetreatments were grouped into two classes on the basis of differences in the effects of treatmentson fluorescence emission at both room temperature and 77K (Table 2):Class 1 treatments (NH4+, anaerobiosis or CCCP):Effects on FM, FM ' and qNp: Anaerobiosis, NH4+ assimilation and CCCP uncouplingall caused large perturbations in room temperature fluorescence and were designated class 1treatments (Figure 5 A,B,C). All of these treatments resulted in an increase in F (thefluorescence induced by the measuring beam) and a decrease in FM relative to dark controllevels. The height of FM ' relative to FM can give a direct approximation of the amount of qNpoccurring (see Appendix 1). After treatment with CCCP, F M ' actually increased briefly relativeto FM before decreasing to 73% of the dark control F M (Figure 5C). This suggests that athylakoid proton gradient occurred even in dark control cells and that this may quench the trueFM level. Non-photochemical quenching of dark fluorescence has also been observed in thediatom Phaeodactylum tricornutum and was attributed to the occurrence of a thylakoid protongradient in the dark in this alga (Ting and Owens, 1993). Indeed, measurements of F o and FM ,in the dark and over a wide range of light intensities in S. minuturrt, indicated that the true Fo(qp=1, qNp=0) and FM (qp=0, qNp=0) occurred at very low light intensities rather than in thedark (see Appendix 1). Quenching of PS2 fluorescence is probably due to a combination of aproton gradient in the dark and the fact that the dark state in green algae is intermediatebetween state 1 and state 2 (Williams and Allen, 1987). In the interest of simplicity,52approximations of the level of qNp and qp were made relative to the dark control, assumingthat these values were 0 and 1 respectively (Appendix 1).The level of qNp after 20 minutes of adaptation to class 1 treatments indicated anincrease of 0.2 relative to the dark control. qNp is thought to result from a variety of non-photochemical processes which have been resolved by relaxation of quenching in the dark or inthe presence of DCMU (Horton and Hague, 1988). These include quenching related to abuildup of trans-thylakoid ApH or energized quenching (qE), quenching resulting fromphotoinhibition of PS2 (%), and quenching related to decreases in PS2 absorbance cross sectiondue to a state 1 to state 2 transition (qT) (Krause and Weis, 1991). In the present study, theamount of qNp was calculated relative to the dark control and only qE and qT were possiblecontributors to the total qNp observed after treatment.The fact that much of the qNp measured by room temperature fluorescence arose fromqT was confirmed by measurements of absolute 77K fluorescence. Complementary changes inPS2 and PS 1 fluorescence peaks have been correlated with the occurrence of a state transition(qT) (Krause and Behrend, 1983; Saito et al, 1983) while qE appears to affect only PS2fluorescence emission at 77K (Krause et al, 1983). In all three treatments, a complementarychange in the amplitude of F686 and F717 peaks was consistent with a state 1 to state 2transition (Figure 6). As a result, the ratio of PS2/PS 1 (F686/F717) fluorescence decreased by20% during all class 1 treatments (Table 2). The implication that these treatments resulted ina state 1 to 2 transition in S. minutum is consistent with the fact that LHC2 phosphorylationincreased in Chlamydomonas reinhardtii after treatment with anaerobiosis (Wollman andDelepelaire, 1984) or CCCP (Bulte et al., 1990; Gans and Rebeille, 1990). It is also possiblethat qE may have contributed somewhat to quenching at room temperature although it isdifficult to determine the magnitude of qE contribution to PS2 quenching.It has been proposed that the state transition may function to regulate the relative ratiosof linear and cyclic electron transport (Allen, 1984; 1992; Turpin and Bruce, 1989). One wayto examine this possibility is to examine the effects of observed changes in qNp on the potential53quantum yield of linear electron transport should cells be illuminated. In illuminated cells, thecorrected quantum yield of linear electron transport, Op, was negatively correlated with qNp(Weis and Berry, 1987; Holmes et al., 1989). In class 1 treated cells, the effect of the increasein qNp (arising from both qT and/or qE) in the dark would be to decrease the quantum yield ofPS2-mediated linear electron transport immediately upon illumination as compared to the darkaerobic control (Table 2). Furthermore, assuming that the qNp occurring in the dark statewould affect the time required to reach a steady-state light state and would also be likely toaffect the light state reached, it is quite possible that the changes in dark qNp could affect Op inthe long term during illumination. A decrease in the quantum yield of PS2-mediated linearelectron transport would decrease the amount of NADPH produced by the PETC at a constantintensity of illumination. In addition, if cyclic electron transport were occurring, decreasing thequantum yield of linear electron transport would increase the contribution of cyclic electronflow to total electron transport which, in turn, would decrease the NADPH/ATP productionratio. The state 1 to 2 transition might also have the additional effect of enhancing the quantumyield of cyclic electron transport via increased PS 1 absorption cross section (Table 2) andassociation with cyt b6f. This, in turn, would further down-regulate NADPH production anddecrease the ratio of NADPH/ATP production.It should be noted that chloroplast respiratory e - transport may down-regulate thepotential production of NADPH via the PETC in two distinct ways. First, by increasing thethylakoid ApH via e - transport to 02, the CRETC could increase qE and result in a decrease inthe potential quantum yield of PS2. A decrease in the quantum yield of linear e - transportwould decrease NADPH production and would subsequently decrease the ratio ofNADPH/ATP produced in the light. Second, by reducing the PQ(cyt b6f) pool, the CRETCwould have the effect of a) decreasing the absorbance cross section of PS2 relative to PS 1 andb) increasing the association of cyt b6f and LHC2 with PS1. Both of these effects woulddecrease the quantum yield of linear electron transport and increase the quantum yield of PS1-54mediated cyclic electron transport resulting in a further decrease in the NADPH/ATPproduction ratio.Effects on Fo, F and qp:^Increases in the level of fluorescence induced by the measuringbeam have been interpreted as resulting either from a decrease in connectivity between LHC2and PS2 or changes in the redox state of QA (Krause and Weis, 1991; Buchel and Wilhelm,1990; Govindjee and Satoh, 1986). Under the conditions used in these experiments, it isunlikely that a decrease in connectivity between LHC2 and PS2 resulted suggesting that theincrease was due to changes in the redox state of QA. The increases in fluorescence observedafter treatment with DCMU (Figure 5D) are most likely due to actinic effects of the measuringbeam due to direct inhibition of QA oxidation. On the other hand, the increases in F observedafter class 1 treatments are not likely to be due to direct inhibition of QA- oxidation but aremore likely to arise because these treatments resulted in reduction of the QA pool which in turnincreased fluorescence (Figure 5 A,B,C). Calculation of changes in qp as a result of class 1treatments indicated that a significant decrease in qp occurred consistent with reduction of theQA pool (Table 2). One potential mechanism whereby the QA pool could become reduced isvia redox equilibration with a more reduced PQ pool. If, as hypothesized, the rate of PQreduction was enhanced by an increase in respiratory carbon flow and reductant, this shouldresult in an overall reduction of the PQ pool. This hypothesis will be examined in more detail inChapter 3. An enhancement in the reduction of the PQ pool is also be consistent with theobservation that a state 1 to 2 transition had occurred during class 1 treatments.It should be noted that it is possible that CCCP may have had some independent effectson QA. In addition to being an uncoupler, CCCP is an ADRY reagent (Accelerates theDeactivation Reactions of Y, the water oxidizing complex of PS2) (Buhkov et al., 1990;Renger, 1972). It has been proposed that CCCP results in inhibition of the back recombinationbetween oxidized and QA- ; this would also result in accumulation of QA and an increase influorescence (Mohanty and Govindjee, 1973).55Class 2 treatments (NO3 - and Pi):In contrast to class 1 treatments, NO3 - and Pi assimilation resulted in very littleperturbation of room temperature fluorescence and were designated as class 2 treatments(Figure 5 E,F). At the same time, only minor (< 5%) changes in qp and qNp were observedwhich resulted in very little effect on the potential quantum yield of PS2 linear electron transportin comparison with dark control cells (Table 2). Comparatively small changes in the ratio ofPS2/PS 1 fluorescence indicated that no state 1 to 2 transition (which would change LHC2allocation between PS2 and PS 1) occurred after either of these treatments (Table 2).SUMMARYSelenastrum minutum provided an ideal system to comprehensively test the hypothesisthat increases in respiratory carbon flow which result in increases in the NAD(P)H/NAD(P)ratio will increase the reduction of PQ (via the NAD(P)H-PQ oxidoreductase) and result in astate 1 to 2 transition. Five metabolically dissimilar treatments including NH4+, NO3 - and Piassimilation, uncoupling with CCCP, and anaerobiosis resulted in large increases in the rate ofrespiratory carbon flow (Table 1). Furthermore, with the possible exception of uncoupling withCCCP (a naturally occurring antibiotic), all of these treatments simulate physiological conditionswhich can occur in nature.This set of five treatments was further sub-divided into 2 classes on the basis of themagnitude of their effects on room temperature and 77K fluorescence. Class 1 treatments(NH4+, anaerobiosis or CCCP) resulted in large changes in fluorescence emission characteristicof a state 1 to state 2 transition. The state 1 to 2 transition has been suggested as a mechanismto decrease the efficiency of PS2-mediated linear electron transport relative to PS1-mediatedcyclic electron transport and would poise the PETC to decrease the production of NADPHrelative to ATP (Allen, 1981; Anderson, 1992). In contrast, class 2 treatments (NO3 - and Pi)resulted in only small changes in fluorescence emission characteristics, implying that a state 1 to562 transition had not occurred, despite the fact that both treatments increased the rate ofrespiratory carbon flow. Since a basic tenet of the hypothesis is that the state 1 to 2 transitionoccurs in response to an over-production of NAD(P)H, the class 1 and class 2 treatmentsprovide an excellent framework within which to begin testing other aspects of the model. Withclass 1 and 2 treatments, it should be possible to a) correlate the occurrence of a state 1 to 2transition with a reduction of the PQ pool (Chapter 3) and b) determine whether reduction ofthe pyridine nucleotide pool is a necessary condition for reduction of the PQ pool and activationof the LHC2 kinase (Chapter 4).57CHAPTER 3: TESTING THE MODEL: THE EFFECTS OF CLASS 1 AND CLASS 2TREATMENTS ON REDUCTION OF THE PQ(CYT B6F) POOL INTRODUCTIONThe PQ pool has been proposed as the intersection point between a chloroplasticrespiratory electron transport chain (CRETC) and the PETC (Goedheer, 1963; Diner andMauzeral, 1973; Bennoun, 1982). Since the redox state of the PQ(cyt b6f) pool can regulatethe activation of the kinase responsible for the state 1 to 2 transition (Allen, 1992) and since theredox state of the PQ pool can be affected by electron flow from NAD(P)H to PQ via theCRETC (Bennoun, 1982), it is critical to determine the effects of increases in respiratory carbonflow on the redox state of the PQ pool. In particular, it is necessary to demonstrate acorrelation between an increase in respiratory carbon flow and PQ reduction both to providedirect evidence consistent with the operation of the CRETC in vivo and to confirmmeasurements of the occurrence of a state 1 to 2 transition.It was demonstrated in Chapter 2 that respiratory carbon flow affected the poise of thePETC for the NADPH/ATP production ratio via a state 1 to 2 transition. Two classes oftreatments which increased respiratory carbon flow were distinguished. Class 1 treatmentsresulted in a state 1 to 2 transition while Class 2 treatments did not. Class 1 treatments alsoincreased F, the fluorescence induced by the measuring beam after the treatment suggesting thatthe QA pool had become more reduced. It was proposed that reduction of the QA pool mightbe a result of redox equilibration with a more reduced PQ pool resulting from class 1treatments. The purpose of this chapter was to determine the effects of increases in respiratorycarbon flow on the redox state of the PQ(cyt b6f) pool in more detail. The sub-hypothesis to betested was that class 1 treatments would result in an increase in PQ(cyt b6f) pool reductionwhile class 2 treatments would not.58Methods which can be used to infer the redox state of the PQ pool:The physical reactions within the pigment beds of PS2 and PS 1 are much faster thanelectron transport processes. Photon capture and exciton migration to the reaction centresoccurs within 10 -15 sec and 5 x 10-12 seconds, respectively (Lawlor, 1987). Chlorophyllfluorescence occurs within 10 -9 seconds if the reaction centre is closed (primary electrontransfer acceptor is reduced), but is faster if the reaction centre is oxidized. At roomtemperature, fluorescence originates mainly from PS2 while fluorescence from PS 1 is minimal.This is due to the fact that, although both P680+ and P700+ can act as traps for excitationenergy (dissipation as heat) and represent "quenchers" of fluorescence, reduction of P700+ isslow (ms to gs) compared to that of P680+ (nsec) and, therefore, P700+ acts as a much moreefficient quencher of fluorescence (Krause and Weis, 1991; Horton and Bowyer, 1991). Therate-limiting step in photosynthetic electron transport is electron transfer from PQH2 to the cytb6f complex. A six-fold increase in this time constant (16 - 90 msec) is observed as thethylakoid proton gradient increases from low to high levels (Lawlor, 1987). On average, anelectron is transferred from water to NADP+ within 20 msec (Wong, 1982; Govindjee andWaseilewski, 1990; Lawlor, 1987).Both time-resolved fluorescence induction and decay can be used to deduce the redoxstate of the PQ pool. In the absence of qNp, the fluorescence yield at any one time isproportional to the redox state of QA; that is, there is a hyperbolic relationship between thefluorescence yield at any one time and the fraction of the QA pool that is reduced (Joliot andJoliot, 1964; Cao and Govindjee, 1990; Gleiter et al., 1993). Fluorescence yield is maximal(FM) when QA is reduced and minimal (F0) when QA is oxidized. The kinetics of fluorescenceinduction from Fo to FM or fluorescence decay from FM to F o are related to competingprocesses which govern the rate of oxidation and reduction of the QA pool (Krause and Weis,1991). The kinetics of these individual processes can be resolved by judicious choice of thetime scale on which measurements are based.59Measurements of fluorescence decay from FM to F o in the pecond time scale can beused to resolve the rate of QA oxidation by QB(PQ). The dark decay of variable fluorescenceback to the Fo state has typically been used to examine the effects of mutations, inhibitors andphotoinhibition on the re-oxidation kinetics of QA (Cao and Govindjee, 1990; Gleiter et al.,1993; Erickson et al., 1989; Govindjee et al., 1992; Robinson and Crofts, 1983). In thismethod, by means of a high intensity single turnover flash, approximately all QA (but not QB orPQ) is reduced, resulting in a maximal level of fluorescence, F M . The decline of fluorescence(induced by a weak, modulated, "non-actinic" measuring light) to the minimal level offluorescence, Fo , in the ensuing dark period reflects the re-oxidation kinetics of QA (Krauseand Weis, 1991). When this decay is curve-fitted, it can be shown to proceed in threeexponential phases with lifetimes of 200 - 900 1.tsec, 2-10 msec, and 1-2 seconds (Cao andGovindjee, 1990; Robinson and Crofts, 1983). The fastest phase has been attributed to electrontransport from QA to QB in centres containing bound QB. The middle phase is thought torepresent the binding kinetics of PQ to the QB site in centres with no QB bound before theflash. The slowest component of the decay has been attributed to PS2 centres that are unable totransmit electrons to the QB pool (PS2(3 centres). QA- re-oxidation apparent from this phase isthought to result from recombination between QA and the S2 state of the water oxidizingcomplex of PS2 (Cao and Govindjee, 1990; Etienne et al., 1990). It should noted, however,that decay models merely quantify results by describing the decay of QA. In reality, theseprocesses may not necessarily be independent of each other and may not follow the first orderreaction kinetics that have been assigned to them (Cao and Govindjee, 1990).Because the rate of re-oxidation can be affected by the redox state of the PQ pool, it isalso possible to infer the reduction state of the PQ pool from fluorescence decay kinetics.However, under conditions where the PQ pool is partially reduced, the QA pool is also likely tobe slightly reduced due to redox equilibration between these two pools (Robinson and Crofts,1983). Thus, before and after the single turnover flash the QA pool will not be fully oxidizedand fluorescence will decay from a state where all QA is reduced to a state where only part of60the QA pool is reduced. In addition, PS2 will exist in a large variety of redox conformationsbefore and during the flash including QAQB, QAQB, QAQB 2 , QAQBH2, QA, and QAleading to considerable complexity as compared to conditions where all QA is oxidized beforethe single turnover flash. However, despite this added complexity, reduction of the PQ poolwill, in addition to slightly reducing the QA pool before the single turnover flash, also decreasethe rate of QA- oxidation because of the decrease in oxidized PQ available to oxidize QA -(Robinson and Crofts, 1983). Thus, one would expect a decrease in the rate of fluorescencedecay when the PQ pool becomes reduced.Measurement, on a msec time scale, of the kinetics of fluorescence induction from F o toFM allows resolution of the kinetics of QA reduction (Krause and Weis, 1991). Since PQ to cytb6f is the rate-limiting electron transfer step in the msec time frame, the availability of oxidizedPQ will determine the kinetics of QA reduction and hence fluorescence induction to FM.Increasing the reduction of the PQ pool should increase the rate of fluorescence induction. Inthis chapter, both fluorescence decay and induction analysis are employed to examine the effectof increased rates of respiration on PQ redox status.MATERIALS AND METHODSExperimental conditions:All experimental conditions were as previously described for steady-state fluorescencesaturation pulse analysis (Chapter 2) unless otherwise specified. Cells were adapted for 20minutes under control or treatment conditions before measurement of fluorescence parameters.Fluorescence:Time-resolved decays:Time-resolved fluorescence decays were measured essentially as described by Schreiber(1986) using the PAM fluorometer (Heinz Walz, Effeltrich, FRG). A single turnover flash (t11261= 8 gsec, 10,000 gEi•m -2 •s-1 ) was provided by the XST 103 (Walz) unit. Three msec beforetriggering of the single turnover flash, a low intensity modulated measuring beam (X = 660 nm,0.5 gEi•m-2 •s-1 ) was electronically switched from 1.6 kHz to 100 kHz. The 100 kHzmeasuring beam was used for a duration of 40 msec to allow resolution of the kinetics of slowerfluorescence decays which resulted from class 1 treatments (in particular NH4+ or anaerobiosistreatment, see Appendix 2). Due to special gating circuitry on the fluorometer detector, thefirst signal point was recorded 120 p.sec after triggering of the single turnover flash. The signaloutput was measured at 25 gsec intervals and stored by the DA100 IBM-compatible interface(Kolbowski and Schreiber, Walz, Effeltrich, 1991). In order to optimize signal to noise ratiosfor curve fitting analysis, 8 consecutive decays (dark time interval between flashes, 15 sec) wereaveraged. The actinic effect of the measuring beam was measured by disconnecting the XST103 flash lamp and electronically triggering the measuring beam to switch from 1.6 to 100 kHz.ASCII files were transferred to Sigma Plot for further analysis. Time zero for thefluorescence decay measurements was fixed at the time the actinic flash reached its maximumintensity (Gleiter et al., 1993). Due to gating circuitry, the first data point used in decay analysiswas at 120 gsec. For curve fitting, data sets were reduced to 150 data points by averagingpoints in the asymptotic part of the decay. Fluorescence decay curves were corrected for theactinic effects of the measuring beam by subtracting the fluorescence induced by the measuringbeam from the observed fluorescence decay (see Appendix 2 for details). [QA1 was calculatedfrom variable chl a fluorescence according to Joliot and Joliot (1964) assuming that theintersystem exciton transfer probability (p) was 0.5 (Cao and Govindjee, 1990). This yields ahyperbolic relationship between fluorescence yield, F(t), and the fraction q(t) of closed reactioncentres at time t such that:q(t) = 2(F(t) - F 0 )/[(F m - F 0 ) + ( F(t) - F 0 )]In curves corrected for the actinic effects of the measuring beam (see Appendix 2), thisrelationship assumed the form:622(F(t)mm_sr — F(t)MB ) q(t) =[(FM — F(t)mB )+ (F(t)mm_sr — F(t)mB )]where F(t)MB+ST was the fluorescence measured at time, t, by the 100 kHz measuring beam inconjunction with a single turnover flash and F(t) MB was the fluorescence measured at time, t, bythe 100 kHz measuring beam in the absence of a single turnover flash. In the case of treatedcells, Fm ' was substituted for FM . The fraction of closed reaction centres at time t, q(t), wasthen be fitted by an iterative least squares method to a sum of exponentials of the form:Nq(t) = q(0) E ai exp -t/tii=1where ai is the amplitude and 'Li is the lifetime of the ith component of the decay. Ti, the tin ofeach decay was calculated as 0.69/Ti.Time-resolved inductions:Fluorescence inductions were