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Wingler A Lea PJ Quick WP et al (1 more author) (2000) Photorespiration metabolicpathways and their role in stress protection Philosophical Transactions Of The Royal Society Of London Series B - Biological Sciences 355 (1402) pp 1517-1529 ISSN 0962-8436

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Photorespiration metabolic pathways

and their role in stress protection

Astrid Wingler1 Peter J Lea2 W Paul Quick3 and Richard C Leegood3

1Department of Biology University College London Gower Street London WC1E 6BT UK2Department of Biological Sciences University of Lancaster Lancaster LA1 4YQ UK

3Robert Hill Institute and Department of Animal and Plant Sciences University of Shecurreneld Shecurreneld S10 2TN UK

Photorespiration results from the oxygenase reaction catalysed by ribulose-15-bisphosphate carboxylaseoxygenase In this reaction glycollate-2-phosphate is produced and subsequently metabolized in thephotorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate During this meta-bolic process CO2 and NH3 are produced and ATP and reducing equivalents are consumed thusmaking photorespiration a wasteful process However precisely because of this inecurrenciency photorespira-tion could serve as an energy sink preventing the overreduction of the photosynthetic electron transportchain and photoinhibition especially under stress conditions that lead to reduced rates of photosyntheticCO2 assimilation Furthermore photorespiration provides metabolites for other metabolic processes egglycine for the synthesis of glutathione which is also involved in stress protection In this review wedescribe the use of photorespiratory mutants to study the control and regulation of photorespiratory path-ways In addition we discuss the possible role of photorespiration under stress conditions such asdrought high salt concentrations and high light intensities encountered by alpine plants

Keywords drought stress glutamate synthase glutamine synthetase glycine decarboxylasehydroxypyruvate reductase serineglyoxylate aminotransferase

1 THE ORIGINS OF PHOTORESPIRATION

Photorespiration is a consequence of the oxygenation ofribulose-15-bisphosphate (RuBP) catalysed by RuBPcarboxylaseoxygenase (rubisco) The ratio of thecarboxylation rate (vc) to the oxygenation rate (vo) isdependent on the CO2 and O2 concentrations theMichaelis constants for these gases (Kc and Ko) and themaximal velocities (Vc and Vo) (Farquhar et al 1980)vcvo ˆVcKoVoKc ([CO2O2]) with the term VcKoVoKc

decentning the specicentcity factor of rubiscoUsing rubisco kinetics it is possible to calculate the

ratio of vc to vo and thereby estimate the rate of photo-respiration at diiexclerent CO2 concentrations (Sharkey1988) In order to calculate the rate of photorespiratoryCO2 release it has to be taken into account that one CO2

is released for every two oxygenation reactions (centgure 1)Sharkey (1988) estimated that under ambient conditionsthe rate of photorespiratory CO2 release is about 25 ofthe rate of net CO2 assimilation (A) With increasingtemperatures the specicentcity of rubisco for CO2 decreases(Brooks amp Farquhar 1985) and the solubility of CO2

decreases relative to that of O2 resulting in enhancedrates of photorespiration at high temperatures Due to thehigh rates of photorespiratory CO2 release photo-respiration is a wasteful process imposing a strong carbondrain on plants When the rate of photorespirationbecomes too high for example when the O2 concentra-tion is increased above the O2 compensation point for a

given CO2 concentration (Tolbert et al 1995) photo-respiration leads to a depletion of carbohydrates and toaccelerated senescence On the other hand long-termgrowth in low O2 (2 kPa) to suppress photorespirationappears to be detrimental to plants and results indecreased rates of photosynthetic CO2 assimilation(measured in air) poor plant growth and alterations in thechloroplast structure (Migge et al 1999) It has beensuggested that photorespiration is important for energydissipation to prevent photoinhibition (Osmond 1981Osmond amp Grace 1995 Osmond et al 1997 Kozaki ampTakeba 1996 Wu et al 1991) In addition photorespirationcan generate metabolites such as serine and glycine whichcan be exported out of the leaf (Madore amp Grodzinksi1984) or used in other metabolic pathways for exampleprovision of glycine for the synthesis of glutathione(Noctor et al 1997 1998 1999) Since glutathione is acomponent of the antioxidative system in plants (Noctor ampFoyer 1998) photorespiration may provide additionalprotection against oxidative damage in high light bysupplying glycine Thus photorespiration in addition tobeing wasteful may also be a useful process in plants