recorded in the same manner as decays with the followingexceptions. A 50 msec, multiple turnover, 10,000 pEi.m-2 .s -1 flash was provided by the XMT103 unit (Walz). This multiple turnover flash has essentially square on/off characteristics andshould not affect the shape of the fluorescence induction curve (Heinz Walz, 1987). A lowerintensity measuring beam (0.05 1.tEi.m-2-s -1 ) was switched from 1.6 to 100 kHz for 50 msecand signal output was recorded at 300 pec intervals. Induction curves were signal-averaged 8times with a frequency of 0.5 min -1 . FM was calculated as the average of the fmal 3 msec of theinduction curve, or the highest point achieved in the induction curve (anaerobiosis, DCMU).The area above the fluorescence induction curve, waswas calculated as the area between thefluorescence curve and its asymptote (FM or FM).63RESULTSTime-resolved fluorescence decays:DCMU and Class 1 treatments (NH4,+ anaerobiosis, CCCP):The effect of class 1 type treatments on fluorescence decays (corrected for the actiniceffect of the measuring beam) is shown in figure 7 (A, B, and C). NH4+ and anaerobictreatment resulted in a large decrease in the rate of decay which was observed as an increase inthe area between the treated decay curve and the dark control decay curve (Figure 7 A, B,).Inhibition, with DCMU, of electron transfer from QA to QB also resulted in a decrease in therate of decay seen as an increase in the area between the treated decay and the dark controlcurve (Figure 7 D). Uncoupling of cells with CCCP did not result in an increase in the areabetween the treated and control decay curves but affected the shape of the fluorescence decay(Figure 7 C). Fluorescence decays in Figure 7 were transformed to q(t) decays as described inMaterials and Methods. The dark control q(t) decay was a sum of two exponential decaycomponents including a fast component with a half-time, T1, of 185 psec and a mediumcomponent with T2 of approximately 5 msec (Table 3). The fast component of the dark controlq(t) decay contributed 64% (as measured by the amplitude of the fast component, a1=0.64)while the slow component contributed only 36% (a2=0.36) to the total q(t) decay. All Class 1treatments resulted in a loss of the fast (lsec) decay component (ai=0) and an increase in thetotal contribution of the medium (msec) decay component (a2=1.0) (Table 3). Although thehalf-time of the msec decay component appeared to increase after treatment with DCMU andNH4+, this increase was not statistically significant as determined by a student t test.Class 2 treatments (NO3 - or Pi):Class 2 type treatments had a much smaller effect compared to class 1 treatments on theobserved rate of fluorescence decay (Figure 7 E, F). NO3 - treatment resulted in a smalldifference between the NO3 - decay and the dark control decay between 0 and 10 msec (Figure1 .21 .00.80.60.40.20.0—0.2• 1.21.00.80▪ 0.6(I)0CC'^ .4V0^0.2V0.0FA: —0.21.21.00.80.60.40.20.0—0.2—10 0^10 20 30 40 50-10 0^10 20 30 40 50Time (msec)64Figure 7: The effect of class 1 and class 2 treatments on corrected time-resolved fluorescence decaykinetics. Decays were measured using a 40 msec duration, 100 kHz measuring beam and were correctedfor the actinic effect of the measuring beam as shown in Appendix 2. Open circles: dark, aerobic controlcells; closed circles: cells treated with A. NH4+; B. anaerobiosis; C. CCCP; D. DCMU; E. NO3 - ; F.Pi. Treatments A-E and F were made to N-limited and Pi-limited cells (3-5 Chl m1: 1 ), respectively.The single turnover flash (t112=8 ilsec) was initiated 3 msec after the measuring beam was switched from1.6 to 100 kHz. Decays were normalized to FM-Fo (control) or F1,;-F (treated) where FM and Fm werethe maximal fluorescence reached during the flash and Fo or F were the fluorescence measured over a 10msec period before flash initiation. All curves were an average of 8 measurements.657 E) while Pi treatment resulted in a difference between the treated and control curves after 10msec of treatment (Figure 7 F). NO3 - assimilation decreased the contribution of the pisec q(t)decay component from 64 to 48% and increased the contribution of the msec decay componentfrom 36 to 52% (Table 3). T1, the half time of the gsec decay component, was also slightlyincreased after treatment with NO3 - (Table 3). The increase in T2, the half-time of the msecdecay component, after treatment with NO3 - was not statistically significant. In Pi-limitedcontrol cells, the 44% contribution of the gsec q(t) decay component (a1) was significantlysmaller than the 64% contribution of this component in N-limited control cells (Table 3).Treatment with Pi resulted in no significant effect on the amplitudes of either the gsec or mseccomponents. The half-time of the msec component (T2), however, was significantly increasedby treatment with Pi.Time-resolved fluorescence inductions (msec time scale):DCMU and Class 1 treatments:Treatment with NH4+, anaerobiosis, or CCCP resulted in an increase in the rate offluorescence induction compared to the dark aerobic control (Figure 8 A, B, C). The areaabove the fluorescence curve is proportional to the rate at which a curve reaches F M . Treatmentwith NH4+ resulted in a rapid increase in fluorescence to FM within 900 p.sec (Figure 8A) and a95% decrease in the Amax , the area above the induction curve (Table 4). Anaerobiosis resultedin an increase to FM within 900 pec but, after reaching a plateau, the fluorescence leveldeclined to a level 10% less than FM (Figure 8B). Amax was decreased by 97% compared tothe control after treatment with anaerobiosis (Table 4). CCCP treatment resulted in a rapidincrease to a level which was 10% below F M and was followed by a slow increase to FM (Figure8C). This corresponded to a 64% decrease in the area above the induction curve as comparedto the dark aerobic control (Table 4). Similarly to treatments with NH4+, treatment withDCMU resulted in induction of fluorescence FM within 900 gsec; subsequently fluorescenceslowly decayed to a level 10% below F M within 50 msec (Figure 8D). In cells treated with0.64 (.07)0.44 (.02) *0.36 (.07)0.56 (.02) *0.0* 1.0*0.0* 1.0*0.0* 1.0*0.0* 1.0*185.3 (24.8) 4.89 (1.59)192.4 (14.6) 6.67 (.34)0.0* 10.8 (1.21)o.o*^9.91 (1.66)0.0* 4.34 (0.34)0.0*^3.35 (0.04)N-limited cellsPi-limited cells+ DCMUClass 1 treatments:+ NH4++ anaerobiosis+ CCCP66Table 3: The effect of DCMU and class 1 and class 2 treatments on the amplitude (ai) and halftimes (Ti = 'Li/0.69) of the fast (iisec; al and T1) and medium (msec; a2 and T2) componentsof time-resolved q(t) decays which were corrected for the actinic effect of the 100 kHzmeasuring beam (see Appendix 2). Cells were adapted to treatments for 20 minutes beforemeasurements of decays. Fluorescence decays (Figure 7) were transformed to q(t) decays andthen curve-fitted for either a single exponential or a sum of two exponentials (see Materials andMethods). tTreatment^al (SE)^a2 (SE)^T1 (.isec) (SE)^T2 (msec) (SE)(corrected for the actinic effect of the measuring beam)Dark aerobic controls:Class 2 treatments:+ NO3 -+ Pi t t0.48 (.01) * 0.52 (.01) * 291.4 (41.9)*0.51 (.04) 0.49 (.04) 201 (13.6)9.09 (1.31)10. 5 (1.0) **t In all cases where curves fitted a single exponential, the r2 for the single exponential fit wasgreater than 0.98 and/or greater than the r 2 for the sum of two exponentials.ft Pi treatment was made to Pi-limited cells, all other treatments were made to N-limited cells.Indicates that this value was significantly different from the N-limited dark aerobic control,i.e. the value was outside the 95% confidence interval as determined by a student t test.** Indicates that this value was significantly different from the Pi-limited dark, aerobic control.0.80.60.40.20.0—0.21.21.00.80.60.40.20.0—0.21.21.00.80.60.40.20.00.2—10 0 10 20 30 40 50 60-10 0Time (msec)671.21.010 20 30 40 50 60Figure 8: The effect of class 1 and class 2 treatments on time-resolved fluorescence inductionkinetics. Inductions were initiated at t=0 with a multiple turnover, 50 msec duration, saturatingflash (10,000 1.1,Ei , m-2 s -1 ). Inductions were normalized to Fm-Fo (control) or FM '-F (treated),where FM and Fm were the maximal fluorescence reached within 50 msec. Open circles: dark,aerobic control cells; closed circles: cells treated with A. NH4+; B. anaerobiosis; C. CCCP;D. DCMU; E. NO3 - ; F. Pi. Treatments A-E and F were made to N-limited and Pi-limited cells(3-5 tg Chl mL -1 ), respectively. All treatments were an average of 8 measurements.68Table 4: The effect of DCMU and treatments which increase respiratory carbon flow on A max ,the area above time-resolved fluorescence induction curves. A max was calculated asFmAm. = I [ t x(Fm — F(t))], where F(t) is the fluorescence at time t and F m ' was substituted fort.0FM in treated cells. Samples were adapted for 20 minutes of treatment before measurement. Pitreatment was made to Pi-limited cells. All other treatments were made to N-limited cells.Treatment (n=) Area above induction curve,Amax (mV.p.sec) (SE)% of control AmaxDark aerobic controls:N-limited cells (19) 3311.5 (208) 100.0Pi-limited cells (3) 637.5 (35) 100.0+ DCMU (2) 47.3 (14.6) 1.4Class 1 Treatments:+ NH4÷ (3) 125.2 (48.3) 3.8+ anaerobiosis (3) 99.4 (5.8) 3.0+ CCCP (3) 1182.8 (172.1) 35.7Class 2 Treatments:+ NO3 - (5) 610.4 (116) 18.4+ Pi (3) 844.3 (41) 132.4*^.^.Significantly different from the dark control as determined by a student t test.69DCMU, the area above the fluorescence induction curve was decreased by 98% compared tothe Amax of dark control cells (Table 4).Class 2 treatments:Treatment with NO3 - resulted in a rapid increase to a level which was 90% of F M andthen a slow increase to FM within 50 msec (Figure 8E). This resulted in an 82% decrease inAmax, the area above the fluorescence curve (Table 4). Pi treatment resulted a slightly slowerrate of fluorescence induction (Figure 8F) and initiated a 30% increase in the area above thefluorescence induction curve (Table 4).DISCUSSIONDCMU and Class 1 treatments:Time-resolved fluorescence decays:Fluorescence decay kinetics are related to QA- re-oxidation kinetics and can be used todeduce the redox state of the PQ pool (Krause and Weis, 1991). Furthermore, the rate of QAoxidation is dependent upon the rate of e - transfer to the QB(PQ) pool and this in turn can beaffected by the redox state of the PQ pool (Robinson and Crofts, 1983). When all QA isoxidized before the single turnover flash, QA oxidation kinetics have been de-convoluted toyield three decay components attributed to PS2 species with QB bound before the flash, QB notbound before the flash and QB-non-reducing centers (Cao and Govindjee, 1990).However, under conditions where QA is not completely oxidized before the flash anumber of redox species would be present and it is not surprising that the fluorescence decays,in this case, can be approximated by 2 components rather than 3 (Table 3). Despite the fact thatQA was likely to have been initially reduced (particularly in the case of class 1 treatments), therate of oxidation of QA- after a single turnover flash should still be affected by the redox stateof the PQ pool. Moreover, decreases in the rate of QA oxidation should affect the contribution70and/or the half-times of the 2 components of the decays. Indeed, all class 1 treatments resultedin a loss of the pec component of QA re-oxidation kinetics and a 100% contribution (a1=1.0)of the slower msec half-time decay component (Table 3) indicating that QA oxidation haddecreased due to reduction of the PQ pool. The loss of the gsec decay component observedafter treatment with DCMU, which prevents binding of QB to PS2 and, therefore, preventsoxidation of QA by QB , further confirms that class 1 treatments resulted in a decrease in therate of QA- oxidation after a single turnover flash (Table 3). The reduction of the PQ poolimplied by the decreased rate of QA- oxidation after class 1 treatments is consistent with theobservation of fluorescence changes indicating the occurrence of a state 1 to 2 transition sincePQ reduction would be required to activate the kinase responsible for the state 1 to 2 transition.Time-resolved fluorescence induction:The reduction state of inter-system electron carriers can also be determined by time-resolved fluorescence induction kinetics on the msec time scale. The area bounded by thefluorescence rise and its asymptote is a measure of the pool size of electron acceptors of PS2which is equivalent to the pool of oxidized PQ available for photochemistry (Schreiber, 1986;Horton and Bowyer, 1991; Krause and Weis, 1991). A faster rise (smaller area above thecurve) is indicative that a smaller proportion of the total pool is being oxidized and is availableto accept electrons from PS2 (Krause and Weis, 1991; Bennoun, 1982). Support for thisinterpretation comes from the observation that the addition of DCMU resulted in a rapidincrease in the rate of QA reduction (Figure 8 D) and decreased the area above the inductioncurve, toto 2% of the dark control level (Table 4). Class 1 treatments (NH4+,anaerobiosis, CCCP ) also resulted in faster rates of fluorescence induction (Figure 8 A, B, C)and decreased the Amax by 96, 97, and 64% respectively (Table 4). Combined with evidencefrom fast fluorescence decays, these data support the hypothesis that class 1 treatments result ina reduction of the PQ(cyt b6f) pool.71An increase in the reduction of the PQ pool, as measured by either fluorescenceinduction or fluorescence decays, as a result of treatment with NH4+, anaerobiosis, or CCCP, isconsistent with the fact that each of these treatments appeared to cause a state 1 to 2 transition(Chapter 2). Reduction of the PQ(cyt b6f) pool is required to activate the protein kinaseresponsible for the state 1 to 2 transition (Allen, 1992). An increase in the reduction of thePQ(cyt b6f) pool, as a result of increased respiratory carbon flow, also provides indirectevidence that respiratory carbon flow can affect electron transport to PQ via an NAD(P)H-PQoxidoreductase (Godde and Trebst, 1980).Class 2 treatments (Pi and NO3 - assimilation):Time-resolved fluorescence decays:In dark control Pi-limited cells, the 44% contribution of the pec component of the QAdecay was significantly smaller than the 64% contribution in N-limited control cells. This mayindicate that the PQ pool was more reduced in Pi-limited control cells than in N-limited controlcells. However, the ratio of PS2/PS 1 fluorescence at 77K was higher in Pi-limited cells than inN-limited cells (Table 2, Chapter 2) suggesting that variations in fluorescence decaycharacteristics may arise from differences in PETC component stoichiometry in cells adapted toN- and Pi-limitation. Pi treatment of Pi-limited cells resulted in no significant change in theamplitudes of either the gsec or msec QA - decay components although an increase in the half-time of the msec component was observed (Table 3). The observation that Pi treatment had noeffect on the contributions of the gsec or msec decay components implies that the rate of QA-oxidation was relatively unaffected compared to class 1 treatments and, therefore, that the redoxstate of the PQ pool was also relatively unaffected.Treatment of N-limited cells with NO3 - resulted in a decrease in the contribution of thegsec q(t) decay component from 64 to 48%, an increase in the half-time of the lisec decaycomponent and an increase in the contribution of the msec decay component from 36 to 52%72(Table 3). Although the contribution of the psec QA decay component decreased after NO3 -treatment, the extent of this change was much smaller than that observed after class 1treatments or treatment with DCMU. This suggests that increases in PQ reduction during NO3 -assimilation were minor compared to class 1 treatments. The observation of only minor changesin PQ reduction, implied by fluorescence decay measurements after treatment with NO3 - , isconsistent with both saturation pulse and 77K fluorescence analysis which indicated that a state1 to 2 transition had not occurred (Chapter 2). Slight discrepancies may have resulted fromactinic effects of repetitive single turnover flashes for signal averaging.Fluorescence inductions:Treatment with Pi slightly increased the area above the fluorescence induction curve andindicated a minor oxidation of the PQ pool during Pi assimilation (Table 4). Comparison ofinduction and j.tsec decay data suggests that Pi treatment had relatively little effect on thereduction of the PQ(cyt b6f) pool. This is consistent with the observation that a state 1 to 2transition was not induced by treatment with Pi (Chapter 2).Treatment with NO3- resulted in an 82% decrease in the area above the fluorescenceinduction curve (Figure 8, Table 4) implying that the pool of oxidized e acceptors became morereduced. This is inconsistent with the minor changes in fluorescence decay kinetics observedand the absence of a state 1 to 2 transition (Chapter 2). The inconsistency between inductionand decay measurements in cells assimilating NO3 - suggests that additional factors may affectQA- oxidation and reduction kinetics after treatment with NO3 - .One possible reason for the increase in PQ reduction observed after treatment withNO3 - is that illumination with a multiple turnover flash may affect the redox state of inter-system e - acceptors. Electron flow can occur from PS2 to NADP within 20 msec (Lawlor,1987). Thus, inhibition of electron transfer from PS1 to NADP would also affect the rate offluorescence induction of the msec time scale. If inhibition of electron transfer from PS1 toNADP were due to a substrate-related phenomenon during NO3 - assimilation, then reduction of73the NADP(H) pool would be expected. However, in N-limited S. minutum, the NADP(H) poolbecame more oxidized during dark NO3 - assimilation (Vanlerberghe et al., 1992).Alternatively, however, it is possible that inhibition of electron transfer from PS 1 to NADPHmight occur as a result of Fd/FNR limitation due to super-complex formation. Precedence forreversible super-complex formation involving Fd and FNR comes from the observation thatFNR, LHC2 kinase, and cyt b6f have been shown to co-purify when isolated from thylakoidmembranes (Joliot et al., 1993, 1989; Gal et al., 1990a; Hodges et al., 1987). It has beenproposed that electrons for NO2 - reduction may come from NADPH via Fd and reverse actionof FNR (Paneque et al., 1967). Although very little is known about the regulation of FNRassociation in the dark, precedent for super-complex association suggests that FNR and nitritereductase may associate during NO3 - assimilation. This, in turn, could decrease the associationand/or availability of Fd/FNR for PS 1 oxidation and result in reduction of the intersystemelectron transport chain which would be further enhanced by repetitive averaging. The decreasein Amax observed during NO3 - assimilation may not be due to direct reduction of PQ byNAD(P)H-PQ oxidoreductase activity but, rather, may reflect the accumulation of reductant atPS1 due to Fd/FNR limitation.SUMMARYThe fluorescence induction and decay data presented above indicate that class 1treatments (NH4+, anaerobiosis, CCCP), which stimulated respiratory carbon flow and resultedin a state 1 to 2 transition, resulted in an increase in the rate of fluorescence induction and adecrease in the rate of fluorescence decay. Both of these results were consistent with anincrease in the reduction of the PQ pool. This would provide a mechanism by which the LHC2kinase could be activated and result in a state 1 to state 2 transition. In addition, reduction ofthe PQ pool, as a result of class 1 treatments, implies that a mechanism exists which can reduce74the PQ pool in the dark and provides indirect evidence for NAD(P)H-PQ oxidoreductaseactivity. A link between an increase in the reduction of the NAD(P)H pool and an increase inthe reduction of the PQ pool would provide strong evidence for interaction between respiratorycarbon flow and the state transition which can regulate photosynthetic NADPH/ATP productionratios. This link will be examined in the next chapter.Treatment with either NO3 - or Pi resulted in negligible changes in room temperature or77K fluorescence after 20 minutes of acclimation indicating that a state 1 to 2 transition did notoccur (Chapter 2). The rate of fluorescence decay on the I.tsec time scale was also relativelyunaffected in either of these treatments in comparison to the affects of DCMU or class 1treatments (Table 3) suggesting that the PQ pool was not greatly reduced. Millisecondinduction kinetics confirmed that the PQ pool was not reduced by Pi treatment. In the case ofNO3 - assimilation, illumination with the multiple turnover flash may have actinic effects whichobscured the effect of NO3 - treatment on PQ redox in the dark. No changes in roomtemperature or 77K fluorescence were observed during NO3 - assimilation (Chapter 2), whichwas consistent with the minor changes in fluorescence decay but inconsistent with inductiondata. Such variability highlights the complexity of factors contributing to the fluorescencecharacteristics of cells assimilating NO3 - .When examining long term induction phenomena, it is obvious that extreme care must betaken, particularly when using induction kinetics to determine rates of QA reduction andoxidation and to deduce the redox state of the intersystem electron transport chain. Becauselight is used to induce and to measure fluorescence, inaccuracy is inevitable in the determinationof QA reduction and oxidation kinetics unless the actinic effects of the measuring beam arecorrected for (see Appendix 2).75CHAPTER 4: THE EFFECTS OF CLASS 1 AND CLASS 2 TREATMENTS ON THEREDOX STATE OF THE PYRIDINE NUCLEOTIDE POOL: INTRODUCTIONIt was proposed in Chapter 1 that respiratory carbon flow may poise the PETC todecrease the NADPH/ATP production ratio by initiating a state 1 to 2 transition. An increase inthe ratio of reduced/oxidized pyridine nucleotides resulting from increased respiratory carbonflow would reduce the PQ(cyt b6f) pool via an NAD(P)H-PQ oxidoreductase (Godde andTrebst, 1980) and result in a state 1 to 2 transition. Treatment of nutrient-limited S. minutumwith NH4+, anaerobiosis, CCCP, NO3 - , or Pi has been shown to result in a rapid increase in therate of respiratory carbon flow as measured by starch degradation and/or CO2 efflux (seechapter 2). However, only the first three treatments (NH4+, anaerobiosis or CCCP) resulted inchanges consistent with the reduction of PQ and the occurrence of a state 1 to 2 transition(chapters 2 and 3). Selective use of these treatments provides an ideal system to test theimplications of the model for interaction between respiratory carbon flow and state 1 to 2transitions. Specifically , this system will allow an examination of whether an increase in theNAD(P)H/NAD(P) ratio is a requirement for an increase in PQ reduction and a state 1 to 2transition.An increase in the cellular NADPH/NADP ratio has been correlated with the occurrenceof a state 1 to 2 transition in the dark by several workers (Bulte et al., 1990; Gans and Rebeille,1990; Mohanty et al., 1990). In Chlamydomonas reinhardtii, Gans and Rebeille (1990)presented evidence consistent with the occurrence of a state 1 to 2 transition after treatment ofdarkened cells with CCCP (uncoupler) or a combination of antimycin A and SHAM (fullinhibition of METC electron transport to either cytochrome or alternative oxidase). Bothtreatments were proposed to result in an activation of respiratory carbon flow due to the releaseof ADP-limitation of PK and activation of glycolysis. Furthermore, both treatments could be76shown to increase NADPH/NADP but not NADH/NAD ratios, suggesting that the former ratiowas important in initiating a state 1 to 2 transition (Rebeille and Gans, 1988; Gans and Rebeille,1990; Bulte et al., 1990). Mohanty et al. (1990) provided evidence consistent with theoccurrence of a state 1 to 2 transition after treatment of darkened, N-limited S. minutum withNH4+ but not NO3-. Mohanty et al. (1990) also suggested that the state 1 to 2 transition wasdue to an increase in the NADPH/NADP ratio although increases in both the NADH/NAD andNADPH/NADP ratios were shown to occur in darkened N-limited S. minutum after treatmentwith NH4+ (Vanlerberghe et al., 1992). In all studies to date, an increase in the cellularNADPH/NADP ratio was proposed to result in an increase in PQ reduction via NAD(P)H-PQoxidoreductase activity in the thylakoid membrane which, in turn, resulted in a state 1 to 2transition.Several pieces of evidence suggest, however, that NADH may be as important as, if notmore important than, NADPH as a substrate for the thylakoid NAD(P)H-PQ oxidoreductase.First, isolated NAD(P)H-PQ oxidoreductase has been shown to utilize NADH with higherefficiency than NADPH (Godde and Trebst, 1980). Second, NADH is commonly associatedwith catabolic respiratory processes and is used primarily for the generation of ATP whileNADPH is primarily associated with reductive biosynthesis (Stryer, 1988). Therefore, in theabsence of reductant utilizing reactions such as NO3 - assimilation, it would be surprising if anincrease in the NADH/NAD ratio did not result as a consequence of increased respiratorycarbon flow. It is equally possible, then, that increases in the NADH/NAD ratio may interactwith NAD(P)H-PQ oxidoreductase to reduce the PQ(cyt b6f) pool and activate the kinaseresponsible for a state 1 to 2 transition.Although an interaction between NADPH and the PQ(cyt b6f) pool is consistent withthe previous work (Mohanty et al., 1990; Bulte et al., 1990; Gans and Rebeille, 1990), nostudies have comprehensively tested the implications of the hypothesis that an increase in theNADPH/NADP but not the NADH/NAD ratio is an absolute requirement for a reduction of thePQ pool and the occurrence of a state 1 to 2 transition. The focus of this chapter will be to77determine whether regulation of the PETC poising for the NADPH/ATP production ratio isaccomplished by increases in the NADPH/NADP and/or NADH/NAD ratios. It is hypothesizedthat class 1 treatments, which increase respiratory carbon flow, PQ reduction, and cause state 1to 2 transitions, will correlate with increases in reduced/oxidized pyridine nucleotide ratios. Onthe other hand, class 2 treatments (which increase respiratory carbon flow but have lesser affectson PQ reduction and do not appear to result in a state 1 to 2 transition) will not increase theratio of reduced/oxidized pyridine nucleotides.Techniques to measure pyridine nucleotides:To test this model, it was necessary to accurately measure changes in both theNADH/NAD and NADPH/NADP ratios, which in turn required measurement of NAD, NADH,NADP and NADPH. Gans and coworkers measured only NAD and NADP and calculated thepools of NADH and NADPH by subtracting the oxidized pool from an estimate of the totalpyridine nucleotide pool (Bulte et al., 1990; Gans and Rebeille, 1990; Rebeille and Gans, 1988).Although this procedure can accurately measure the quantities of oxidized pyridine nucleotides,resolution of changes in reduced pools, which contribute only a small amount to the overall poolsize, can be difficult. In particular, the contribution of the NADH pool to the total NAD(H)pool in most green algae and higher plants is less than 10% (Vanlerberghe et al., 1992; Huppe etal., 1992; Bulte et al., 1990; Takahama et al., 1980; Bonzon et al., 1983; Matsumura-Kadota etal., 1982; Muto and Miyachi, 1981). Resolution of a 50% change in the NADH pool (whichwould constitute only a 5.6% change in the NAD pool) would be limited by experimental error.To accurately measure changes in the reduced pyridine nucleotide pools in the present work,simultaneous measurements of oxidized and reduced pyridine nucleotides were undertakenusing rapid, parallel sampling in which one sample was quenched in acid (destroys reducedpyridine nucleotides) and the other in base (destroys oxidized pyridine nucleotides). Highlyspecific enzymatic cycling assays allowed resolution of changes in NADP, NADPH, NAD andNADH (Passonneau and Lowry, 1974).78Measurements of all pyridine nucleotides using this method have been made in S.minutum after treatment with NH4+, NO3 - (Vanlerberghe et al., 1992) and Pi (Gauthier andTurpin, 1993). The purpose of this chapter was to measure NAD, NADP, NADH and NADPHafter treatment with anaerobiosis and CCCP to provide a complete pyridine nucleotide data setfor analysis. The complete data set was then used to test the positive and negative implicationsof the hypothesis that increases in respiratory carbon flow, which increase the NADH/NAD orNADPH/NADP ratios, will result in an increase in PQ reduction and a state 1 to 2 transition.The advantages of the present study are twofold. First, the simultaneous measurementof all four forms of pyridine nucleotide (NAD, NADH, NADP, NADPH) allowed accurateresolution of changes in both NADH/NAD and NADPH/NADP ratios. Second, unlike previouswork (Bulte et al., 1990; Rebeille and Gans, 1988; Gans and Rebeille, 1990; Mohanty et al.,1990) which has used only a small (n 5_ 2) data set to correlate increases in the NADPH/NADPratio with an increase in PQ reduction, the combination of data sets from all five treatments inthe present study allowed a more rigorous examination of the model for interaction betweenrespiratory carbon flow and state transitions.MATERIALS AND METHODSExperimental:Experimental conditions were the same as those described in chapter 1. Cells wereconcentrated to 20 lag Chl mL -1 and dark-adapted for 20 minutes before the initiation ofsampling.Pyridine nucleotide determinations:Sampling:^Pyridine nucleotides were determined essentially as described previously(Vanlerberghe et al., 1992). Duplicate cell samples (200 gL) were killed simultaneously by79injection into 900 IaL of acid (Ch1C13:MeOH:1N HC1, 7.5:17.5:1, v/v) or alkali(Ch1C13:MeOH:1N KOH, 7.5:17.5:1, v/v). All samples were kept on ice until the experimentwas complete. Basic samples were then heated for 5 minutes at 60 °C to ensure completedecomposition of oxidized pyridine nucleotides. Acid treated samples retain oxidized pyridinenucleotides with decomposition of reduced forms. All samples were subsequently neutralizedwith KOH or HCI (for acid- and base-treated samples, respectively) and mixed with 400NaF (12.5 mM). After centrifugation, the aqueous layer was removed and concentrated to lessthan 500 p.L in a Speed Vac centrifuge to ensure removal of Me0H from the samples. Samplevolume was then adjusted to 1 mL with distilled H2O.Enzymatic cycling: Enzymatic cycling of pyridine nucleotides was performed as describedpreviously (Vanlerberghe et al., 1992; Passonneau and Lowry, 1974). In cycling, thenucleotides act as catalysts for an enzymatic dismutation between two substrates. Thenucleotide concentrations are far below the KM values of the two enzymes and the reactionrates (and, consequently, the final products) are proportional to the initial nucleotideconcentrations. One of the products is determined after several thousand cycles. Thistechnique allows extremely small physiological concentrations of pyridine nucleotides (10 -14mol) to be "amplified" via cycling into a measurable quantity of an end-product. By usingenzymes which are extremely specific for either the phosphorylated or non-phosphorylatedforms of pyridine nucleotides, it is possible to accurately measure NADH, NAD, NADP andNADPH.Pyridine nucleotide samples were stored in liquid N2 for less than 24 hours beforecycling. Cycling assays (fmal volume = 120 gL) consisted of 1001.L assay reagent and up to 20of sample. The sample volumes used for each nucleotide were 2.5 (NAD), 10 - 20(NADH), 7.5 (NADP) and 5 pL (NADPH). Phosphorylated pyridine nucleotides were cycledusing an assay mix (0.2M Tris, pH 8.4;10 mM lactate; 50 mM ammonium acetate; 5 mM 2-OG(BMC 127850) and 0.3 mM ADP) containing glutamate dehydrogenase (BMC 127078, 90 U)80and G6P dehydrogenase (BMC 127663, 70 U). Non-phosphorylated pyridine nucleotides werecycled in an assay mix (0.1M Tris, pH 8.0;1 mM G6P; 5 mM 2-OG (BMC 127850); 30 mMammonium acetate; and 0.1 mM ADP) containing lactate dehydrogenase (Sigma L7755, 120 U)and glutamate dehydrogenase (BMC 127078, 180 U). All enzymes were treated with activatedcharcoal prior to cycling. Samples were cycled simultaneously with standards for one hour at37 °C. Standards were 0 to 1 pmol NADP(H) and 0 to 1.5 pmol NAD(H) of authentic pyridinenucleotides (BMC 775754, 107735, 128031, 107824). Standard curves always had an r 2 of0.98. The product of the NADP(H) assay was 6-phosphogluconate while the product of theNAD(H) assay was pyruvate. Cycling products were stored in liquid N2 until measurement.Products of the cycling assays were measured using standard coupled enzymatic assaysand a dual wavelength spectrophotometer (ZFP22, Sigma, Berlin, FRG) as described previously(Quick et al., 1989; Wirtz et al., 1980, see also Appendix 3). All metabolite measurements werenormalized to chlorophyll.Fluorescence:Room temperature fluorescence was measured as described in Chapter 1.RESULTSFor clarity and completeness, the results presented here will summarize the effects of all5 treatments on both phosphorylated and non-phosphorylated pyridine nucleotides. These datainclude original research by this author and other published work from Dr. Turpin's lab. Pleaserefer to figure legends for the original source of data. Results presented are the average of aminimum of 3 separate experiments; variation around each point was less than 5%.81NADP, NADPH, and the NADPH/NADP ratio:1) NH4+:NADPH levels changed less than 5% for the first 1.5 minutes and less than 12% for thefollowing 3.5 minutes after treatment with NH4+ (Figure 9A). After 10 minutes, NADPHlevels decreased by 25%. In contrast, NADP decreased by 12% within 5 seconds and by 20%within 30 seconds of treatment with NH4+. NADP decreased to 36% of the control levelwithin 2 minutes (Figure 9A). The NADPH/NADP ratio increased by 20% within 30 secondsand reached a peak of 1.6-fold the dark control ratio within 2 minutes (Figure 9D). TheNADPH/NADP ratio then declined to a plateau level of approximately 40% greater than thedark control within 5 minutes after treatment.2) Anaerobiosis:Anaerobiosis doubled NADP within 5 seconds and reduced NADPH by 23% (Figure9B). NADP oscillated to one half the control level within 1 minute, increased to 30% above thecontrol after 2 minutes, and then returned to a level 5 to 10% below control levels within 3minutes. NADPH mirrored this oscillation with a 20% decrease after 1 minute, a 10% decreaseafter 2 minutes and a return to a level 10% greater than the control (Figure 9B). TheNADPH/NADP ratio oscillated to a level less than one half the control within 5 seconds,increased to a level 70% greater than the control within 1 minute, dropped to 30% of thecontrol level within 2 minutes, and then continued to oscillate at a level slightly higher than thecontrol for the remainder of the experiment (Figure 9E).Re-supply of oxygen to anaerobic cells: If oxygen was re-supplied to dark anaerobic cells,NADPH increased by 12% within 30 seconds and 20% within 2 minutes of re-supply. NADPdecreased approximately 5% within 1 minute of re-supply (data not shown). TheNADPH/NADP ratio increased by approximately 15% within 30 seconds of re-supply and 25%within 3 minutes of re-supply before returning to control levels within 10 minutes of re-supply(Figure 9E, inset).if+ anaerobiosis820—20-10 0 10 20 30 40 50-20-10 0 10 20 30 40 50Time (min)35o- 30rnE 25▪ 201510a_5035.••—■-C30rnE 2520151 0CL.5020On 150105a_z4.5a_z3.5a_2.54.53.5 <2.5 0z1.53.52.5 °¢za_1.5 0z0.5Figure 9: The effect of class 1 treatments on NADP, NADPH and the NADPH/NADP ratio in S. minutum.Figures A-C: NADPH, closed circles; NADP, open circles. Figures D-F, NADPH/NADP ratio. N-limited cells(30 Chl m1: 1 ) were dark adapted for 20 minutes before beginning sampling. Cells were treated at time 0 with:A,D: NH4+; B,E: anaerobiosis; C,F: CCCP. Figure 5E inset, the effect of 02 re-addition after 20 minutes ofanaerobic adaptation. NH4+ data reproduced with permission from Vanlerberghe et al. (1992).833) CCCP:The data in Figure 9C illustrate the identical trends of 9 such experiments. Within 5seconds of treatment with CCCP, NADP increased by 50%, NADPH decreased by 15% and theratio of NADPH/NADP dropped by almost half. NADP reached a maximum some 2.5-foldgreater than the dark control level within 1 minute of treatment while NADPH decreasedgradually to approximately 30% of dark control levels within 5 minutes (Figure 9C). Within 30seconds, the NADPH/NADP ratio dropped to 20% of its original level and remained lower than15% of the original level for the remainder of the experiment (Figure 9F).4) NO3- :Treatment with NO3 - resulted in a 240% increase in NADP, a 30% decrease in NADPHand 70% decrease in the NADPH/NADP ratio within 5 seconds (Figure 10A, C). Within 30seconds, this ratio decreased to a level 2.1-fold less than the dark control and remained at thislevel for the first 20 minutes. NADPH retained the 30% decrease for approximately 2 minutesbefore recovering to a level slightly higher than the dark control level within 20 minutes.5) Pi:Within 20 seconds of treatment with Pi, NADPH peaked at a level 60% greater than thedark control level whereupon it decreased back to a level slightly below the dark control within5 minutes (Figure 10B). NADP dropped immediately upon treatment with Pi and reached alevel approximately half that of the control within 5 minutes. Within 5 seconds of treatment, theNADPH/NADP ratio increased by 1.5-fold and reached a plateau at approximately twice thedark control level after 2.5 minutes (Figure 10D).NAD, NADH, and the NADH/NAD ratio:1) NH4+:Within 5 seconds of treatment with NH4+, NADH increased 2-fold, NAD decreased by9% and the NADH/NAD ratio increased by 2.3-fold (Figure 11A). NADH increased to a level54a_a3 1I2 °-z10214540353025201510518161412108642—20 —10840 10 20 30 40-20 —10 0 10 20 30 40Time (m in)Figure 10: The effect of class 2 treatments on NADP, NADPH and the NADPH/NADP ratio inS. minutum. Figures A and B: NADPH, closed circles; NADP, open circles. Figures C and D,NADPH/NADP ratio. Cells (30 pg Chl mL -1 ) were dark adapted for 20 minutes beforebeginning sampling. Cells were treated at time 0 with: A,C: NO3 - ; B,D: Pi. NO3 - and Pitreatments were made to N- and Pi-limited cells, respectively. Data represents the mean of 3experiments. Figures reproduced with permission from Vanlerberghe et al. (1992) and Gauthierand Turpin (1993).+ NH4+NADHA .70_c0 60rn 50E400E 3020100L-I-10-Cc:n155E1050Er-1550 5z—451250cn 105E 850 6545250z5—150.2 0z0.1 z0zo . 10z0zo . 10z0.0850.30.00.20.00.2—20 — 1 0 0 10 20 30 40 50-20 —10 0 10 20 30 40 50Time (min)Figure 11: The effect of class 1 treatments on NAD, NADH and the NADH/NAD ratio in S. minutum.Figures A-C: NADH, closed circles; NAD, open circles. Figures D-F, NADH/NAD ratio. N-limited cells(30 g.tg Chl niL- 1 ) were dark adapted for 20 minutes before beginning sampling. Cells were treated at time0 with: A,D: NH4+; B,E: anaerobiosis; C,F: CCCP. Figure 7E inset, the effect of 02 re-addition after20 minutes of anaerobic treatment. NH4+ data reproduced with permission from Vanlerberghe et al.(1992).864 times that of the control within 1 minute and remained at a level 3-fold greater than the darkcontrol for the remainder of the experiment. The NADH/NAD ratio reached a maximum of 4.8-fold greater than the dark control level after 1 minute. The ratio gradually decreased to a level3-fold greater than the dark control within 15 minutes (Figure 11D).2) Anaerobiosis:NAD and NADH levels were relatively unaffected for the first 20 seconds after initiationof anaerobiosis (Figure 11B). After 2 minutes, NADH increased 2.5-fold and NAD was 10%smaller than the dark control. After 5 minutes, NADH decreased and remained at a level 2-foldgreater than the control for the remainder of the experiment. Anaerobiosis had no effect uponthe NADH/NAD ratio until 30 seconds after treatment whereupon the ratio increased by 25%.After 2 minutes, this ratio reached a maximum which was 2.8-fold greater than the dark controland then returned to a plateau level of approximately 2.4-fold greater than the control (Figure11E).Re-supply of anaerobic cells with oxygen: Re-supplying steady-state anaerobic cells with 02decreased NADH by 60% within 5 seconds of re-supply. NADH recovered to control levelsafter 3 minutes. NAD increased by approximately 5% within 30 seconds of 02 re-supply (datanot shown). The NADH/NAD ratio was halved within 5 seconds of 02 re-supply and returnedto control levels within 3 minutes of oxygen re-supply (Figure 11E, inset).3) CCCP:Within 5 seconds of uncoupling with CCCP, NAD decreased 12% , NADH increased2.4-fold and the NADH/NAD ratio increased 2.7-fold (Figure 11C, F). NAD remained 10 to15% lower than the dark control for the remainder of the experiment while NADH increased to3.5-fold the control levels within 1 minute. The NADH/NAD ratio peaked at a levelapproximately 4-fold greater than the dark control ratio within 1 minute of treatment and87remained more than 3-fold greater than the control for at least 8 minutes after treatment (Figure11 F).4) NO3- :NAD and NADH and the NADH/NAD ratio were relatively unchanged for the firstminute after treatment with NO3 - (Figure 12A). NADH increased by 60% within 2 minutes andcontinued to increase to approximately 1.8-fold greater than dark control levels within 15minutes of treatment. NAD decreased by approximately 10% after 1.5 minutes (Figure 12A).The NADH/NAD ratio increased to 1.7-fold control levels within 1.5 minutes and remainedabove this level for the remainder of the experiment (Figure 12C).5) Pi:Treatment with Pi resulted in a 20% decrease in NAD within 5 seconds after which timeNAD recovered to a level 20% greater than the control within 5 minutes (Figure 12B). NADHincreased 1.5-fold after 20 seconds, dropped to control levels within 1.5 minutes, and thengradually increased to a level 30% greater than the control within 5 minutes of treatment. TheNADH/NAD ratio oscillated with a 40% increase within 20 seconds, a 10% decrease after 1.5minutes and then reached a plateau at a level 5 to 10% greater than the dark control within 2.5minutes (Figure 12D).Fluorescence:Re-supply of 02 to darkened, steady-state anaerobic cells resulted in a rapid decrease inF, the level of fluorescence induced by the low intensity measuring beam. Within 5 seconds, Fdecreased by 40% and within 1 minute F recovered to a level close to dark aerobic controllevels (Figure 13). The fluorescence induced by a saturating flash (F M) required approximately5 to 10 minutes to recover (Figure 13).NADNADH4\+ NO 3A.^0NAD0 . 