We have used a range of barley mutants with reducedphotorespiratory enzyme activities to study the followingaspects of photorespiratory metabolism (i) the controlexerted by photorespiratory enzymes on photosyntheticpoundux (ii) the eiexclect of photorespiratory metabolites onphotosynthetic metabolism (iii) regulation of the expres-sion of photorespiratory enzymes (iv) the occurrence ofalternative photorespiratory pathways and (v) the signif-icance of photorespiration under stress conditions

Phil Trans R Soc Lond B (2000) 355 1517^1529 1517 copy 2000 The Royal Society

doi 101098rstb20000712

Author for correspondence (rleegoodshecurreneldacuk)

2 PHOTORESPIRATORY METABOLISM

In the oxygenase reaction catalysed by rubisco onemolecule each of glycerate-3-phosphate and glycollate-2-phosphate are formed (centgure 1) Seventy-centve per cent ofthe carbon of glycollate-2-phosphate is recycled in thephotorespiratory pathway (Leegood et al 1995) In thispathway glycollate-2-phosphate is centrst hydrolysed toglycollate by a chloroplastic phosphoglycollate phospha-tase After transport into the peroxisomes glycollate isoxidized to glyoxylate by glycollate oxidase Glyoxylatecan be transaminated to glycine by serineglyoxylateaminotransferase (SGAT) or by glutamateglyoxylateaminotransferase Half of the glycine molecules areconverted to N5N10-methylene tetrahydrofolate (THF) inthe reaction catalysed by glycine decarboxylase (GDC) inthe mitochondria In this reaction CO2 and NH3 arereleased The other half of the glycine molecules can reactwith N5N10-methylene THF in the serine hydroxymethyl-transferase (SHMT) reaction to form serine After trans-port from the mitochondria to the peroxisomes serine isconverted by SGAT to hydroxypyruvate which isreduced to glycerate by hydroxypyruvate reductase (HPR)Glycerate is then phosphorylated by glycerate kinase inthe chloroplasts and the resulting glycerate-3-phosphate is

converted to RuBP in the Calvin cycle Due to thetransamination of glyoxylate to glycine and the formationof NH3 in the GDC reaction photorespiratory carbonmetabolism is intimately linked to nitrogen metabolism inthe leaf Since photorespiration proceeds at very highrates it has been estimated that the production of NH3 byphotorespiration is an order of magnitude greater thanthe primary assimilation of nitrogen resulting fromnitrate reduction (Keys et al 1978) Therefore thereassimilation of photorespired NH3 by plastidic gluta-mine synthetase (GS-2) and ferredoxin-dependent gluta-mate synthase (Fd-GOGAT) is essential for maintainingthe nitrogen status in plants This process is necessarilyvery ecurrencient the rate of NH3 emission in wild-type barleyis about 001 of the rate of photorespiratory NH3

release (Mattson et al 1997) assuming a rate of photo-respiratory CO2 release of 25 of A

(a) The use of mutants to study the control

of photorespiratory metabolism

The involvement of most of the enzymes in photo-respiratory metabolism has been concentrmed by work withthe respective mutants A mutant screen was centrst devisedby Somerville amp Ogren (1979) to isolate photorespiratorymutants of Arabidopsis It is based on the fact that

1518 AWingler and others Photorespiration and stressprotection

PhilTrans R Soc Lond B (2000)

malatemalate

2-OG 2-OG

Glu Glu

Gln

Glu

2 RuBP2 glycollate-2-P

2 glycollate 2 glycollate NAD malate

NADH

OH-pyruvate2 glyoxylate

2 glycine serine

2 glycine serine

OAA

NAD malate

NADH

CO2

NH3

OAA

glycerate glycerate

PEROXISOME

CHLOROPLAST MITOCHONDRION

3 glycerate-3-P

Calvincycle

ADP ATP

2Pi

H+

OH-

2O2

NH3

2O2

2H2O2

Figure 1 Photorespiratory metabolism shown with oxygenation of two molecules of RuBP This oxygenation gives rise to two

molecules of glycerate-3-P (C3) and two molecules of glycollate-2-P (C2) The latter are converted into a further molecule ofglycerate-3-P in the photorespiratory cycle Since one molecule of CO2 is liberated by GDC only nine carbons of the original ten

carbons in the two molecules of RuBP (C5) are recycled in the Calvin cycle Metabolite transporters are indicated by solid

rectangles though in certain cases these are speculative as for NH3 transport between organelles (although see Gazzarrini et al1999) and for glycine and serine transport across the mitochondrial membrane Glu glutamate Gln glutamine