31600.2 o00.1 z_c0rn 120E0E 80c400z0.000.2_c, 30rnE-3 20+ Pi0z0.1zNADH^•B.0.00^10 20 30 40—20 —10 0 10 20 30 40-20 —1088Time (min)Figure 12: The effect of class 2 treatments on NAD, NADH and the NADH/NAD ratio in S.minutum. Figures A and B: NADH, closed circles; NAD, open circles. Figures C and D,NADH/NAD ratio. Cells (30 lag Chl mL -1 ) were dark adapted for 20 minutes before beginningsampling. Cells were treated at time 0 with: A,C: NO3 - ; B,D: Pi. NO3 - and Pi treatments weremade to N and Pi-limited cells, respectively. Data represents the mean of 3 experiments. Figuresreproduced with permission from Vanlerberghe et al. (1992) and Gauthier and Turpin (1993).Fm -Fo —...891,--43 minFigure 13: The effect of 02 re-addition on room temperature fluorescence from cells (3 lag ChlmL-1 ) that were anaerobically adapted for 20 minutes. Up arrow indicates removal of 02 andonset of anaerobiosis, down arrow indicates re-addition of 02. F o , minimal control level offluorescence; FM maximal control level of fluorescence induced by a saturating flash; F,fluorescence induced by the fluorescence measuring beam after treatment; F M ', maximal level offluorescence induced by a saturating flash after treatment.90DISCUSSIONVery little is known about the kinetic properties of the NAD(P)H-PQ oxidoreductase. Itis not clear if this enzyme is a rate-limiting enzyme subject to allosteric regulation, or if it is anequilibrium enzyme. All of the following analysis assumes that the NAD(P)H-PQoxidoreductase is an equilibrium enzyme and is regulated by changes in the product/substrateratios.NADP, NADPH, and the NADPH/NADP ratio:Class 1 Treatments (NH4+, anaerobiosis, CCCP):Treatment of N-limited S. minutum with NH4+, anaerobiosis, and CCCP resulted in anincrease in respiratory carbon flow which was correlated with PQ reduction and a subsequentstate 1 to 2 transition (Chapters 2 and 3). Although these treatments consistently increased PQreduction, they had widely varying effects upon the NADPH/NADP ratio (Figure 9 D-F).Treatment with NH4+ decreased NADP and NADPH to 1/2 and 9/10 of their original levels,respectively (Figure 9A). This resulted in an increase in the NADPH/NADP ratio by 1.6-foldwithin 2 minutes (Figure 9D). In the case of anaerobiosis, large oscillations in NADP andNADPH were transitory in nature only lasting approximately 2 minutes (Figure 9B). TheNADPH/NADP ratio dropped rapidly, increased to a level greater than the control and thenreturned to equilibrium within 2 minutes. (Figure 9E). After uncoupling with CCCP, NADPincreased 2.6-fold while NADPH dropped to 2/5 of its original level (Figure 9C). This resultedin an 80% decrease in the NADPH/NADP ratio within 30 seconds and a 10-fold decrease within5 minutes (Figure 9C). On the other hand, uncoupling nutrient-sufficient Chlarnydomonasreinhardtii with CCCP was reported to cause a 45% increase in the contribution of NADPH tothe total NADP(H) pool corresponding to an increase in the NADPH/NADP ratio (Bulte et al.,1990; Gans and Rebeille, 1990; Rebeille and Gans, 1988). The reasons for the differencebetween NADPH/NADP ratios observed in the present study and previous work are unclear. It91is possible, though, that the nutrient status of the alga may affect the path of carbon flow afteruncoupling to NAD- or NADP-utilizing enzymes and hence result in different effects on theNADPH/NADP ratio. It is also possible that methodology may account for these differences.The advantage of the present work is that it measures NADPH and NADP separately todetermine the NADPH/NADP ratio while the measurements of Gans and coworkers (Rebeilleand Gans, 1990; Bulte et al., 1990; Gans and Rebeille, 1988) were based on measurements ofoxidized pyridine nucleotides and suffer the limitations previously discussed.Class 2 Treatments (NO3 - and Pi):Treatment of S. minutum with NO3 - or Pi also resulted in an increase in respiratorycarbon flow but, in contrast to the three treatments outlined above, these two treatmentsresulted in relatively minor changes in PQ reduction and did not appear to cause a state 1 to 2transition (Chapters 2 and 3). However, these two treatments did not have consistent effects onthe NADPH/NADP ratio (Figure 10C, D). Treatment with NO3 - caused a doubling of NADPand a small transient (< 2 min.) drop in NADPH (Figure 10A). The overall effect upon theNADPH/NADP ratio was a 2-fold decrease within 2 minutes of treatment (Figure 10C). On theother hand, treatment with Pi halved NADP, doubled NADPH (Figure 10B), and resulted in a2-fold increase in the NADPH/NADP ratio within 2 minutes of treatment (Figures 10D).Increases in the NADPH/NADP ratio do not appear to be correlated with PQreduction:From the previous discussion it appears that class 1 and class 2 had no consistent effectupon the NADPH/NADP ratio. Pi treatment did not result in a state 1 to 2 transition butincreased the NADPH/NADP ratio while CCCP treatment resulted in a state 1 to 2 transitionbut decreased the NADPH/NADP ratio. These observations suggest that NADPH may not beresponsible for reducing PQ and the resulting state 1 to 2 transitions.92NAD, NADH, and the NADH/NAD ratio:Class 1 Treatments (NH4,+ anaerobiosis, CCCP):Treatments with NH4+, anaerobiosis, and CCCP all resulted in relatively rapid decreasesin NAD, increases in NADH, and an overall increase in the NADH/NAD ratio of greater than2.8-fold within 2 minutes of treatment (Figure 11). Treatment with NH4+ resulted in a 2.3-foldincrease in the NADH/NAD ratio within 5 seconds of treatment and a level 4.7-fold higher thanthe dark control within 1 minute (figure 11D). The effects of anaerobiosis were slightly delayedand no effect on the NADH/NAD ratio was observed until 30 seconds after treatmentwhereupon the level increased to 2.8-fold the dark control level within 2 minutes (Figure 11E).Within 5 seconds of CCCP treatment, NADH increased 2.4-fold, NAD decreased 9%, and theNADH/NAD ratio reached a level 2.7-fold larger than the dark control level (Figure 11C, F).Within 2 minutes, the NADH/NAD ratio peaked at 4-fold the dark control level (Figure 11F).The observation that large increases in the NADH/NAD ratio resulted after all class 1treatments (which were correlated with increased PQ reduction and a state 1 to 2 transition)implies that NADH may be responsible for PQ reduction via the NAD(P)H-PQ oxidoreductase.Re-supply of 02 to anaerobic cells: One implication of the proposal that increases in theNADH/NAD ratio result in reduction of PQ and a state 1 to 2 transition is that re-oxidation ofPQ should be correlated with a decrease in the NADH/NAD ratio. Re-supply of 02 to steady-state anaerobic cells would allow oxidation of PQ by removing substrate limitation for theterminal oxidase for the CRETC. Indeed, within 5 seconds of 02 re-supply, the level offluorescence induced by the measuring beam (F) decreased by 40% and completely recoveredwithin 1 minute (Figure 13). F has been shown to be a good estimate of the oxidation state ofQA and hence the PQ pool (Chapter 2). The observed decrease in F, therefore, is consistentwith an oxidation of QA(PQ) due to the removal of substrate limitation. The differencebetween FM ' and FM indicates the level of non-photochemical quenching due, in part, to a state 1to 2 transition (Chapter 2). Only after F had recovered (implying PQ oxidation) did FM'93increase, indicating that the LHC2 kinase had been deactivated and a state 2 to state 1 transitionhad occurred (Figure 13). The rapid oxidation of PQ was correlated with a slow increase in theNADPH/NADP ratio (Figure 9E, inset) and a rapid 60% oxidation of the NADH/NAD ratio(Figure 11E, inset). If NADPH were responsible for reducing PQ, one would not expect theobserved accumulation of reductant as the PQ pool was oxidized. Instead, oxidation of PQwould be expected to result in increased flow of accumulated pools of reductant to PQ. Theobserved decrease in the NADH/NAD ratio is consistent with this hypothesis although it ishighly likely that the METC, which would also be 02-limited, would contribute significantly tothe 60% decrease in the NADH/NAD ratio. An increase in the NADPH/NADP ratio after 02re-supply to anaerobic cells is inconsistent with NADPH being responsible for the reduction ofPQ and provides more evidence to suggest that NADH is responsible for PQ reduction and thestate 1 to 2 transition observed during class 1 treatments.Changes in the NADH/NAD ratio after CCCP treatment:The increase in the NADH/NAD ratio observed in S. minutum after treatment withCCCP contrasts with data from nutrient-sufficient Chlamydomonas reinhardtii where nochange in the NADH/NAD ratio was observed upon uncoupling with CCCP (Gans and Rebeille,1988; Rebeille and Gans, 1990; Bulte et al., 1990). The discrepancy between the results fromwork with S. minutum and C. reinhardtii likely stems from the inaccuracy inherent in themethod used to calculate NADH levels in work with C. reinhardtii (Bulte et al., 1990; Gans andRebeille, 1988; Rebeille and Gans, 1990). This method involves calculation of NADH as thedifference between the total NAD(H) pool and measured levels of NAD. Since NADHcontributes less than 10% to the total pool, the 240% increase in NADH, seen in S. minutumafter treatment with CCCP (Figure 11C), would only cause a 12 to 15% decrease in the NADpool. Experimental error in the measurement of the NAD pool could easily obscure changes ofthis magnitude and result in no observed change in the contribution of NADH to the totalNAD(H) pool in C. reinhardtii (Bulte et al., 1990; Rebeille and Gans, 1990; Gans and Rebeille,941988). Direct measurement of both NADH and NAD, however, has allowed an accurateresolution of large changes in the NADH/NAD ratio after CCCP treatment of S. minutum(Figure 9C)Overall, the observation that class 1 treatments resulted in an increase in theNADH/NAD ratio but had no consistent effect on the NADPH/NADP ratio implies that NADHmay be responsible for reduction of the PQ pool and a state 1 to state 2 transition.Class 2 Treatments (Pi and NO3 -):Treatment with Pi resulted in rapidly oscillating changes in the NAD(H) pool for the first2 minutes after treatment with Pi (Figure 12 B, D). After 2.5 minutes of Pi treatment, however,the NADH/NAD ratio reached a plateau at a level which was only 10% higher than the controland remained at this level for the first 20 minutes of treatment (Figure 12D). Pi treatment wasnot observed to result in a large reduction of the PQ pool or cause a state 1 to 2 transition. Theobservation that the NADH/NAD ratio was relatively unaffected while the NADPH/NADP ratioincreased after Pi treatment is supports the contention that NADH is responsible for thereduction of the PQ pool and a state 1 to state 2 transition.NO3 - treatment resulted in changes in the NAD(H) pool which were slower and ofsmaller magnitude than changes induced by class 1 treatment. NADH, NAD and theNADH/NAD ratio were relatively unchanged for the first minute after treatment with NO3 - .Subsequently, NAD decreased, NADH increased, and the NADH/NAD ratio peaked at a levelapproximately 1.7-fold the dark control level (Figure 12A, C). The increased NADH/NADratio, observed during NO3 - treatment, is not completely consistent with the hypothesis thatNADH is responsible for PQ reduction and a state 1 to 2 transition. Such inconsistencyhighlights the complexity of the case of NO3 - assimilation. NO3 - resulted in only minorchanges in qNp, the F686/F717 77K fluorescence ratio and fluorescence decay kinetics whichsuggested that PQ reduction was only marginally effected and that a state 1 to 2 transition hadnot occurred (Chapter 2 and 3). The situation was complicated, however, because induction95kinetics were faster during NO3 - assimilation, suggesting that PQ reduction might haveoccurred. The increases in the NADH/NAD ratio observed (1.7-fold) were intermediatebetween those observed for class 1 treatments ( 2.8 - 4.7-fold) and the other class 2 treatment,N addition (1.1-fold). There are several plausible explanations for the inconsistencies notedbetween increases in the NADH/NAD ratio during NO3 - assimilation and the proposedinteraction between increased NADH/NAD ratios, PQ reduction and a state 1 to 2 transition.It is possible that NO3 - may represent an intermediate between the class 1 and class 2definition. This, however, does not explain why only small changes in 77K fluorescence wereobserved suggesting that a state 1 to 2 transition had not occurred. Alternatively, it is possiblethat a threshold increase in the NADH/NAD ratio is required to increase the electron flow ratefrom NADH to PQ such that it becomes limited by terminal oxidase activity and results in PQreduction. It has been suggested that the terminal oxidase is the rate-limiting step in thecyanobacterial respiratory electron transport chain, which shares PQ with the PETC (Myers,1986; Peltier and Schmidt, 1991). If the amount of NADH present in dark control cells werewell below the KM (NADH) of the CRETC, electron flow would not be limited and PQ wouldremain mostly oxidized. Increases in NADH, though, could increase the number of electronsflowing to the terminal oxidase and result in a buildup of reduced PQ once the terminal oxidasebecame saturated with electrons from PQ. The amount of NADH required to saturate theterminal oxidase and result in significant reduction of PQ might be greater than the increasenoted after treatment with NO3 - but less than that observed after class 1 treatments. Finally, itis possible that increases in the chloroplastic NADH/NAD ratio after treatment with NO3 - arecompartmentalized. In such a scenario, the observed increases in the NADH/NAD ratio wouldresult from mitochondrial activity and would not reflect increases in the chloroplast. Sinceincreases in chloroplastic NADH/NAD ratio would be required to increase PQ reduction, a lackof change in chloroplastic NADH/NAD ratios during NO3 - treatment would be consistent withthe observations that a state 1 to 2 transition had not occurred. These issues will be dealt within more depth in Chapter 6.96SUMMARYThe present work has the advantage of employing a large number of different typestreatments (n=5) and a consistently measured pyridine nucleotide data set from the sameorganism to test a proposed model for interaction between respiratory carbon flow and statetransitions. Using treatments which rigorously tested the implications of this model, it appearedthat increases in the NADPH/NADP ratio could not be correlated with increases in PQ. Thus,during class 1 treatments, the NADPH/NADP ratio was observed to increase (NH4+), oscillate(anaerobiosis) and decrease (CCCP). Similarly, during class 2 treatments, the NADPH/NADPratio either decreased (NO3-) or increased (Pi). This led to the contention that NADPH wasnot responsible for PQ reduction and the occurrence of a state 1 to 2 transition. On the otherhand, accurate measurement of both NADH and NAD allowed resolution of increases in theNADH/NAD ratio which could, for the most part, be correlated with PQ reduction. Class 1treatments resulted in large increases in the NADH/NAD ratio of 4.7-fold (NH4+), 2.8-fold(anaerobiosis) and 3.8-fold (NH4+). In addition, Pi treatment (class 2) caused a rapidoscillation followed by almost no change in the NADH/NAD ratio after 2.5 minutes. Sinceclass 1 treatments resulted in PQ reduction and a state 1 to 2 transition and class 2 treatmentsdid not, these results were consistent with increases in NADH being responsible for PQreduction and a state 1 to 2 transition. Intermediate increases in the NADH/NAD ratio duringNO3 - assimilation were somewhat inconsistent with this model and highlighted the complexityof the factors involved during NO3 - assimilation. Several plausible explanations were proposedto explain these inconsistencies and these will be dealt with in a later chapter.The observation that increases in NADH/NAD ratios were largely correlated with anincrease in PQ reduction suggests that the physiological significance of the CRETC may betwofold. First, it would appear that the CRETC functions to recycle reductant for respiratorycarbon flow in the chloroplast much as the METC in the mitochondria recycles reductant fromTCA cycle activity. Second, the rate of e - flow through the CRETC can affect the redox stateof the PQ(cyt b6f) pool. This, in turn, appears to result in a state 1 to 2 transition and poises97the PETC for a decrease in the NADPHIATP production ratio. It would appear, then, that theCRETC may also function to allow changes in respiratory carbon flow status to communicatewith the PETC and down-regulate the NADPH/ATP production ratio.98CHAPTER 5: INTERACTION BETWEEN RESPIRATORY CARBON FLOW ANDTHE STATE TRANSITION AFTER UNCOUPLING WITH CCCPINTRODUCTIONChapters 2 through 4 involved developing and testing a general model which correlatedan increase in the reduction state of the pyridine nucleotide pool with the occurrence of a state 1to 2 transition. Treatments which increased the rate of respiratory carbon flow resulted inchanges in fluorescence characteristics which were classified into two groups. Class 1treatments (NH4+, anaerobiosis or CCCP) resulted in large changes in steady-state fluorescenceand PS2/PS 1 fluorescence ratios indicative of a state 1 to 2 transition and these changes werecorrelated with reduction of the PQ pool. Class 2 treatments (NO3 - or Pi) resulted in minimalchanges in fluorescence indicating that a state 1 to 2 transition had not occurred. Changes inPQ reduction, resulting from Class 2 treatments, were much smaller than those observed afterclass 1 treatment. Increases in the NADPH/NADP ratio were not correlated with a reduction ofthe PQ(cyt b6f) pool and a state 1 to 2 transition suggesting that increases in the NADH/NADratio might be the factor responsible for reduction of the PQ(cyt b6f) pool. In support of thiscontention, class 1 treatments resulted in increases in the NADH/NAD ratio of 2.8-fold orgreater while Pi treatment (class 2) resulted in only a minimal (10%) increase after 2 minutes oftreatment. Some inconsistency was introduced by the observation that NO3 - assimilationresulted in an increase in the NADH/NAD ratio which was intermediate in magnitude betweenthat of class 1 treatments and Pi treatment. Several plausible explanations were suggested forthis inconsistency and these will be dealt with in more detail in the subsequent chapter.In order to fully understand how interactions occur between respiratory carbon flow andthe state transition, it is necessary to understand how respiration is activated. Thisunderstanding can provide an insight into the specific mechanism responsible for the increases incellular NADH/NAD ratios observed during class 1 treatments and may help explain the smallerincreases observed after class 2 treatments. In green algae it appears that much of glycolytic99carbon flow occurs in the chloroplast. Indeed, it has been proposed that starch is metabolizedto the level of 3-PGA in the chloroplast before being exported to the cytosol. In support of thiscontention, the NAD- and NADP-utilizing forms of GAPDH were observed to be localized 86and 100%, respectively, in isolated chioroplasts of Chlamydomonas reinhardtii (Klein, 1986).Although it is possible that other minor pathways may produce NADH in the chloroplast, Ihypothesize that chloroplastic NAD-GAPDH is the major source of the NADH that results inPQ reduction.The regulation of respiratory carbon flow in S. minutum has been extensively studiedand detailed metabolite data exist for the kinetics of changes in respiratory metabolites andcofactors after treatment with NH4+ (Turpin et al., 1990; Vanlerberghe et al., 1992),anaerobiosis (Vanlerberghe et al., 1989), Pi (Gauthier and Turpin, 1993), and NO3 - (Fell andTurpin, unpublished; Vanlerberghe et al., 1992). However, changes in key respiratorymetabolites and cofactors have not been resolved after treatment with CCCP in S. minutum.The purpose of the present chapter was to examine the metabolic sequence of events leadingfrom activation of respiratory carbon flow to the reduction of PQ and initiation of a state 1 to 2transition in the specific case of CCCP uncoupling. This information was then used todetermine the enzymes potentially responsible for NADH production and PQ reduction in thechloroplast. In the subsequent chapter, this information will be compiled with available datafrom the other 4 treatments to develop a more complete model for the regulatory sequenceresponsible for activation of a state transition by respiratory carbon flow.MATERIALS AND METHODSExperimental:Refer to Chapter 1. The algal suspension was aerobically dark adapted for 30 minutesbefore the initiation of sampling.100Starch degradation:Aliquots for determination of starch degradation were taken over a 3 hour period asdescribed in Vanlerberghe et al. (1990). Long sampling periods were required because the largeinitial pool size of starch in these cells made it impossible to accurately measure changes overshort time periods. Cells were used directly from the chemostat (1.2 1.tg mg -1 Chl). Samples(100 lit) were removed at 20 minute intervals, frozen in liquid N2, and lyophilized. Freecontaminating glucose was fully oxidized and all starch solubulized by resuspending the freezedried sample in 400 pL of 0.02N NaOH, autoclaving for 100 minutes (121°C, 14 psi),vortexing, and autoclaving for an additional 100 minutes. Solubulized starch was then degradedto glucose by treating samples with a-amylase (20 units, Sigma A-1278) and amylogucosidase(2 units, Sigma A-3042) for 15 hours at 55°C in 200 mM Na-acetate (pH 5.0).Amylogucosidase and a-amylase were dialyzed in 200 mM Na-acetate (pH 5.0) for 24 hoursprior to use. Glucose was determined using standard metabolite analysis (see below).Metabolites:Cells were harvested and concentrated to 40 lag Chl mL -1 . Samples (1 mL) were killedin 10% HCLO4 (fmal v/v) and rapidly frozen in liquid N2. Samples were then slowly thawed onice, neutralized with 5M KOH/1M triethanolamine, and centrifuged. The supernatant wasbrought up to 1.5 mL with dH2O. All neutralized samples were stored in liquid N2 untilanalysis. All metabolites were measured using standard, coupled enzymatic assays (Quick et al.,1989; Wirtz et al., 1980; see also Appendix 3) and a dual wavelength spectrophotometer(ZFP22, Sigma Instruments, FRG). All assay reagents were obtained from BoehringerMannheim Co.Gas exchange:CO2 release:The rate of CO2 release from cells (5 1.tg Chl mL -1 ) was measured in an open gasexchange system with an infra red gas analyzer (IRGA, ADC 225 MK3, Analytical101Development Co. Ltd., Hoddesdon, England). CO2-free air was bubbled at a constant rate of200 mL•min -1 through cells contained in a sealed cuvette. The air from the cuvette wasreturned to the IRGA where any CO2 release from the cells was detected. The rate of CO2release was calculated from the chlorophyll content of the cuvette, the flow rate, and measuredconcentration of CO2. Subsequently, 10 .tM CCCP was injected via a serum-stoppered portand increases in the rate of CO2 release were monitored. 90 ppm CO2 in N2 was used as astandard.02 consumption:Net oxygen exchange after uncoupling with CCCP was measured using a Clark typeoxygen electrode (Hansatech Ltd., King's Lynn, England). Cells (1.2 p.g Chl mL -1 ) were dark-adapted 20 minutes prior to placing them in the darkened oxygen electrode chamber. Rates ofcontrol respiratory 02 consumption were measured for 3 minutes prior to uncoupling withCCCP.Fluorescence:Fluorescence was measured as described previously in Chapters 1 and 2.RESULTSThe effect of CCCP on key respiratory metabolites:The data reported are the mean of 3 separate experiments. The variation about eachdata point was less than 10% and in most cases was due to slight concentration differencesbetween experiments. In all cases, the trend observed in the mean data set was apparent in eachreplicate. Time points for fast sampling were 5, 20, 35, 50, 75, and 90 seconds.102Starch degradation:^Dark aerobic starch breakdown occurred at a rate of 12.3 p.molglucose equivalents mg -1 Chl 11 -1 . The rate of starch breakdown in dark CCCP treated cellswas 25.0 wnol glue mg-1 Chl h-1 (Figure 14).Adenylates:^Uncoupling of S. minutum with CCCP resulted in complementary changes inATP, ADP and AMP (Figure 15A). Within 5 seconds, the concentration of ATP decreased 2-fold and reached a minimum within 15 seconds of uncoupling. ATP recovered slightly after 2minutes but remained below control levels for the duration of the experiment. ADP and AMPincreased 2- and 5-fold above the control level within 1 minute. ADP gradually returned tocontrol levels while AMP recovered to a level approximately 2-fold greater than the controlafter 40 minutes. The adenylate energy charge dropped from 0.8 to 0.4 within 15 seconds andrecovered only marginally for the duration of the experiment (Figure 15B).PEP and Pyr:^Within 5 seconds of CCCP treatment, pyruvate increased 100% and PEPdecreased 40% which resulted in a 3.2-fold decrease in the PEP/PYR ratio (Figure 16A, B).PEP leveled off at a tenth of its original concentration within 5 minutes while Pyr dropped backto control levels and subsequently increased for the remainder of the experiment. ThePEP/PYR ratio did not recover for the duration of the experiment (Figure 16B).F6P, FBP, TP: The levels of F6P, FBP and TP were unaffected for the first 20 seconds aftertreatment with CCCP (Figure 17 A). After a lag of 20 seconds, F6P decreased slowly from 160to 40 nmol mg-1 Chl with an approximate half-time of 5 minutes. After a similar lag, FBPincreased to a level 4-fold greater than the control with a half-time of 5 minutes. Triosephosphates (TP) increased 2- to 3-fold with the same kinetics as FBP. After 20 seconds, theratio of FBP/F6P increased slowly over the course of the experiment from 2 to 30 (Figure 17B).103400300_crnE.—3CT3503Cn0E3„.E-6E_c250—50 2502000^50^100^150Time (min)Figure 14: The effect of CCCP treatment on the long term rate of starch degradation in S.minutum. Cells were dark adapted for 20 minutes before beginning sampling. Arrow indicatestreatment with CCCP. Data represent the mean of 3 experiments. Rate of starch degradationbefore and after treatment with CCCP was 12.3 and 25.0 .tmol gluc equiv mg -1 CM h-1,respectively._crnEOEca_L003002502001501005000.80.7O:1^6=; 00 •ADPadenylate energycharge104400300 1-cnE200 3E10000.50.4ATP^1, + CCCP0-A+ CCCP—10^0^10^20^30^40^500.3—20Ti me(min)Figure 15: The effect of CCCP treatment on the cellular concentration of adenylates in S.minutum. Cells were dark adapted for 20 minutes before beginning sampling. Arrow indicatestreatment with CCCP. Data represent the mean of 3 experiments. A. ATP, open circles; ADP,[ATP]+0.5[ADP][ATP]+[ADP]+[AMP]closed circles; AMP, open squares. B. Adenylate energy charge,105140_c120rnE1000E80>0 60a_ 40L._eL 20a_LJ0Time (min)Figure 16: The effect of CCCP treatment on the cellular concentration of pyruvate (Pyr) andphospho enol pyruvate (PEP) in S. minutum. Cells were dark adapted for 20 minutes beforebeginning sampling. Arrow indicates treatment with CCCP. Data represent the mean of 3experiments. A. Pyr, open circles; PEP, closed circles. B. PEP/Pyr ratio.5 0 0 70_c0 40 0CP6 3 0 0Eet- 200 F-L_0Cl_CO 100 L--I0302520078- 151050—5605 0 CScn4030 E20 PaLL100+ CCCP0• 7= FBP106p\ ^0.* °• TP):1F6PA+ CCCPFBP/F6PP-CYCIB—20^— 10^0^10^20^30^40Time(min)Figure 17: The effect of CCCP treatment on cellular levels of fructose bisphosphate (FBP),fructose-6-phosphate (F6P) and triose phosphate (TP) in S. minutum. Cells were dark adaptedfor 20 minutes before beginning sampling. Arrow indicates treatment with CCCP. Datarepresent the mean of 3 experiments. A. FBP, closed circles; TP, open circles; F6P, opensquares. B. FBP/F6P ratio.107G6P, G1P: G6P decreased 4-fold with the same lag and kinetics observed in F6P (Fig 18A).After addition of CCCP, G1P increased slightly but returned to near control levels within 5minutes. The ratio of G1P/G6P tripled with a half-time of 5 minutes (Figure 18B).6PG: Treatment with CCCP had no effect on 6PG levels (data not shown).PEP/TP, ATP/ADP, NADH/NAD, F _ ( [PEITATPIENADM ):^Within 5 seconds ofITPIIADPIINADICCCP treatment, the PEP/TP ratio decreased 25% and decreased steadily to a level which wasonly 1% of the initial control value within 5 minutes (Table 5). Similarly, the ATP/ADP ratiodecreased by 70% within 5 seconds of CCCP treatment and remained at or below this level forthe first 20 minutes after treatment with CCCP (Table 5). In contrast, the NADH/NAD ratioincreased 2.6-fold after 5 seconds of CCCP treatment and continued to increase for the first 2minutes after treatment (Table 5, see also Figure 11E, Chapter 4). The overall effect of theindividual ratio changes was to decrease the mass action ratio (F) by 43% within 5 seconds andby 94% within 2 minutes. The mass action ratio continued to decline for 20 minutes aftertreatment with CCCP (Table 5).Gas exchange:CO2 release: The average (n=3) rate of dark CO2 release measured was 137 limo' CO2 mg -1Chl h-1 . After CCCP treatment CCCP, this rate increased within 1 minute to 257 gmol CO2mg-1 Chl h -1 , an approximately 2-fold increase (Figure 19). After 15 minutes, the rate of CO2release slowly decreased and reached control levels within 45 minutes.02 exchange: The dark rate of 02 consumption was 151.2 wnol 02 mg -1 Chl h -1 . After 2minutes of CCCP treatment, the rate of 02 exchange doubled to 343.4 wnol 02 mg -1 Chl h-1.ii + CCCPG1P•4^-•10830200,--.__c 1500uiE-.,6 100Ec...._.,f-Ia_coo 50.__.sp-C4)----------0G6P10 041 + CCCPA0.30.--eg 0.20.10.0—20 —10 0 1 0 20 30 40Ti me (m in)Figure 18: The effect of CCCP treatment on cellular levels of glucose-6-phosphate (G6P) andglucose- 1-phosphate (G1P) in S. minutum. Cells were dark adapted for 20 minutes beforebeginning sampling. Arrow indicates treatment with CCCP. Data represent the mean of 3experiments. A. G6P, open circles; G1P, closed circles. B. G1P/G6P ratio.109Table 5: The effect of CCCP on the PEP/TP, ATP/ADP, NADH/NAD and[PEP][NADH][ATP]/[TP][NAD][ADP] ratios compared to the dark aerobic control. Valueswere normalized to the dark aerobic control.Treatment PEP/TP ATP/ADP NADH/NAD [PEP] [NADH] [ATP]]'[TP] [NAD] [ADP]+ CCCP0 sec 1.00 * 1.00 * 1.^* Loo *5 sec 0.75 0.30 2.58 0.5730 sec 0.31 0.22 2.98 0.212 min 0.09 0.19 3.42 0.065 min 0.01 0.25 3.38 0.0120 min 0.01 0.33 2.00 0.005t An approximation of the mass action ratio of the reaction sequence TP + NAD + ADP + Pi —›(1,3 bisPGA, 3PGA, 2-PGA) PEP + NADH + ATP +* Actual values were 1.33, 3.75, 0.45 and 0.225 for PEP/TP, ATP/ADP, NADH/NAD andmass action ratio, respectively.300c„.250200CVo 15010050110—10^0^10^20^30^40^50^60Time (min)Figure 19: The effect of CCCP treatment on the rate of dark CO2 efflux in S. minutum asmeasured by an open gas exchange IRGA system. Cells (3.5 lag Chl m1: 1 , pH 6.0) were darkadapted for 45 minutes to allow equilibration before treatment with CCCP. Cells were treated attime 0 with 10 p.lvl CCCP.111Within 7 minutes, the rate of 02 consumption increased to a level greater than 4-fold thecontrol rate (Figure 20).Fluorescence measurements:Steady-state saturation pulse analysis:^The effect of CCCP treatment was to initiallyquench F, the level of fluorescence measured by the low intensity measuring beam (Figure 21A).After 2 minutes, F began to increase and reached a level 1.5-fold greater than the dark controlwithin 11 minutes. Similarly, qNp was initially decreased by 15% relative to the dark control(Figure 21B). After 2 minutes, qNp began increasing relative to the dark control and reached amaximum value of 0.23. Treatment with CCCP resulted in an initial increase in qp relative tothe dark control. After 2 minutes of CCCP treatment qp decreased slowly by 40% (Figure21B).77K fluorescence:^The initial effect of CCCP treatment was to slightly increase theF686/F717 ratio of fluorescence measured at 77K (Figure 21C). The F686/F717 ratio began todecrease only after 2 minutes of treatment with CCCP. Within 8 minutes, the F686/F717 ratiohad decreased from 2.35 to 1.55.DISCUSSIONMetabolic changes observed after uncoupling with CCCP:After treatment with CCCP, the rate of starch degradation doubled (Fig 14). Thekinetics of the increase in starch degradation could not be resolved by the technique usedbecause the amount of degraded starch was small compared to the large starch reserve innutrient-limited cells. The rate of CO2 release, however, increased by approximately 2-foldwithin 1 minute (Figure 17), suggesting that the rate of respiratory carbon flow was enhanced_c700rnE600(-N400E 500400ct_E3000200cnO1000a)^—10 —5 0 5 10 15112Time (min)Figure 20: The effect of CCCP treatment on the rate of dark, 02 consumption in S. minutummeasured using a Hansatech oxygen electrode. Cells (3 pg Chl mL - l) were dark adapted for 20minutes to allow equilibration before treatment with CCCP.O\-o0N 1.5+ CCCP I^• ai^ C1PCCCPcINF00-0-0-0)O1.21.00.80.60.40.20.0—(3.02.5 -N 0 -coCO 2.0 -(0\fr + CCCP113Fm 'F—10 —5 0^5^10 15 20 25 30Time (min)Figure 21: The effect of CCCP treatment on A. room temperature saturation pulse analysis (seealso Figure 5C, Chapter 2), B. Photochemical (qp) and non-photochemical (q Np) quenchingcoefficients calculated from A, and C. 77K fluorescence F686/F717 ratios.114within 1 minute. The following discussion will outline a model to explain a) how respiratorycarbon flow is activated by treatment with CCCP and b) specifically how this increase inrespiratory carbon might affect the chloroplastic NADH/NAD ratio, increase the redox state ofthe PQ(cyt b6f) pool, and result in a state 1 to 2 transition. A detailed model for this interactionis presented in Figure 22.Activation of respiratory carbon flow:Based on what is known about the use of intermediates in the study of metabolic control(Rolleston, 1972) and specific studies on the activation of the key respiratory enzymes PK andPFK in S. minutum (Turpin et al., 1990; Vanlerberghe et al., 1989; Botha and Turpin, 1990a;Lin et al., 1989), the following mechanism was hypothesized for the activation of respiratorycarbon flow after treatment with CCCP.Activation of "lower glycolysis" and respiratory control: In isolated mitochondria and invivo, it has been observed that respiratory 02 consumption by the METC can be controlled bythe availability of ADP (Lambers, 1985; 1990). Adenylate control, however, may function atone of two levels. It appears that both mechanisms may operate in vivo in a manner whichseems to be tissue-specific and varies with developmental stage and substrate availability(Lambers, 1990). In the first case, the rate of e flow through the METC is directly limited byADP because ADP-limitation of the ATP synthase increases the proton motive force andrestricts e - transport to 02 (Day and Lambers, 1983). The second possibility is that the rate ofrespiratory carbon supply is ADP-limited and this in turn limits reluctant supply to the METC(Azcon-Bieto et al., 1983). Pyruvate kinase (PK), in particular, has been observed to functionfar from equilibrium; this fact has been deduced from estimates of substrates and products invivo (Rolleston, 1972; Turner and Turner, 1980; Douce, 1985). In addition, experimentalevidence suggests that PK is ADP-limited in vivo (Turpin et al., 1990; Vanlerberghe et al.,1989; Turner and Turner, 1980).4PEPstarch c G 113G6PF6PPFK>C. ADPt FBPATPchloroplast I ATP, TPA^t ADP, PGATriose-P I ^®<NAD-GAPDHNADHcytosol3-PGAPi3-PGAOAA4 PEPtADP<PK 1 I;)0 RATP^ 4NAD(P)H/PQ +^> • PQH.^PYR NADF 1,8 state 1kinase > ATPI state 2NADH OAA4,7FADH2acetylV^coAcitrate+ CCCPmalate TCA cycle2 OGt FADH2NADHNADHNADH^O IADP + PiPYR t mitochondrionNADH ATP02 4ATPFigure 22: The proposed mechanism for interaction between respiratory carbon flow and poisingof the PETC via the state 1 to 2 transition after uncoupling with CCCP. See text for details andlist of abbreviations for abbreviations.116The initial effect of CCCP treatment was to dissipate the mitochondrial proton gradientand prevent chemiosmotic production of ATP (Figure 22, 1). This, in turn, resulted in a 50%decrease in ATP within 5 seconds with concurrent 2- and 3-fold increases in ADP and AMP(Figure 15A). Changes in adenylate pools served to decrease the adenylate energy charge from0.8 to 0.6 within 5 seconds (Figure 15B). These changes in adenylates appeared to releaserespiration from adenylate control because the rates of respiratory 02 consumption and CO2efflux rapidly increased after CCCP treatment (Figures 19 and 20). If ADP were directlylimiting the rate of e- flow through the METC, this should have resulted in a rapid decrease inthe NADH/NAD ratio followed by a decrease in pyruvate as carbon flow was activated torespond to "draw-down" from the TCA cycle. However, the rapid increase in ADP wasaccompanied by a 3-fold increase in the NADH/NAD ratio (See Figure 11C, Chapter 3),implying that direct ADP-limitation of the METC was not occurring. On the other hand, a 2-fold increase in pyruvate, a 40% decrease in PEP and a 3.2-fold decrease in the PEP/PYR ratiowere observed within 5 seconds of CCCP treatment (Figure 16A and B). The decrease insubstrate for PK (PEP) and increase in product (Pyr) which occurred immediately aftertreatment with CCCP may constitute a "cross-over" point (Chance and Williams, 1965;Williamson, 1966) and implies that respiration is activated by a release of ADP-limitation onpyruvate kinase activity (Figure 22, 2). The observation of increased carbon flow through PK(measured as an enhancement of CO2 efflux and the NADH/NAD ratio), despite the fact thatthe increased ratio of products/substrates would make the reaction less thermodynamicallyfeasible, is a characteristic of rate-limiting or regulatory enzymes (Rolleston, 1972). Theestimated concentration of ADP in vivo (33 11M, Turpin et al., 1990) is below the kM for boththe plastidic (kM =200 11.M) and cytosolic (km= 50 gM) isozymes of PK (Lin et al., 1989) andis consistent with ADP-limitation of PK in vivo in S. minutum. On the basis of metabolitechanges after uncoupling with CCCP, it appears that adenylate control functions at the level ofADP-limitation of PK in N-limited S. minutum which confirms studies done with this algaduring anaerobiosis and NH4+ assimilation (Turpin et al., 1990; Vanlerberghe et al., 1989).117GAPDH: The enzymatic reactions governing the conversion of TP to PGA and PEP (NAD-GAPDH, PGA kinase, phosphoglyceromutase and enolase) are thought to be close toequilibrium under normal conditions (Turner and Turner, 1980; Douce, 1985; Stryer, 1988).The rate of flow through these enzymes, then, is thought to be governed largely by theconcentrations of cofactors, substrates and products (Rolleston, 1972; Stryer, 1988).Thermodynamically, the rate of flow through equilibrium enzymes is related to the ratio ofproducts/substrates, or the mass action ratio. In general, the smaller the mass action ratio for anequilibrium enzyme, the greater the flow through the enzyme (for thermodynamic rationale, seethe next chapter). The mass action ratio for NAD-GAPDH is difficult to determine becauselevels of 1,3 bisPGA are extremely low in vivo and, consequently, GAPDH and PGA kinase aregenerally considered as a combined reaction (Rolleston, 1972; Takahama et al., 1981). Themass action ratio for the combined reaction is G = [PGA][ATP][ NADH][H ] . However, it[T13][ADP][NAD][Pi]was not possible to measure PGA after treatment with CCCP and PEP was used as anapproximation of the PGA component of the mass action ratio. Thus, the mass action ratioreflected the combined reactions from TP to PEP. This interpretation was justified by theobservation that, of other cases in which respiratory carbon flow changed, the magnitude andkinetics of decreases in PEP were mirrored by changes in PGA consistent with these reactionsbeing close to equilibrium (Turner and Turner, 1980; Turpin et al., 1990; Vanlerberghe et al.,1990). The mass action ratio was further approximated by removing the [H+] component fromthe equation.Within 5 seconds of CCCP treatment, the PEP/TP ratio decreased by 25% and theATP/ADP ratio decreased by 70% (Table 5). The rapid decrease in PEP observed aftertreatment with CCCP (Figure 16A) initially decreased the PEP/TP ratio. Rapid decreases inATP and increases in ADP after treatment with CCCP (Figure 15A) would both havecontributed to the reduction of the ATP/ADP ratio within 5 seconds (Table 5). Thus, despite118the fact that a 2.6-fold increase in the NADH/NAD ratio occurred within 5 seconds, the massaction ratio for TP conversion to PEP (F) had almost halved (Table 5). The PEP/TP ratiocontinued to decline until it reached a value which was only 1% of the dark control within 5minutes of CCCP treatment (Table 5). The continued decline of this ratio was subsequentlymaintained by increases in TP that resulted from PFK activation after 30 seconds (see discussionbelow). The continued decline of the PEP/TP ratio had the effect of decreasing F by 79% after30 seconds and by 95% after 2 minutes, despite the fact that the NADH/NAD ratio continued toincrease as a result of CCCP treatment (Table 5). The 100-fold decrease in the mass actionratio for carbon flow from TP to PEP after 5 minutes of treatment with CCCP was, therefore,driven decreases in the ATP/ADP and PEP/TP ratios. In turn, decreases in the PEP/TP ratiowere driven first by activation of PK (within 5 seconds) and subsequently by activation of PFK(within 30 seconds, see below).The flow through equilibrium enzymes is thermodynamically related to the mass actionratio such that decreases in the mass action ratio increase the rate of flow (See next chapter; seealso Rolleston, 1972; Stryer, 1988). An increase in carbon flow from TP to PEP would bethermodynamically favoured by decreased levels of PEP and ATP and increased levels of TPand ADP. In turn, increased carbon flow via NAD-GAPDH should result in increased NADHproduction and an increased NADH/NAD ratio. NAD-GAPDH was shown to be 86% localizedin the chloroplast in Chlamydomonas reinhardtii (Klein, 1986). Although little is known aboutthe localization of NAD-GAPDH in S. minutum, similarities between FBPase isozymes and PFKbetween S. minutum and Chlamydomonas suggest that glycolytic compartmentation is similarbetween these two algae (Botha and Turpin, 1990a, b). Assuming that the same enzymelocalization occurs in S. minutum, the increase in carbon flow implied by the increased massaction ratio should result in an increase in the chloroplastic NADH/NAD ratio (Figure 22, 4).Thus, it is proposed that carbon flow via NAD-GAPDH would increase within 5 to 15 secondsof uncoupling because of a decrease in the product of this reaction (PGA) resulting fromobserved decreases in PEP (Figure 16A). Since NAD-GAPDH produces NADH, this would119also contribute to the increase in the cellular NADH/NAD ratio observed (Figure 11C, Chapter3).Upper glycolysis (Phosphofructokinase): Within the first 30 seconds after CCCP treatment,very little change occurred in either F6P, FBP, or TP levels. After 30 seconds there was a 75%decrease in the level of F6P and a 2- and 4-fold increase in FBP and TP, respectively (Figure17). These changes were similar to the "cross-over" effect observed for PK and are consistentwith the contention that PFK is an important regulatory enzyme in glycolysis (Turner andTurner, 1980; Turpin et al., 1990; Botha et al., 1988). In vivo levels of PEP have been shownto strongly inhibit PFK in S. minutum (Botha and Turpin, 1990; Turpin et al., 1990) and otherorganisms (Botha et al., 1988; Knowles et al., 1990; Garland and Dennis, 1980; Kelly andLatzko, 1977). The 40% decrease in PEP levels observed within 5 seconds could, therefore, beimportant in the activation of PFK and the top half of glycolysis. Increases in the FBP/F6P ratiooccurred after the drop in PEP (compare Figures 16 and 17) and suggested that activation ofPK was responsible for the activation of PFK (Figure 22, 3). Pi has also been shown to be apotent activator of S. minutum PFK (Turpin et al., 1990; Botha and Turpin, 1990a). It ispossible that PFK was further activated by the uncoupler-mediated decline in ATP andconsequent increase in Pi. One consequence of an activation of PFK activity is an increase inTP levels. An increase in TP would further activate NAD-GAPDH by augmenting increases inthe TP/PGA ratio brought about by activation of PK (see previous discussion; see also Figure22, 4).The lack of change in 6PG after treatment with CCCP could imply that the majority ofcarbon flow from starch occurs via glycolysis and not through the oxidative pentose phosphatepathway.Phosphoglucomutase:^Phosphoglucomutase catalyses the conversion of G1P, a directproduct of starch degradation, to G6P and appears to be an equilibrium enzyme (Douce, 1985).120G6P, G1P, and the G1P/G6P ratio remained relatively unchanged for the first 30 seconds aftertreatment with CCCP whereupon G6P decreased approximately 4-fold, serving to triple theG1P/G6P ratio within 10 minutes (Figure 18B). The 30 second lag in activation ofphosphoglucomutase suggests that an increase in the rate of starch degradation occurred onlyafter PK and PFK were activated.The kinetics of PQ reduction and the state 1 to 2 transition:In the chloroplast, the first effect of uncoupling with CCCP was an immediate decreasein F (the level of fluorescence induced by the measuring beam after treatment) and an increase inFM ' (Figure 21A). This resulted in a decrease in qNp from dark control levels and a slightincrease in qp (Figure 21B). The speed with which qNp relaxed suggests that the quenchingwas caused mostly by qE which is related to the thylakoid ApH (Horton and Hague, 1988). Onthe basis of thermoluminescence (Bennoun, 1982) and room temperature fluorescencemeasurements (Ting and Owens, 1993), it has been suggested that chloroplastic respiratoryelectron transport from NAD(P)H to 02 may result in proton translocation across the thylakoidmembrane and a trans-thylakoid ApH. The rapid release of qNp observed after uncoupling withCCCP (Figure 21B) is consistent with the occurrence of a proton gradient in the dark due toCRETC activity in aerobically adapted control cells. The slight increase in qp observedindicated that QA became more oxidized immediately after treatment and suggests thatthylakoid electron transport may have been limited by the trans-thylakoid ApH in the dark(Figure 21B).Two minutes after treatment with CCCP, F M ' began to decrease while F began toincrease. F increased 2-fold which resulted in a concurrent decrease in qp with a half-time ofapproximately 4 minutes (Figure 21A, B). Since CCCP entered the cell and uncoupled protongradients within 5 seconds, it is unlikely that the changes in F were related solely to thephotochemical effects of CCCP on PS2 (ADRY effects, Renger, 1972). Instead, it is more121likely that changes were correlated with an increase in PQ reduction due to an increase in thechloroplastic NADH/NAD ratio. Thus, changes in PQ reduction occurred with a half-time ofapproximately 4 minutes. FM ' decreased by 20% within 20 minutes (412= 7 minutes) whichresulted in an increase in qNp (Figure 21B). It is likely that the majority of this increase in qNpresulted from an increase in qrr (a state 1 to state 2 transition) since uncoupling would havereleased qE associated with the thylakoid ApH.A determination of the kinetics of metabolic change makes it possible to more accuratelyresolve the kinetics of a state transition. It was observed that changes in the F686/F717 ratio(figure 21C) were offset