2-OG 2-oxoglutarate OAA oxaloacetate

photorespiratory mutants are conditional lethals In high(4 02) CO2 photorespiration is suppressed and thegrowth of the photorespiratory mutants is indistinguish-able from the wild-type However when the mutants aretransferred from high CO2 into air they show severesymptoms of stress such as chlorosis After transferringthe plants back into high CO2 photorespiratory mutantsrecover This method was also used to isolate photo-respiratory mutants of barley (Kendall et al 1983) Overthe years Arabidopsis barley tobacco and pea mutantswith mutations in a large range of photorespiratoryenzymes and transporters (phosphoglycollate phospha-tase catalase SGAT GDC SHMT NADH-dependentHPR GS-2 Fd-GOGAT dicarboxylate transport) havebeen isolated (for reviews see Somerville 1986 Blackwellet al 1988 Leegood et al 1995)

While in the long term the homozygous photo-respiratory mutants are not viable at ambient CO2

concentrations heterozygotes can be grown in air Wehave used heterozygous mutants of GS-2 Fd-GOGATGDC and SGAT to study the control exerted by photo-respiratory enzymes on photosynthetic and photo-respiratory metabolism (HIgraveusler et al 1994ab 1996Wingler et al 1997 1999ab) The concept of control theorywas developed by Kacser amp Burns (1973) They showedthat the control of poundux through a metabolic pathway isshared by the enzymes of the pathway and that for eachenzyme a control coecurrencient (the fractional change in thepoundux through a pathway divided by the fractional changein the amount of enzyme) can be calculated As it is dicurren-cult to quantify the rate of photorespiration the controlexerted by photorespiratory enzymes on photorespirationcannot easily be determined Accumulation of substratesof the respective reactions can however serve as a centrstindication for an impairment of poundux through the photo-respiratory pathway In addition restrictions on oxygena-tion of RuBP should also aiexclect carboxylation so thatphotosynthetic poundux should be decreased in a mannersimilar to the photorespiratory poundux The eiexclects ofreduced photorespiratory enzyme activities can beexpected to be most severe under conditions that lead to ahigh rate of oxygenation of RuBP Such conditions arehigh light low external CO2 concentrations highexternal O2 concentrations high temperatures and stressconditions that lead to stomatal closure and a decline inintercellular CO2 concentrations (Ci) We thereforestudied the eiexclect of a variety of conditions on the perfor-mance of photorespiratory barley mutants

In contrast to the homozygous mutants heterozygotesshow only minor eiexclects of the lowered enzyme activitieson photosynthesis Of the heterozygous mutants studiedthe decrease in photosynthesis was most severe in plantswith reduced activities of Fd-GOGAT (HIgraveusler et al1994b) Even in moderate light and in ambient CO2these plants exhibited reduced rates of CO2 assimilationIn plants with reduced activities of GDC (Wingler et al

1997) and GS-2 (HIgraveusler et al 1994b) the eiexclect on CO2

assimilation was negligible in ambient CO2 but becamemore severe in low CO2 Plants with reduced activities ofSGAT on the other hand did not show a signicentcantreduction in CO2 assimilation even when photosynthesiswas measured under conditions of high rates of photo-respiration (high light and low CO2 Wingler et al 1999a)

However the rates of CO2 assimilation were reducedcompared with the wild-type when the stomata closedduring moderate drought stress (Wingler et al 1999b)Therefore photorespiratory enzymes such as SGAT andGDC that are `in excessrsquo under normal growth condi-tions can in the long term exert appreciable controlunder stress conditions that lead to increased rates ofphotorespiration

In plants with reduced photorespiratory enzyme activ-ities the following alterations could lead to reduced ratesof photosynthesis (i) an impairment of the recycling ofthe carbon in the photorespiratory pathway could resultin a depletion of Calvin cycle metabolites (ii) an impair-ment of photorespiratory nitrogen reassimilation couldresult in a decline in the nitrogen status of the leaf and areduction in the amount of photosynthetic proteins and(iii) accumulation of photorespiratory metabolites couldhave a feedback eiexclect on Calvin cycle activity