slightly (1 to 2 minutes) from changes in PQ redox indicated byincreases in F and decreases in qp (Figure 21A B) but occurred with similar kinetics onceinitiated. This implied that once PQ was reduced, activation of the LHC2 kinase andreallocation of LHC2 occurred within 1 to 2 minutes. These state transitions kinetics areconsiderably faster than those reported for green algae and higher plants (412 = 5 - 7 minutes,Williams and Allen, 1987). Although some of this change may occur because thylakoids are lessstacked in N-limited cells (Plumley and Schmidt, 1991), it is equally possible that the kinetics ofPQ reduction determine the kinetics of a state transition. Unless PQ redox changes occurimmediately, calculation of t112 using initiation of treatment as a starting point will be an over-estimate of the actual kinetics of the state transitionThe relationship between the NADH/NAD ratio and PQ redox changes aftertreatment with CCCP:In chapter 4 it was proposed that increases in the NADH/NAD ratio would result inreduction of PQ. A 3-fold increase in the NADH/NAD ratio occurred within 5 seconds oftreatment with CCCP (Figure 11C, Chapter 4). Since the kinetics of PQ reduction showed a 2minute lag after changes in the NADH/NAD ratio, it is unlikely that the cellular ratio isresponsible for increasing the reduction of PQ. Changes in the rate of carbon flow throughNAD-GAPDH were approximated by the kinetics of changes in the mass action ratio for the122conversion of TP to PEP (Table 5, see discussion above). The full activation of NAD-GAPDHappeared to occur as a result of a combination of decreases in the ATP/ADP and PEP/TP ratioswhich were initiated first by PK activation and subsequently by PFK activation. On the basis ofchanges in the mass action ratio, the half-time for increases in carbon flow through NAD-GAPDH (and increased chloroplastic NADH production) was between 5 and 30 seconds (Table5). Changes in F and qp (indicating PQ reduction) did not begin to occur until approximately 2minutes after treatment with CCCP (Figure 21A, B). This suggests that increased flow throughNAD-GAPDH and increased chloroplastic NADH/NAD ratios might be responsible forreduction of the PQ(cyt b6f) pool and the transition from state 1 to state 2.SUMMARYMetabolite changes accompanying uncoupling with CCCP treatment were examined todetermine the mechanism responsible for the activation of respiratory carbon flow and themetabolic sequence of events leading to reduction of PQ and the initiation of the state 1 to 2transition. The immediate effect of uncoupling was to decrease ATP and increase ADP andAMP levels. This was accompanied by rapid increases in the rate of CO2 efflux and 02consumption, indicating that an increase in respiratory carbon flow had occurred. Long-termrates of starch degradation doubled and, combined with gas exchange data, suggested that anincrease in respiratory carbon flow occurred as a result of CCCP treatment. Increases in ADPwere correlated with a rapid increase in Pyr and a decrease in PEP consistent with the activationof PK by increasing ADP availability. Decreases in F6P and increases in FBP and TP wereobserved to occur within 30 seconds of CCCP treatment and were consistent with the activationof PFK. The lag in changes in the FBP/F6P ratio after the activation of PK was consistent withthe activation of PFK activity by decreases in PEP (brought about by PK activation). Activationof PK and PFK resulted in a subsequent decrease in G6P and an increase in the G1P/G6P ratio,123implying that activation of starch degradation occurred as a result of "draw-down" of carbon viaglycolysis.In addition, uncoupling with CCCP was observed to greatly decrease the combined massaction ratio for the reactions resulting in conversion of TP to PEP (all equilibrium reactions).This decrease in the mass action ratio occurred initially as a result of decreases in the ATP/ADPratio and decreases in PEP resulting from activation of PK. Subsequently, the mass action ratiowas further decreased by increases in TP resulting from activation of PFK. Decreases in themass action ratio can be related to increases in the flow through equilibrium enzymes. It wasproposed that increased flow through NAD-GAPDH (one of the enzyme reactions included inthe combined mass action ratio) could result in increased NADH production. On the basis ofenzyme localization studies in Chlamydomonas, it was proposed that NAD-GAPDH activitywas localized predominantly in the chloroplast (Klein, 1986). Assuming that a similar enzymelocalization occurred in S. minutum, it was proposed that increases in NAD-GAPDH activitywould lead to an increase in the chloroplastic NADH/NAD ratio. Increases in carbon flow viaNAD-GAPDH, indicated by decreases in the mass action ratio, were followed by increases inPQ reduction and resulted in a state 1 to state 2 transition. The slight lag between increases incarbon flow via NAD-GAPDH and PQ reduction implied that increases in the chloroplasticNADH/NAD ratio via NAD-GAPDH activity were responsible for PQ reduction and a state 1to state 2 transition.124CHAPTER 6: DEVELOPMENT OF A COMPREHENSIVE MODEL FOR THEINTERACTION BETWEEN RESPIRATORY CARBON FLOW AND POISING OFTHE STATE TRANSITIONINTRODUCTIONThe primary hypothesis of the present work is that increases in the NADH/NAD ratioare responsible for increased PQ reduction and the occurrence of a state 1 to 2 transition. Insupport of this contention, class 1 treatments resulted in increases in the NADH/NAD ratio of2.8-fold or greater while Pi treatment (class 2) resulted in only a minimal (10%) increase after 2minutes of treatment. However, the observation that NO3 - treatment resulted in an increase inthe NADH/NAD ratio which was intermediate in magnitude between that of class 1 treatmentsand N treatment was inconsistent with the contention that increases in the NADH/NAD ratioresulted in PQ reduction and a state 1 to 2 transition. Several plausible explanations weresuggested for this inconsistency including a) the fact that a threshold increase in theNADH/NAD ratio might be required to result in increased PQ reduction and b) the increases inthe cellular NADH/NAD ratio observed did not reflect changes in the chloroplasticNADH/NAD ratio and, therefore, did not affect PQ reduction. This chapter will focus on thesecond possibility. The rationale for this approach will be developed below.Reduction of PQ in the thylakoid membrane requires that the chloroplastic NADH/NADratio increases as a result of respiratory carbon flow. It is quite possible that increases in themitochondrial NADH/NAD ratio would not directly affect chloroplastic NADH/NAD ratios oraffect the redox state of PQ. This is because it is unlikely that reductant would be shuttled fromthe mitochondria (where NADH can be efficiently oxidized either by the cytochrome oralternative pathway of the METC) to the chloroplast where chlororespiratory flow has beenestimated to comprise only 10 to 20% of the full respiratory electron transport capacity of a cell(Bennoun, 1982; Peltier et al., 1987). This suggests that chloroplastic respiratory carbon flowmust be responsible for the increase in PQ reduction observed after treatment with class 1effectors.125The interpretation of changes in cellular pyridine nucleotide ratios is limited by the factthat changes in compartmental ratios may not be reflected by changes in cellular levels. Theouter membranes of the chloroplast and mitochondrium are impermeable to pyridinenucleotides. Movement of redox potential between compartments appears to be limited toindirect pyridine nucleotide shuttles such as the proposed malate/oxaloacetate shuttle or triosephosphate/phosphoglycerate shuttle (Heber, 1974; Ebbighausen et al., 1985;1987). Preliminaryestimates of the NADH/NAD ratio in the cytosol and mitochondria suggest that the ratio ismuch higher in the mitochondria than in the cytosol (Kromer and Heldt, 1991) and implies thatgradients in reduced pools of pyridine nucleotides may be maintained between cellularcompartments.To date, no method exists to rapidly fractionate green algal cells making it impossible todirectly measure compartmental NADH/NAD ratios. However, on the basis of enzymelocalization work in the chloroplasts of the green alga Chlarnydomonas reinhardtii (Klein,1986), some information is known about the compartmentation of NADH and NADPHproducing and consuming enzymes. In turn, information about the activity of these enzymes canbe determined by examining changes in the pools of substrates and products of each enzyme(Turpin et al., 1990; Rolleston, 1972).One major source of NADH in the chloroplast is from the activity of NAD-GAPDHwhich catalyzes the conversion of TP to PGA. 86% of NAD-GAPDH was localized in thechloroplast in the green alga Chlamydomonas reinhardtii (Klein, 1986). In the previouschapter, it was proposed that increases in NAD-GAPDH activity could lead to an increase inchloroplastic NADH/NAD ratios and result in PQ reduction. The purpose of the presentchapter is to examine this hypothesis in more detail in order to develop a consistent and detailedmodel for the interaction between respiratory carbon flow and the PETC in vivo. This involvedtesting the hypothesis that class 1 treatments should result in a large increase in respiratorycarbon flow via chloroplastic NAD-GAPDH whereas class 2 treatments should not. To do this,a large metabolite data set was assembled from previously published work from this lab and126analyzed to compare general trends in metabolite and cofactor ratios between class 1 and class 2treatments. Thermodynamic considerations of key respiratory enzymes revealed some largedifferences between class 1 and class 2 treatments.RESULTSIn the interests of clarity, previously published data will be summarized herein. Tablefootnotes indicate the source of data.Kinetics of changes in ADP, Pyr/PEP and FBP/F6P:Table 6 is a summary of the effects class 1 and class 2 treatments on ADP, Pyr/PEP andFBP/F6P ratios over 20 minute period.Class 1 treatments:NH4:+ ^Treatment of N-limited cells with NH4+ resulted in a 1.6-fold increase in ADP and a6.6-fold increase in the Pyr/PEP ratio within 5 seconds. The increase in ADP was maintainedfor 30 seconds but decreased to control levels within 2 minutes. The Pyr/PEP ratio remained 7-fold higher than the dark control for at least 20 minutes after treatment. The FBP/F6P ratioincreased by 66% within 5 seconds and achieved an 8.5-fold increase within 5 minutes (Table6). The TP/PGA ratio doubled within 5 seconds and increased to 12.4-fold the dark controlwithin 5 minutes after treatment with CCCP (Table 6).Anaerobiosis: Anaerobiosis resulted in a 1.8-fold increase in ADP and 2-fold increase in thePyr/PEP ratio within 30 seconds. ADP remained at this level while the Pyr/PEP ratio increasedto a level 4-fold greater than the control within 5 minutes. The FBP/F6P ratios was unchangedfor the first 2 minutes after which the ratio increased and reached 3-fold the dark control ratio127Table 6: A comparison of the effects of class 1 and class 2 treatments on changes in key respiratoryintermediates and their ratios. Pi treatment was made to Pi-limited cells, all other treatments were madeto N-limited cells. All values are normalized to the respective dark, aerobic control value.Time after treatment ADP^Pyr/PEP FBP/F6P PGA/TPClass 1 treatments:+ NH4+^(Turpin et al, 1990; Feil and Turpin, unpublished)0 sec 1.00* 1.00* 1.00* 1.00*5 sec 1.60 6.62 1.66 0.4730 sec 2.16 7.60 3.48 0.222 min 1.04 12.15 5.04 0.195 min 1.00 7.85 8.48 0.0820 min 1.00 7.08 8.88 0.08+ anaerobiosis^(Vanlerberghe et al, 1989)0 sec 1.00 1.00 1.00 1.005 sec 1.45 1.10 1.04 0.8130 sec 1.75 2.00 1.05 0.502 min 1.70 3.00 2.53 0.185 min 1.75 3.75 2.93 0.1120 min 1.80 4.50 3.67 0.08+ CCCP (see Chapter 5)0 sec 1.00 1.00 1.00 1.00 **5 sec 1.72 2.87 0.81 0.75 **30 sec 1.54 3.83 2.44 0.31^**2 min 1.96 8.30 3.94 0.09 **5 min 1.35 46.2 6.78 0.02 **20 min 1.48 106.4 12.72 0.01 **Class 2 treatments:+ N01 (Vanlerberghe et al, 1992; Feil and Turpin, unpublished)0 sec 1.00 1.00 1.00 1.005 sec 1.17 1.00 1.57 0.5130 sec 0.85 1.09 1.62 0.392 min 0.67 1.46 3.08 0.195 min 0.84 2.05 3.27 0.2920 min 0.75 2.25 3.32 0.11+ Pi (Gauthier and Turpin, 1993)0 sec 1.00*** 1.00*** 1.00*** 1.00***5 sec 1.18 1.52 0.55 0.6630 sec 1.32 0.88 0.65 0.942 min 1.11 0.30 0.70 1.115 min 1.09 0.36 0.90 0.9320 min 1.11 0.40 2.50 0.72* The actual values in N-limited cells were 108.3 (± 10.4) nmol mg -1 Chl, 0.53 (± 0.26), 0.86 (± 0.65) and 18.0(± 10.7) for ADP, Pyr/PEP, FBP/F6P and PGA/TP, respectively.** These values are approximated from the PEP/TP ratio because PEP and PGA remain in equilibrium.*** The actual values in Pi-limited cells were 57 nmol mg -1 Chl, 2.5, 2.0, 1.25 for ADP, Pyr/PEP, FBP/F6Pand PGA/TP, respectively.128within 5 minutes. (Table 6). The TP/PGA ratio doubled within 30 seconds and increased 9-foldwithin 5 minutes of anaerobic treatment (Table 6).CCCP:^The kinetics of changes in ADP, and the Pyr/PEP and FBP/F6P ratios aresummarized in Table 6 and have already been described (see results, Chapter 5). TheTP/PGA(=TP/PEP) ratio tripled within 30 seconds and increased 87-fold within 5 minutes oftreatment with CCCP (Table 6)Class 2 treatments:NO3-: ADP increased by 15% within 5 seconds of treatment with NO3 - and then recoveredto a level which was 15% less than the dark control for the remainder of treatment. ThePyr/PEP ratio was relatively unaffected until 2 minutes where upon it increased by 50% andreached a maximum of 2.3-fold the dark control level within 20 minutes. The FBP/F6P ratioincreased by 60% within 5 seconds and tripled within 2 minutes (Table 6). The TP/PGA ratiodoubled within 5 seconds and increased 3.5-fold within 5 minutes of treatment with NO3 -(Table 6).Pi: Treatment with Pi resulted in a 30% increase in ADP within 30 seconds whereupon ADPdecreased but remained at a level 10% greater than the dark control for at least 20 minutes.Within 5 seconds of treatment, Pyr/PEP ratios increased by 50%. However, within 2 minutes,the Pyr/PEP ratio declined to half the dark control level and remained lower than the control forthe next 20 minutes. The FBP/F6P ratio decreased to half the control level within 5 secondsand remained lower than the control for 5 minutes. After 20 minutes, the FBP/F6P ratiorecovered and reached a level 250% greater than the dark control (Table 6). The TP/PGA ratioincreased 50% within 5 seconds of treatment with Pi and then declined to dark control levelswithin 30 seconds after treatment with Pi (Table 6).129PGA/TP, NADH/NAD, ATP/ADP and F ([PGA][NADH][ATP] / [TP][NAD][ADP]):Table 7 presents a summary of the effects of class 1 and class 2 treatment on the ratiosof PGA/TP, NADH/NAD and ATP/ADP after 5 minutes of treatment. These ratios were thenused to make an approximation of F, the mass action ratio for the conversion of TP to PGA byNAD-GAPDH and PGA kinase.Class 1 treatments (NH4+, anaerobiosis and CCCP):The PGA/TP ratio decreased by 92, 89, and 97% within 5 minutes of treatment with NH4+,anaerobiosis and CCCP, respectively. Similarly, the ATP/ADP ratio decreased by 20, 64, and75% after treatments with NH4+, anaerobiosis and CCCP. On the other hand, the NADH/NADratio increased by 4-, 2.8-, and 3.6-fold after 5 minutes of treatment with NH4+, anaerobiosisand CCCP, respectively. The mass action ratio for the conversion of TP to PGA decreased by71, 90, and 99% after 5 minutes of treatment with NH4+, anaerobiosis and CCCP, respectively(Table 7).Class 2 treatments (NO3 - and Pi):After 5 minutes of treatment with NO3 - , the PGA/TP ratio decreased by 71%, theNADH/NAD ratio increased by 70% and the ADP/ATP ratio increased by 23%. This decreasedthe combined mass action ratio (for conversion of TP to PGA) by 38% within 5 minutes (Table7). All 4 ratios remained relatively unchanged after 5 minutes of treatment with Pi (Table 7).Effects of illumination:Illumination resulted in a 70% increase in the PGA/TP ratio, a 30% increase in theATP/ADP ratio and a 24% increase in the NADH/NAD ratio(Table 7). In turn, this resulted ina 2.8-fold increase in the combined mass action ratio.4.52 0.292.80 0.103.60 0.011.70 0.62 ttt1.00 0.99 tttClass 1 treatments:+ NH4+^0.08^0.80+ anaerobiosis^0.11 0.34+ CCCP 0.03 tt^0.25Class 2 treatments:+ NO3 -^0.29^1.23+ Pi 0.93 1.08130Table 7: The effect of illumination and treatments which increase the rate of respiratory carbonflow on the PGA/TP, ATP/ADP, NADH/NAD and [PGA] [NADH][ATP]/ITPIINAD][ADP]ratios compared to the dark aerobic control. Values were taken 5 minutes after treatments andwere normalized to the dark aerobic control.Treatment^PGA/TP^ATP/ADP^NADH/NAD [PGA][NADH][ATP] *[TP] [NAD] [ADP]illuminated, aerobic **^1.72^1.28^1.24^2.78dark, aerobic control^1.00 ***^1.00 ***^Loo ***^Loo t(N or Pi-limited)* An approximation of the mass action ratio of the reaction sequence TP + NAD + ADP + Pi^(1,3bisPGA) -> 3PGA + NADH + ATP** Values from N-limited cells, data for Pi-limited cells not available.*** Actual values for N-limited cells were 18.0 (± 10.7), 4.16 (± 0.8) and 0.059 (± 0.024) for thePGA/TP, ATP/ADP and NADH/NAD ratios, respectively. Actual values were 1.25, 0.96, 0.125 and0.125 for PGA/TP, ATP/ADP, NADH/NAD and the mass action ratio in Pi-limited cells.t Actual value for r in the dark N-limited control was 4.37 (± 1.53). This corresponds to a AG' value of-2.13 kcal/mol calculated from AG'= RT1n(171C eq). AG°' for this reaction is -3.0 kcal mot -1 (Stryer,1988). Values for Keq calculated from K., = 10 -°G° " 36 (Stryer, 1988).t t This is an approximation and represents the PEP/TP ratio (see Chapter 5).11t These values were not significantly different from the dark aerobic control values.131DISCUSSIONActivation of respiratory carbon flow : the effect of class 1 and 2 treatments on ADP, andthe Pyr/PEP, FBP/F6P and PGA/TP ratios:Class 1 treatments (NH4+, anaerobiosis or CCCP):The changes in key respiratory metabolites previously observed with CCCP treatmentwere remarkably similar to the changes in metabolites observed after treatment with NH4+ andanaerobiosis. After treatment with NH4+ or anaerobiosis, an increase in ADP was closelycorrelated to an increase in the Pyr/PEP ratio (Table 6) indicating that respiratory flow wasincreased by activation of PK due to relief of ADP-limitation of PK (Turpin et al., 1990;Vanlerberghe et al., 1989). In turn, the FBP/F6P ratio increased after to increases in thePyr/PEP ratio suggesting that the decrease in PEP resulting from PK activation served toactivate PFK in all class 1 treatments (Turpin et al., 1990; Vanlerberghe et al., 1989). In allcases, the PGA/TP ratio appeared to decrease initially as a result of PK activation (whichdecreased PGA) and this decrease was further augmented by PFK activation (which increasedTP) indicated by increases in the FBP/F6P ratio (Table 6).Class 2 treatments (NO3 - or Pi):Increases in ADP were not correlated with an increase in the Pyr/PEP ratio or PKactivation after treatment with NO3 - or Pi. ADP increased slightly and then decreased by 15%within 30 seconds of treatment with NO3 - while the Pyr/PEP ratio was unchanged until 2minutes after treatment with NO3 - after which the ratio doubled within 5 minutes (Table 6).These observations were inconsistent with an activation of PK by relief of ADP-limitation. Onthe other hand, the FBP/F6P ratio increased by 60% within 5 seconds of NO3 - treatment andcorresponded to a 2-fold decrease in the PGA/IY ratio (Table 6). It has been suggested thatrespiratory carbon flow through the oxidative pentose phosphate pathway is activated by adecrease in the cellular NADPH/NADP ratio after treatment with NO3- (Vanlerberghe et 0,1992; Figure 8A). Thus, it is thought that respiratory carbon flow is activated in response to the132reductant demands for NO3 - reduction rather than a release of ADP-limitation of PK(Vanlerberghe et al., 1992). PK activation occurred after PFK activation and it is thought thatPK may be activated, in this case, by a decline in glutamate, which is an inhibitor of PK in vivo(Turpin et al., 1990).After treatment with Pi, there was an 18% increase in ADP and a 50% increase in thePyr/PEP ratio within 5 seconds. However, the Pyr/PEP ratio subsequently dropped rapidly andreached a level which was only 30 % of the dark control level within 2 minutes of treatmentwith Pi (Table 6). The FBP/F6P ratio dropped relative to the dark control for the first 5minutes of treatment with Pi and only increased by 2.5-fold within 20 minutes (Table 6). Thefact that neither PK or PFK appeared to be activated is consistent with the observation that thePGA/TP ratio remained relatively unchanged by Pi treatment for the first 20 minutes (Table 6).Although the activation of respiration by Pi treatment is not well understood, it is thought thatrespiratory carbon flow may be controlled by the ATP requirements of a plasmalemma H-F-ATPase which is activated to maintain intracellular pH and provide the proton motive force topower Pi uptake (Gauthier and Turpin, 1993). It has also been proposed that cells respond toPi-limitation by developing enzymatic pathways to bypass glycolytic enzymes which require Pi(Theodorou et at, 1990). If these enzyme bypasses were functioning in cells re-supplied withPi, the rate of carbon flow would increase but flow through NAD-GAPDH (which requires Pi)might remain minimal. Thus, the minimal change in the PGA/TP ratio observed after Ntreatment might reflect the operation of Pi conserving carbon shunts during the initial period ofPi assimilation.The effect of class 1 and 2 treatments on the combined mass action ratio for NAD-GAPDH and PGA kinase:The enzymatic reaction governing the conversion of TP to PGA are described by thefollowing equations:133TP+NAD + Pi < NAD- GAPDH >1,3 bis PGA + NADH + H +1,3 bis PGA + ADP <  PGA kinase > 3 PGA + ATPwhich, when summed, yield the overall reaction of:TP + NAD + ADP + Pi <  GAPDH and PGA kinase > 3 PGA + NADH + H+ + ATPThe enzymes responsible for the conversion of TP to 3-PGA (NAD-GAPDH and PGA kinase)are thought to be close to equilibrium and, hence, are freely reversible (Turner and Turner,1980; Douce, 1985; Stryer, 1988). The rate of flow through "equilibrium" reactions isdependent to a large extent on the extent to which they are displaced from equilibrium and thisis described by the free energy of the reaction (AG'). AG' is related to the ratio of theequilibrium constant for a reaction Keg and the mass action ratio, F, by the equation AG' = RTln(F/Keg). Since reactions are thermodynamically favoured when AG' is negative, as the massaction ratio (F) for an equilibrium enzyme decreases the flow through the enzyme increases(Rolleston, 1972).In the previous chapter it was proposed that increases in respiratory carbon flow via theNAD-GAPDH would increase the chloroplastic NADH/NAD ratio and result in PQ reduction.This proposal was made assuming that localization of NAD-GAPDH in S. minutum is similar tothat observed in Chlamydomonas, where 86% of NAD-GAPDH was localized in thechloroplast. Furthermore, it was hypothesized that increases in NAD-GAPDH activity in thechloroplast would be correlated to increases in NADH/NAD ratios and would result in PQreduction in class 1 but not class 2 treatments. In order to examine this hypothesis rigorously, itwas necessary to determine if increases in flow through NAD-GAPDH occurred. This wasdone by a thermodynamic consideration of the effects of changes in key respiratory metabolites.The mass action ratio for NAD-GAPDH is difficult to determine because levels of 1,3-bisPGA are extremely low in vivo and consequently, GAPDH and PGA kinase are generallyconsidered as a combined reaction (Rolleston, 1972; Takahama et al., 1981). The mass action[PGA][ATP][NADH][11+] ratio for the combined reaction is F = ^However, because [Pi] and[TP][ADPJ[NAD][Pi]134[I-1+] were not actually measured, the mass action ratio was approximated asr [PGA][ AT" NADH]   Although compartmentation may certainly affect the accuracy of[TP][ADPRNAD]this approximation, it was useful to demonstrate certain trends in vivo.Effects of Class 1 treatments on the mass action ratio:The mass action ratio decreased by 70, 90 and 99% after 5 minutes of class 1 treatmentwith NH4+, anaerobiosis or CCCP, respectively (Table 7). During all class 1 treatments, largedecreases in the PGA/TP and ATP/ADP ratios were responsible for the decrease in the massaction ratio for TP to PGA conversion despite increases in the NADH/NAD ratio. Decreases inthe TP/PGA ratio occurred as a result of activation of both PK and PFK (see previousdiscussion). In turn, large decreases in the mass action ratio after 5 minutes of class 1treatments would thermodynamically favour an increase in flow from TP to PGA (Rolleston,1972). In particular, decreases in this combined mass action ratio suggest that carbon flowthrough NAD-GAPDH would increase as a result of class 1 treatments. Assuming that 86% ofNAD-GAPDH is chloroplastic, this increase in activity would serve to enhance NADHproduction in the chloroplast and could be responsible for the reduction of the PQ poolobserved after class 1 treatments.Effects of Class 2 treatments on the mass action ratio:On the other hand, the mass action ratio for conversion of TP to PGA was notsignificantly affected by treatment with either Pi or NO3 - (Table 7). This suggests that the flowof carbon from TP to PGA during Pi assimilation was not thermodynamically favoured incomparison to the dark control and that flow through NAD-GAPDH and PGA kinase wasrelatively unchanged within 5 minutes of either class 2 treatment. In the absence of an increasein flow through NAD-GAPDH, chloroplastic NADH/NAD ratios should also be relativelyunaffected and would be consistent with the absence of large changes in PQ reduction or a state1351 to 2 transition during class 2 treatments. The intermediate changes in cellular NADH/NADratios observed after NO3 - treatment were likely to reflect increases in mitochondrial ratios dueto TCA cycle activity. Thus, the case of Pi treatment strongly supports the contention that class2 treatments should not result in an increase in the chloroplastic NADH/NAD production ratiovia increased flow through NAD-GAPDH. With the exception of fluorescence induction data,the case of NO3 - treatment also appears consistent with the model suggesting regulation of thestate 1 to 2 transition by increases in chloroplastic NAD-GAPDH activity.Does ATP play a role in the regulation of the state transition?:Bulte et al. (1990) have suggested that, in addition to the effects of PQ reduction,decreases in [ATP] play an important role in the regulation of state transitions. Although theydid not speculate as to the exact role of ATP, these authors indicated that oxidation of PQ wasnot sufficient for the recovery of a state 1 to state 2 transition, and that increases in [ATP] werealso required. These authors used far red light and DCMU to oxidize PQ in cells that weretreated with CCCP or ATPase inhibitors. However, it is quite possible that NAD(P)H mightreduce PQ in the light via the NAD(P)H-PQ oxidoreductase (see literature review section onH2 photoevolution, p. 23). Since the redox state of PQ was only inferred, and not directlymeasured by Bulte et al. (1990), it is likely that oxidation of PQ is indeed sufficient for therecovery of a state 1 to state 2 transition and that PQ remains reduced under conditions wherethe ATP pool is decreased from control.From the present work, it would appear that ATP does not have a direct role inregulating the state transition. Instead, it seems that decreases in the ATP/ADP ratio releaserespiratory carbon flow from ADP limitation at the level of PK. This indirectly results in anincrease in carbon flow via NAD-GAPDH and production of NADH in the chloroplast. Theincreased chloroplastic NADH/NAD ratio resulting from increases in carbon flow via NAD-GAPDH then results in reduction of PQ via the NAD(P)H-PQ oxidoreductase and activates thekinase responsible for the state 1 to 2 transition. Hence, if the ATP/ADP ratio remained low136upon illumination, PK would remain activated, chloroplastic NADH production would continueand PQ would remain reduced in the light. Thus, I suggest that decreases in ATP per se do notdirectly affect the redox state of PQ, but rather form part of a signal transduction pathwaywhich leads to an increase in chloroplastic NADH/NAD ratios.The physiological significance of interaction between respiratory carbon flow andphotosynthesis via the CRETC in vivo:The present study has clearly shown that interaction occurs between respiratory carbonflow and the PETC in the dark. Specifically, NAD(P)H-PQ oxidoreductase activity appeared toallow NADH produced by respiratory carbon flow (via NAD-GAPDH) to reduce the PQ(cytb6f) pool and result in a state 1 to 2 transition. It has been proposed that the CRETC may bephysiologically significant in vivo in darkened cells because it may maintain a transthylakoidproton gradient in the dark which allow rapid induction of ATP production upon illumination(Peltier et al., 1987), and it may allow oxidation of reductant produced by starch degradation inthe dark (Bennoun, 1982). The observation that CRETC activity also appears to allowregulation of the state 1 to 2 transition by changes in respiratory carbon flow through NAD-GAPDH suggests that an additional important function for the CRETC may be to allowcommunication between respiration and photosynthesis in both the dark and the light.In the present study, experiments were undertaken in the dark to reduce the complexityof processes contributing to both biochemical and physiological measurements of respiratorycarbon flow, PQ reduction and the state 1 to 2 transition. Although interactions betweenrespiratory carbon flow and the PETC were observed in the dark, the ramifications forinteraction between respiratory carbon flow and photosynthesis in the light are considerable.First, the regulation of the state transition in the dark by respiratory carbon flow (via theCRETC) may be important to poise the light harvesting reactions of cells for subsequentillumination. Class 1 treatments were shown to result in large decreases in the combined massaction ratio for the conversion of TP to PGA (Table 7). On the other hand, illumination137resulted in a 2.7-fold increase in the combined mass action ratio (Table 7) consistent withthermodynamic enhancement of photosynthetic carbon fixation which requires carbon flow inthe opposite direction to glycolysis. It is conceivable that the large changes in the mass actionratio brought about by class 1 treatments could make photosynthetic carbon flow lessthermodynamically favourable and reduce carbon flow in the photosynthetic direction uponillumination. This, in turn, could result in over-reduction of PS2 due to NADP-limitation of thePETC. Over-reduction of PS2 may cause photodamage unless NADPH production could bedown-regulated. The state 1 to 2 transition occurring during class 1 treatments in the darkcould, therefore, provide a mechanism to photoprotect PS2 upon illumination under theseconditions. In addition, the state 1 to 2 transition would decrease the amount of LHC2associated with PS2 and could enhance the amount of cyclic e - flow and ATP production whichwould directly increase the mass action ratio and tend to favour carbon flow in thephotosynthetic direction. These proposals could be examined using illumination of class 1treated cells in the presence of inhibitors of the NAD(P)H-PQ oxidoreductase (e.g. rotenone).If control- and inhibitor-treated cells were treated with class 1 or class 2 treatments andsubsequently illuminated, the presence of the inhibitor should increase the susceptibility of class1 treated cells to photodamage upon illumination in comparison to control and class 2 treatedcells.Perhaps more importantly, however, the CRETC would also allow direct interactionbetween respiratory carbon flow and the PETC in the light. Increases in respiratory carbon flowoccur in the light in response to increased biosynthetic requirements (Weger et al., 1989) and inresponse to decreased ATP/ADP ratios (Turpin and Weger, 1990). CRETC activity wouldallow increases in NADH production (which would specifically signal an increase in respiratorycarbon flow) to directly regulate the poising of the PETC for linear and cyclic electron transportvia the state transition in the light. As mentioned above, this regulation may be important toallow photoprotection of PS2 if respiratory carbon flow were to affect photosynthetic carbonflow. Preliminary research has indicated that the 5 treatments used in the present study also138result in increases in respiratory carbon flow in the light (Holmes and Turpin, unpublished).Furthermore, class 1 treatments also appear to result in a state 1 to 2 transition in the light whileNO3 - treatment does not. This is clearly a fruitful area for future research to extend the presentstudy's observations about the interactions between respiratory carbon flow and the PETC.In conclusion, it appears that regulation of the PETC NADPH/ATP production ratio byrespiratory carbon flow through NAD-GAPDH may be physiologically significant. NAD-GAPDH utilizes metabolites which are shared by both the reductive reactions of the Calvincycle and the oxidative reactions of glycolysis. The status of glycolytic carbon flow through thisenzyme can affect TP/PGA ratios and is likely to affect photosynthetic carbon fixation inilluminated cells. The significance of the CRETC may be that it allows NADH produced byNAD-GAPDH to signal the status of glycolytic carbon flow to the PETC and, in turn, couldallow the PETC to compensate via the state 1 to 2 transition.139CHAPTER 7: GENERAL SUMMARY AND CONCLUSIONS: The interaction between photosynthesis and respiration has previously been studiedmainly with respect to the regulation of respiration by photosynthesis upon illumination.However, little is known about the regulation of photosynthesis by respiration. The fact thatrespiration has both a catabolic function and is necessary to provide carbon skeletons forbiosynthesis implies an intimate co-regulation with photosynthesis.On the basis of previous studies on the mechanism of the state transition and the factthat NAD(P)H-PQ oxidoreductase has been observed to affect PQ(cyt b6f) pool reduction invitro, a hypothesis was developed to examine the interaction between respiratory carbon flowand the regulation of the NADPH/ATP production ratio by the PETC (via the state transition).It was hypothesized that increases in respiratory carbon flow that lead to increases in the ratio ofreduced/oxidized pyridine nucleotides would result in reduction of the PQ(cyt b6f) pool and astate 1 to 2 transition. In contrast, increases in respiratory carbon flow which did not lead toincreases in reduced/oxidized pyridine nucleotide ratios were not expected to change PQ(cytb6f) redox or result in a state 1 to 2 transition.Five treatments were shown to increase respiratory carbon flow in the green alga,Selenastrum minutum. Dark assimilation of NH4+ or NO3 - by N-limited cells, anaerobiosis,uncoupling with CCCP and Pi assimilation by Pi-limited cells all resulted in a 2- to 10-foldincrease in respiratory carbon flow as measured by respiratory CO2 release and/or starchdegradation. These treatments were then sub-divided into two classes on the basis of theirability to cause a state 1 to 2 transition. Treatment with NH4+, anaerobiosis or CCCP resultedin large perturbation of room temperature and 77K fluorescence parameters indicative of a state1 to 2 transition and were termed class 1 treatments. Class 1 treatments were also observed todecrease the potential quantum yield of linear e - transport consistent with poising of the PETCfor down-regulation of NADPH/ATP production ratio. NO3 - assimilation (N-limited cells) andPi assimilation (Pi-limited cells) were designated as class 2 treatments because they resulted in140only minor changes in room temperature and 77K fluorescence parameters indicating theabsence of a state 1 to 2 transition. In turn, the potential quantum yield of linear electrontransport was relatively unaffected by either of these treatments.On the basis of the original hypothesis, it was expected that class 1 treatments wouldincrease PQ(cyt b6f) pool reduction whereas class 2 treatments would not. The reduction of thePQ(cyt b6f) pool was deduced from measurements of time-resolved fluorescence decay andinduction kinetics. The rate of fluorescence decay was significantly decreased and the rate offluorescence induction was greatly increased indicating that the PQ(cyt b6f) pool had beenreduced by class 1 treatments. On the other hand, fluorescence decay and induction parameterswere relatively unaffected by Pi treatment (class 2) suggesting that PQ reduction wasunaffected. Similarly, NO3 - treatment resulted in only minor changes in fluorescence decaykinetics. These observations were consistent with the original hypothesis. However, NO3 -treatment resulted in a large increase in the rate of fluorescence induction. The magnitude ofchanges in induction kinetics during NO3 - assimilation were inconsistent with changes in decaykinetics and the absence of a state 1 to 2 transition during NO3 - assimilation. It was proposedthat a decrease in Fd/FNR association with PS 1 due to super-complex formation with nitritereductase could result in reduction of the PETC upon induction due to Fd/FNR limitation ofPS1 in NO3 - treated cells.On the basis of these observations, it was hypothesized that class 1 treatments wouldresult in large increases in the reduced/oxidized ratio of pyridine nucleotides whereas class 2treatments would have a much smaller effect. The present study used a highly specific methodto measure pyridine nucleotides that allowed accurate measurement of changes in both theNADPH/NADP and NADH/NAD ratios. Increases in the NADPH/NADP ratio were notcorrelated with an increase in PQ(cyt b6f) pool reduction or a state 1 to 2 transition in thepresent study. Large increases in the NADH/NAD ratio occurred after all class 1 treatmentsand were correlated with PQ reduction and a state 1 to 2 transition. Only small changes in theNADH/NAD ratio were observed during Pi assimilation. These results suggested that increases141in the NADH/NAD ratio were responsible for reduction of the PQ pool and a state 1 to 2transition. NO3 - assimilation, however, resulted in an intermediate increase in the NADH/NADratio which was inconsistent with the observed absence of a state 1 to 2 transition during NO3 -assimilation.One plausible explanation for the inconsistency posed by the increased NADH/NADratio during NO3 - assimilation was that, due to compartmentation, increases in theNADH/NAD ratio might not occur in the chloroplast and would not affect PQ(cyt b6f) poolreduction. It was proposed that class 1 treatments resulted in increases in chloroplasticNADH/NAD ratios whereas class 2 treatments did not. This possibility was explored bycomparing the mechanism by which respiratory carbon flow was activated during class 1 and 2treatments. In all class 1 treatments, respiration appeared to be activated by increases in ADPwhich released PK from ADP limitation. PFK was then subsequently activated by decreases inPEP due to PK activation. In turn, all class 1 treatments resulted in large decreases in the massaction ratio for the conversion of TP to PGA via NAD-GAPDH and PGA kinase despiteincreases in the cellular NADH/NAD ratios. NAD-GAPDH has been shown to be localized86% in the chloroplast of Chlamydomonas reinhardtii (Klein, 1986). Assuming a similarenzyme localization in S. minutum, the increase in carbon flow through NAD-GAPDH impliedby a decreased mass action ratio strongly suggested that this enzyme was responsible for anincrease in chloroplastic NADH/NAD ratios, reduction of PQ and the occurrence of a state 1 to2 transition.On the other hand, activation of respiratory carbon flow after class 2 treatments did notinvolve release of PK from ADP-limitation. Instead, class 2 treatments resulted in a decrease(NO3 -) or only a minor increase in ADP (Pi). The mass action ratio for TP to PGA conversionwas not significantly affected by either treatment, consistent with only minor changes in PQreduction and absence of a state 1 to 2 transition during class 2 treatments. On the whole, theseresults are consistent with the hypothesis that NADH produced by NAD-GAPDH wasresponsible for PQ reduction and a state 1 to 2 transition.142The observation that state 1 to 2 transitions occurred in the dark in response to increasesin respiratory carbon flow implies that regulation of photosynthesis by respiratory carbon flowthrough the CRETC may be physiologically significant in the light. First, the state 1 to 2transition would allow for photoprotection of PS2 in class 1 treated cells upon illumination.Photoprotection of PS2 might be necessary due to changes in glycolytic flow which, in turn,could affect carbon flow in the photosynthetic direction upon illumination. Second, increases inrespiratory carbon flow which resulted in increases in chloroplastic NADH/NAD ratios in thelight could potentially directly regulate the redox state of the PQ(cyt b6f) pool and theactivation of a state 1 to 2 transition. Preliminary studies of the effect of class 1 and 2treatments in the light suggest that this may be a fruitful area for future research.In conclusion, the present study clearly indicates that interaction occurs betweenrespiratory carbon flow and poising of the PETC for NADPH/ATP production ratios via thestate 1 to 2 transition. This interaction is likely to be a necessary consequence of the fact thatboth anabolic (Calvin cycle) and catabolic (glycolysis) processes occur in the same compartment(the chloroplast) and share common intermediates. 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Nature 201: 725-726Wilhelm C, Duval JC (1990) Fluorescence induction kinetics as a tool to detect achlororespiration activity in the prasinophycean alga Mantionella squamata. BiochimBiophys Acta 1016: 197-202Willeford KO, Gombos Z, Gibbs M (1989) Evidence for chloroplastic succinatedehydrogenase participating in the chloroplastic respiratory and photosynthetic electrontransport chains of Chlamydomonas reinhardtii. Plant Physiol 90: 1084-1087Williamson P (1966) Glycolytic control mechanisms. II. Kinetics of intermediate changesduring the aerobic-anoxic transition in perfused rat heart. J Biol Chem 241: 5026-5036162Williams WP, Allen JF (1987) State 1/state 2 changes in higher plants and algae. PhotosynthRes 13: 19-45Wirtz W, Stitt M, Heldt HW (1980) Enzymatic determination of metabolites in thesubcellular compartments of spinach protoplasts. Plant Physiol 66: 187-193Wiskich T (1980) Control of the Krebs cycle. In D.D. Davies, ed, Metabolism andRespiration, Vol 2. Academic Press, New York, pp 244-276Wollman FA, Bulte L (1989) Towards an understanding of the physiological role of statetransitions. In D.O. Hall, G Grassi, eds, Photosynthetic Processes for Energy andChemicals. Elsevier Publishers, London, pp 198-207163APPENDIX 1: APPROXIMATION OF QNp AND QpAs can be seen in Figure 23, the level of fluorescence measured by the measuring beamin the dark was not a true Fo (qp=1, qNp=0) because it was quenched relative to the level of F omeasured at low light intensities in S. minuturn. Similarly, the true FM (qp=0, qNp=0) occurredat low light intensity and implies that FM measured in the dark in this alga is quenched.However, in the interests of simplicity, calculations of qp and qNp were made relative to thedark control assuming that qp=1 and qNp=0.Approximation of qNp and qp from room temperature PAM traces1) qNp:according to van Kooten and Snel (1990):qNp = 1 - (FM' - Fo ')/(FM - Fo )= [(FM - FM')-(Fo - F0 ')]/(Fm - Fo)qp = (FM' - F)/(FM' - F0 ')where FM is the fluorescence intensity after a saturating flash in dark aerobic controlcells, FM' is the fluorescence intensity after a saturating flash in treated cells, Fo is the level offluorescence induced by the measuring beam in dark aerobic cells (qp = 0), Fo ' is the trueminimal level of fluorescence induced by after treatment (qp = 0) and F is the actualfluorescence intensity measured at any time t.Since it is not possible to accurately measure true F o ' in the dark under conditions whereQA is reduced (i.e. qp # 0)assume that the true Fo ' S Fo if qT or qE occurs. That is, (F 0 -F0 ') 0.a minimum estimate of the amount of qNp and qp occurring would be:qNp = (FM - FM')/(FM - Fo)164qp = (FM' - F)/(FM'- Fo) MF 00.90.8■-■cn0.7a)0.6—a)L._0.5a)cc)a)L_0.4LT1650.31000 2000 3000 4000 5000 6000Light Intensity (pEi/rn a/s)Figure 23: The effect of light intensity on the measurement of room temperature fluorescenceparameters, Fo , the minimal level of fluorescence measured by the 1.6 kHz measuring beam andFM , the maximal level of fluorescence induced by a saturating flash. Cells were adapted for 10minutes to each light intensity. FM was the maximal level of fluorescence obtained with a 50 msecmultiple turnover flash at each light intensity. Fo , was the minimal level of fluorescence inducedby the 1.6 kHz measuring beam within 5 seconds of darkening. N.B. Illumination with far redlight immediately before Fo measurement had no effect of the value of F o obtained.166APPENDIX 2: CORRECTION OF TIME-RESOLVED FLUORESCENCE DECAYSFOR THE ACTINIC EFFECTS OF THE MEASURING BEAMINTRODUCTION The decay of variable fluorescence from a maximal level of fluorescence, FM to aminimal level of fluorescence, F o , can be used to determine the rate of re-oxidation of QA- (Caoand Govindjee, 1990; Krause and Weis, 1991). In this method, all QA (but not QB or PQ) isreduced by the means of a high intensity single turnover flash. The decline of fluorescence,which reflects the re-oxidation of QA- to QA, is measured by a weak, modulated "non-actinic"measuring light in the following "dark" period. In cells where all QA is oxidized before theflash, QA oxidation kinetics have been deconvoluted to yield three decay components whichare attributed to QA oxidation by QB in PS2 centers a) with QB bound before the flash and b)with QB not bound before the flash and c) by recombination between QA and the S2 state ofthe water oxidizing complex in centres that are unable to transmit electrons to the QB pool (PS2(3 centres) (Cao and Govindjee, 1990; Etienne et al, 1990).Because the rate of re-oxidation of QA can be affected by the redox state of the PQpool, it is also possible to infer the reduction state of the PQ pool from fluorescence decaykinetics. However, if the PQ pool is reduced before the single turnover flash, the QA pool willalso be somewhat reduced due to redox equilibrium with the PQ pool. Thus, PS2 will exist in anumber of redox conformations and QA will not be completely oxidized either before or afterthe single turnover flash. Despite this added complexity, the rate of QA oxidation will still beaffected by PQ redox and the rate of QA oxidation will decrease as the PQ pool becomesreduced. The accurate determination of the rate of QA re-oxidation, however, is dependentupon the fact that the measuring beam itself is non-actinic, that is, that the measuring beam doesnot cause QA reduction and/or have any effect upon QA re-oxidation kinetics (Krause andWeis, 1991). In measurements of fluorescence decay to date (Cao and Govindjee, 1990;Etienne et al., 1990; Gleiter et al., 1992; Robinson and Crofts, 1983; Schreiber, 1986;167Govindjee et al., 1992) no attempt has been made to examine the actinic effects of the lowintensity measuring beam used to measure the kinetics of the fluorescence decay.It has already been observed that even the low intensity 1.6 kHz measuring beam usedfor steady-state saturation pulse measurements had actinic effects which were observed as a risein fluorescence after treatment with DCMU (Figure 5, Chapter 2) However, resolution offluorescence decay in the [tsec time scale requires that the frequency of the measuring beam beincreased from 1.6 to 100 kHz. In turn, this increases the light intensity of the measuring beamand increases the likelihood that the measuring beam will have actinic effects. In cells where theQA pool was already reduced, it was, therefore, quite likely that an increase in light intensity ofthe MB would further reduce the QA pool. Thus, it was anticipated that resolution of QA- re-oxidation kinetics after class 1 treatments simply using fluorescence decay measurements wouldbe difficult. That is, accurate measurement of the rate of QA- re-oxidation would becomplicated by the fact that the measuring beam would be contributing to QA reduction inaddition to being used to measure QA oxidation. We have examined this question bymeasuring the actinic effects of the higher frequency measuring beam after both class 1 and class2 type treatments.MATERIALS AND METHODSRefer to Chapter 2, methods for time -resolved fluorescence decays.RESULTS Uncorrected fluorescence decays:Figure 24 shows the effect of class 1 and class 2 treatments on fluorescence decays thathave been measured essentially as previously described (Schreiber, 1986; Ettiene et al. , 1990;168Cao and Govindjee, 1990; Govindjee et al. , 1992). The 100 kHz measuring beam was used fora duration of 40 msec to allow resolution of the kinetics of fluorescence decays in cells treatedwith either NH4+ or anaerobiosis. Decays shown were normalized to Fv max = Fm-Fo (control)and Fvmax i = FM'-F (treated cells) where FM and Fm ' were the values extrapolated to t= 0 infitted decay curves. With the exception of anaerobically treated cells, all the decays wereobserved to reach a quasi steady-state plateau within 5 to 10 milliseconds which was higher thanthe initial Fo level (Figure 24). The quasi-stationary level reached in cells treated with class 1treatments (NH4+ or CCCP) or DCMU was considerably higher than that reached by darkcontrol cells (Figure 24 A, B, D). Class 2 treatments (NO3 - or Pi) resulted in quasi steady-statelevels which were only slightly higher than control cells (Figure 24 E, F). The differencebetween the plateau and the initial F o (control) or F (treated) induced by the measuring beamremained for as long as the measuring beam remained at 100 kHz.Fluorescence decays shown in Figure 24 were transformed to q(t) decays as described(Chapter 3) and curve-fitted to asymptote to the F o (control) or F (treated) measured previousto the single turnover flash. The dark control decay was de-convoluted to a sum of twoexponentials with a fast component (T1= 288 [tsec) comprising 65% of the decay (al= 0.65),and a slower component (T2 = 34.5 msec) comprising 35% of the decay (a2= 0.35) (see Table8). DCMU treatment resulted in a loss of the pec decay component and the appearance of arapid (T1= 7 gsec) rise component as indicated by the negative amplitude of al (Cao andGovindjee, 1990). The half-time of the msec component, T2 was increased tenfold over thedark control (Table 8). NH4+ assimilation increased the t112 of both the pec and msec q(t)decay components (T1 and T2) by approximately 2- and 4.5-fold and increased the contributionof the msec decay component from 35 to 90% (Table 8). Anaerobiosis resulted in completeloss of the fast component of the decay and a 100% contribution of the msec decay component(Table 8). Uncoupling with CCCP decreased the contribution of the 1..tsec component from 65to 42% and increased the contribution of the msec component from 35 to 58 % (Table 8). The1.21.00.80.60.40.20.0,---,-Do 1.2.15a)^1.0LLoo 0.8CD,......, 0.6CU0a)C 0.4(.)u)a.) 0.2I._oD 0 . 0I:: 1.21.00.80.60.40.210 20 30 40 50-10 0^10 20 30 40 50Time (msec)169Figure 24: The effect of treatments which increase respiratory carbon flow on uncorrected time-resolvedfluorescence decay kinetics. Decays were measured using a 40 msec duration, 100 kHz measuring beamand were not corrected for the actinic effect of the measuring beam. Open circles: dark, aerobic controlcells; closed circles: cells treated with A. NH4+; B. anaerobiosis; C. CCCP; D. DCMU; E. NO3 - ; F.Pi. Treatments A-E and F were made to N-limited and Pi-limited cells (3-5 jig Chl/mL), respectively. Thesingle turnover flash (t1/2=8 j.tsec) was initiated 3 msec after the measuring beam was switched from 1.6 to100 kHz. Decays were normalized to F 1, max = Fm-Fo (control) or Fvmax =Fm -F (treated). All curveswere an average of 8 measurements.170Table 8: The effect of DCMU and treatments which increased respiratory carbon flow on theamplitude (ai) and half-times (Ti = tit0.69) of the fast (pee; al and T1) and medium (msec;a2 and T2) components of time-resolved q(t) decays which were not corrected for the actiniceffect of the 100 kHz measuring beam. Cells were adapted to treatments for 20 minutes beforemeasurements of decays. Fluorescence decays (Figure 25) were transformed to q(t) decays andthen curve-fitted for either a single exponential or sum of two exponentials (see Materials andMethods). tTreatment al^(SE)^a2^(SE)^T1 (1.tsec) (SE)^T2 (msec) (SE)(uncorrected for the actinic effect of the measuring beam)Dark aerobic controls:N-limited cells 0.65 (.02) 0.35 (.02) 288.3 (23.14) 34.98 (8.28)Pi-limited cells 0.45 (.01) * 0.55 (.01) * 258.8 (11.2) 51.4 (4.8)+ DCMU -1.17^(.03) * .17^(.03) * 7.2 (2.92) * 392.9 (23.3) *Class 1 treatments:+ NH4+ 0.1^(.01) * 0.9 (.01) * 658.0 (61.5) * 156.1^(11.24) *+ anaerobiosis 0.0* 1.0* 0.0 * 25.8 (2.26)+ CCCP 0.42 (.01) * 0.58^(.01) * 98.7 (1.83) * 38.9 (2.19)Class 2 treatments:+ NO3 - 0.48^(.01) * 0.52 (.01) * 332.0 (25.5) 47.1^(0.99)+ Pi t t 0.48 (.04) 0.52 (.04) 197.3^(6.0) 118.3 (9.69) **t In all cases where curves fitted a single exponential, the r2 for the single exponential fit wasgreater than 0.98 and/or greater than the r2 for the sum of two exponentials.t Pi treatment was made to Pi-limited cells, all other treatments were made to N-limited cells.Indicates that this value was significantly different from the N-limited dark aerobic control,i.e. the value was outside the 95% confidence interval as determined by a student t test.** Indicates that this value was significantly different from the Pi-limited dark, aerobic control.171half-time of the i.tsec component (T1) decreased 3-fold after CCCP treatment while T2 wasunaffected (Table 8). NO3 - assimilation resulted in a decrease in the contribution of the fastdecay by 17% (Table 8). Pi assimilation resulted in a 2.4-fold increase in T2, the half-time ofthe msec decay component.The actinic effects of the 100 kHz measuring beamThe actinic effect of the 100 kHz measuring beam was measured by disconnecting thesingle turnover flash unit and measuring only fluorescence induced by the change in frequencyfrom 1.6 to 100 kHz. After 20 minutes of dark acclimation in control or treated cells, initiationof the 100 kHz measuring beam resulted in a fluorescence induction curve which reached aplateau within approximately 40 msec (Figure 25). The actinic effect of the 100 kHz measuringbeam was minimal in N-limited dark control cells (Figure 25 A-E) but the 100 kHz measuringbeam produced a distinct induction curve in Pi-limited control cells (Figure 25 F). The 100 kHzmeasuring beam resulted in significant fluorescence induction after DCMU or Class 1 treatmentas compared to the dark control (Figure 25 A, B, C, D). Both NO3 - and Pi treatment resultedin only minor changes in fluorescence induction as a result of the 100 kHz measuring beamcompared to the dark control (Figure 25 E, F).Correction factors for fast decays:In all cases, the fluorescence decayed to the level of fluorescence induced by themeasuring beam within 40 msec (data not shown). All decay curves were corrected bysubtracting the induction curves from the observed fluorescence decay before transformingfluorescence data to q(t) oxidation data.The effect of correcting fluorescence decays on q(t) decay parameters:Correcting fluorescence decay curves for actinic effects of the measuring beam had nosignificant (as determined by a student T test) effect on the contribution of the q(t) [tsec and1.00.80 . 60a)rn.(/)0a)EN00vc-0-oa)U-c)a)0,.a)0Lt._0 . 40 . 20 .00.80.60 . 40 .20. 00.80 .60.40.2172—-_+ NH4control, e, opp.vourowtoweci.oism . •_+ DCMU-awl?-^ control_^t II\ 100 kHztooceploAA0PWA/001:00/0)MB, 1.6 kHzA.1^1^I^t^1 1 D.I^I^I^I_ -+ anaerobic __ _ + NO 3_^_ „.., 4,....stomoxfpiopeolovoleIP4,0)•"e_ - ....---a.^....._^_^. , „Alt, aPyr.ozOreceeerf....P'„,op •^416 .^. •^-^• • • • , • ■ 'control control-^11\ -^tI^I^I^I^I^B. I I^1^I^I^E.+ CCCP ...adromeamsagaiwie_control+ Pi.^..4 • . 410404e04.010v111‘— 40 ffewlgOVOIAVAWAPPYOWV) — 4},y•'W4,0 • • . e4 loot"%%IVO °^ control_ _11\I^1^I^I^I^C. I I^I^I^I^F.Figure 25: The effect DCMU and class 1 and 2 treatments on the level of fluorescence induced by the 100kHz measuring beam. Open circles: dark, aerobic control cells; closed circles: cells treated with A.NH4+; B. anaerobiosis; C. CCCP; D. DCMU; E. NO3-; F. Pi. Treatments A-E and F were made toN-limited and Pi-limited cells (3-5 pg Chl m1: 1 ), respectively. The 1.6 kHz measuring beam was switchedon at time 0 (large arrow, A.) and was switched to 100 kHz for a 40 msec duration as indicated by thesmall arrow. All curves were an average of 8 measurements.0.0—10 0^10 20 30 40 50^0^10 20 30 40 50Time (milliseconds)173msec decay components in N- or Pi-limited control cells or in cells treated with anaerobiosis,NO3 - or Pi (Compare Table 8 and Table 3, Chapter 3). In cells treated with NH4+ or CCCPcorrection of fluorescence decay curves resulted in a loss of al, the ilsec q(t) decay componentand 100% contribution of a2, the msec decay component. Correction of fluorescence decaysresulted in loss of the fluorescence rise component and a 100% contribution of a2 aftertreatment with DCMU (Table 3 and 7). In all cases, correction of the fluorescence decayresulted in a large decrease in T2, the half-time of the msec decay component ranging from 5 to40-fold. T1 decreased slightly after correction in all cases except Pi treatment although thisdecrease was not significant in the case of NO3 - (Compare Tables 3 and 7).DISCUSSIONUncorrected fluorescence decays:In uncorrected fluorescence decays from N- and Pi-limited control or treated cells,fluorescence decays reached a plateau that was higher than the initial F o level within 40 msec.This resulted in a slow q(t) component with half-times (T2) which were greater than 25 msecfor all treatments (Table 8). The steady state level reached after treatment with DCMU, NH4+and CCCP was considerably higher than the level reached in the dark control (Figure 24). Thiswas consistent with a slower half-time of the msec component of the decay in each of thesecases (Table 8) and suggested that the PQ pool might be more reduced under thesecircumstances. In addition, NH4+ assimilation and anaerobiosis resulted in either a decrease orcomplete loss of the ilsec decay component which supported the suggestion that the PQ poolhad been reduced as a result of these treatments (Table 8).Treatment with NO3 - resulted in a 17% decrease in the contribution of the fastcomponent of the uncorrected q(t) decay suggesting that the rate of QA oxidation wasdecreased and that the PQ pool had become slightly reduced (Table 8). Pi treatment resulted inno significant change in the contribution of either component but doubled the half-time of the174msec q(t) decay component (Table 8). The effects of NO3 - or Pi treatment on uncorrected q(t)decays were significant and suggested some effect of each of these treatments on the oxidationkinetics of QA but were relatively minor compared to the effects of DCMU, NH4+ oranaerobiosis. This observation is consistent with the hypothesis that class 1 treatments wouldreduce the PQ pool while class 2 treatments would not.However, the half-time of the pee decay component, T1 decreased after the class 1CCCP treatment suggesting that the rate of QA oxidation had increased and that oxidation ofthe PQ pool might have occurred. This was inconsistent with the observation that CCCPresulted in a state transition (Chapter 2). In order to resolve this apparent discrepancy and todetermine whether the measuring beam was affecting the rate of fluorescence decay, the effectof the measuring beam was measured in the absence of the single turnover flash.Actinic effects of the measuring beam:It has been proposed that the PQ pool was reduced during class 1 treatments leading toreduction of the QA pool due to redox equilibrium between these two pools (Chapter 2). Thissuggested that the higher frequency (and intensity) measuring beam required to resolvefluorescence decay kinetics might also have actinic effects and affect QA reduction/oxidationkinetics. With the exception of anaerobically treated cells, the fluorescence decays reached astable plateau level which was higher than F o (fluorescence level before the single turnoverflash, control) or F (fluorescence level before the single turnover flash, treated) within 10 msecand remained close to this level as long as the measuring beam was on (Figure 24). Inparticular, the stable plateau was much greater than the original F level in cells treated withDCMU, NH4+ and CCCP (Figure 24 A, C, D). This observation suggested that the measuringbeam was actinic and resulted in QA reduction, preventing accurate resolution of QA oxidationkinetics from fluorescence decays. To test this hypothesis, the effect of the measuring beam inthe absence of a single turnover flash was measured.175The actinic effect of the measuring beam was measured as the extent of fluorescenceinduced by conversion of the measuring beam from 1.6 to 100 kHz in the absence of the singleturnover flash. A small induction curve occurred in N-limited control cells (Figure 25). Theamount of fluorescence induced by the 100 kHz measuring beam after treatments with DCMU,NH4+ anaerobiosis and CCCP was particularly pronounced when compared to the dark control(Figure 25 A, B, C,). These effects suggested that the 100 kHz measuring beam did, indeed,have an actinic effect in class 1 treated cells and caused reduction of the QA pool in excess ofthat due purely to redox equilibration between QA and a more reduced PQ pool. On the otherhand, NO3 - and N resulted in very little increase in fluorescence induced by the 100 kHzmeasuring beam as compared to the respective dark controls suggesting that the measuringbeam had a relatively small actinic effect (Figure 25 E, F). These results tend to confirm theproposal that the QA and PQ pool were relatively oxidized in class 2 treated cells while thesepools were more reduced in class 1 treated cells.The large increase in fluorescence induced by the 100 kHz measuring beam after DCMUand class 1 treatments was strongly correlated with the large difference between F and thequasi-plateau level reached in the fluorescence decays. That is, all decays from either control ortreated cells decayed to the level of fluorescence induced by the measuring beam within 40 msec(data not shown). This suggested that the observed decay curve was actually a sum of QA-oxidation after the single turnover flash and QA- reduction due to actinic effects of themeasuring beam. Thus, to resolve the kinetics of QA oxidation, the induction curve (due to the100 kHz measuring beam alone = QA reduction) was subtracted from the observed decay curve(the effects of the single turnover flash in the presence of the 100 kHz measuring beam = sum ofQA oxidation and QA reduction).Effects of correcting fluorescence decays:In comparison to uncorrected curves where the fluorescence decayed to a quasi-plateauwhich was much higher than Fo or F, correcting the fluorescence decay curves resulted in a176decay to 0 within 40 msec after all treatments (see Figure 8, Chapter 3). In addition, the half-time of the msec component of the q(t) decay was decreased in all cases to a half-time rangingfrom 3.5 to 8 msec (compare Table 3 and 8). This is much more consistent with the half-timesof the msec components observed in nutrient sufficient Chlamydomonas (2-11 msec)(Govindjee et al. , 1992) and in spinach and soybean (6-7 msec) (Cao and Govindjee, 1990).One significant effect of correcting the fluorescence decay for the actinic effects of themeasuring beam is that it changed the shape of the fluorescence decay curve in cells treated withCCCP, DCMU and NH4+ and resulted in a loss of the rapid fluorescence decay componentassociated with the uncorrected decay (compare table 3 and 8).The advantage of correcting the fluorescence decay for the actinic effects of themeasuring beam is that it was possible to isolate QA oxidation kinetics (as a result of the singleturnover flash) from QA reduction kinetics (due to the effects of the measuring beam). OnceQA oxidation kinetics were isolated, it was then possible to deduce the redox status of the PQpool on the basis of slower QA- re-oxidation kinetics after a variety of treatments whichincreased the rate of respiratory carbon flow. It is obvious from the above discussion thatextreme care must be taken when using fluorescence decays to determine rates of QA reductionand oxidation and, further, to infer the redox state of the intersystem electron transport chain.By virtue of the fact that light is being used to a) induce and b) measure fluorescence,inaccuracy in determination of QA reduction and oxidation kinetics is likely to occur unless theactinic effects of the measuring beam are corrected for.APPENDIX 3: METABOLITE ASSAYSMetabolite Assays:Metabolite assays were enzymatically coupled to the reduction or oxidation of pyridinenucleotides and measured at 334 nm on a Sigma dual wavelength spectrophotometer (ZFP22,Sigma Instruments, FRG). All assay reagents were provided by Boehringer Mannheim Co.Pyr, PEP: PEP and pyruvate were assayed using a 100 mM Tris buffer (pH 7.5) containing 5mM MgC12, 2.5 mM NADH and 5 mM ADP. Pyruvate was measured as NADH oxidation dueto pyruvate reduction to lactate and was started by lactate dehydrogenase (1U, BMC 127230).PEP was coupled to this reaction with pyruvate kinase (1.3 U, BMC 128155).Hexose phosphates and ATP (F6P, ATP, G6P, GIP): Hexose phosphates and ATP weremeasured sequentially in an assay mixture (100 mM Tris, pH 8.1; 5 mM MgC12) containing 2.5mM NADP and 10 mM glucose. G6P was coupled to NADP reduction via conversion of G6Pto 6PG by G6PDH (.14 U, BMC 127663). F6P was coupled to this reaction using phospho-glucose isomerase (0.7 U, BMC 128 139). G113 was coupled to the G6P reaction withphosphoglucomutase (0.4, BMC 108375). ATP was measured by coupling glucosephosphorylation via hexose kinase (.42 U, BMC 127809) to the G6PDH reaction.ADP, AMP: ADP and AMP were measured in a 50 mM Hepes buffer (pH 7.6, 10 mMKH2PO4; 2.5 mM NADH and 5 mM PEP and LDH (.4U BMC 127 230). PEP and ADP wereconverted to Pyr and ATP via pyruvate kinase (3 U, BMC 128 155) and coupled to pyruvateoxidation to lactate via lactate dehydrogenase. AMP was coupled to this reaction by conversionof AMP and ATP via myokinase (5U, BMC 107 506).177178TP and FBP: Triose phosphate was measured in a 100 mM Tris (pH 8.1; 5 mM MgC12; 2.5mM NADH). Triose phosphates were converted to glycerine 3-P using GDH/TPI (0.3 U, BMC127 787). FBP was coupled to this assay using aldolase (0.018U, BMC 102652).6PG: 6PG was measured by coupling 6PG oxidation to NADPH production in an assay mixcontaining .25 mM NADP and 100 mM Tris, pH 8.1 and 5 mM MgC12. The reaction wasstarted by adding 6PGDH (0.2 U, BMC 108 391).Glucose: Glucose was measured in an assay mix containing 100 mM Tris (pH 8.1); 5 mMMgC12; 0.25 mM NADP, 2.5 mM ATP and G6PDH (0.14 U, BMC 127663). Glucose wasphosphorylated to G6P by hexokinase (.42 U, BMC 127809) and coupled to G6P oxidation byG6PDH.

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