(i) First indications that reduced rates of photosynthesisin homozygous photorespiratory mutants are due toa depletion of metabolites were obtained byproviding carbon and nitrogen in metabolites thatcannot be formed at sucurrencient rates in the mutantsFor example the supply of glutamine to a GS-2mutant of barley (Blackwell et al 1987) of serine to aGDC mutant of barley (Blackwell et al 1990) or ofsucrose to cell cultures of an SGAT mutant ofNicotiana sylvestris (McHale et al 1989) partiallyrestored photosynthetic activity However the poolsof RuBP which should directly aiexclect photosynthesisappear to be very stable in the mutants Transfer ofthe homozygous barley mutant lacking GS-2 fromhigh CO2 into air did not lead to a decline in RuBPcontent (Leegood et al 1995) and heterozygousmutants with reduced activities of GS-2 SGAT orGDC did not contain less RuBP than wild-typeplants (Wingler et al 1999b) On the other handother metabolites of the Calvin cycle such as fructose-16-bisphosphate did decrease in the mutantsindicating a feedback regulation of rubisco activity

(ii) Since the photorespiratory pathway and nitrogenassimilation are closely linked one might expect thatreduced photorespiratory enzyme activities couldlead to a depletion of metabolically availablenitrogen Clear alterations in nitrogen metabolismbecame apparent in the heterozygous photo-respiratory mutants in the Fd-GOGAT mutants thecontent of glutamine increased while the content ofglutamate decreased (HIgraveusler et al 1994a) in theGS-2 mutants NH3 production increased while thecontent of glutamine decreased (HIgraveusler et al 1994aMattson et al 1997) in the SGAT mutants thecontent of serine increased (Wingler et al 1999ab)(centgure 2c) and in the GDC mutants the content ofglycine increased under conditions of high photo-respiratory poundux (Wingler et al 1997 1999b) In plantswith reduced activities of GS-2 and more severelyin plants with reduced activities of Fd-GOGAT theprotein content in the leaves and the total activity ofrubisco were reduced It is therefore likely that thereduced rates of photosynthesis in plants withreduced Fd-GOGAT activities were partly caused by

Photoresp iration and stress protection AWingler and others 1519

Phil Trans R Soc Lond B (2000)

lower amounts of photosynthetic enzymes due to areduced availability of nitrogen Plants with reducedactivities of SGATor GDC did not however exhibitchanges in the total protein content In order toincrease the possibility of a depletion of physio-logically available nitrogen plants with reducedSGAT activities were grown with a low supply ofnitrogen Under these conditions the protein content

was reduced to the same extent as in wild-typeplants and there was no eiexclect of reduced SGATactivities on photosynthesis (centgure 2ab) eventhough serine also accumulated under these condi-tions (centgure 2c)

(iii) An accumulation of metabolites could also directlylead to a feedback regulation of photosyntheticactivity This topic has been extensively discussed byLeegood et al (1995 1996) There is little evidencethat NH3 accumulating in the GS-2 mutants directlyinhibits photosynthesis by uncoupling of photo-synthetic electron transport This was shown byfeeding glutamate to homozygous GS-2 mutants(Blackwell et al 1987) Supply of external glutamateincreased the accumulation of NH3 while at thesame time it restored photosynthesis to wild-typerates Accumulation of serine is also unlikely toinhibit photosynthesis In cell cultures of the Nicotiana

sylvestris mutant lacking SGAT an almost ninefoldincrease in serine had no eiexclect on photosynthesiswhen sucrose was supplied as a carbon source toprevent the depletion of carbon stores (McHale et al1989) Of the metabolites we measured in themutants glyoxylate is the most likely to exert a feed-back eiexclect on photosynthesis In in vitro studies it hadbeen shown that glyoxylate can inhibit the activationof rubisco (Campbell amp Ogren 1990) Using hetero-zygous GS-2 mutants HIgraveusler et al (1996) haveshown that there is a negative relationship betweenthe glyoxylate content in the leaves and the activationstate of rubisco indicating that glyoxylate can act as afeedback inhibitor of photosynthesis in vivo Otherphotorespiratory metabolites that have been shown toinhibit enzymes of the Calvin cycle are glyceratewhich inhibits fructose-16-bisphosphatase andsedoheptulose-17-bisphosphatase (Schimkat et al1990) and glycollate-2-phosphate which inhibitstriose-phosphate isomerase (Anderson 1971) In addi-tion to inhibiting activities of certain enzymes it hasbeen proposed that photorespiratory metabolitesmight also act as signals in the regulation of theexpression of photorespiratory and other enzymes

3 REGULATION OF THE EXPRESSION

OF PHOTORESPIRATORY ENZYMES

Expression of most of the photorespiratory enzymesie glycollate oxidase catalase HPR SGAT P- H- and

1520 AWingler and others Photorespiration and stress protection


wt HNsgat HNwt LNsgat LN

(a)30

20

10

0

2000

PFD (mmol m-2

s-1

)

CO

2 a

ssim

ilati

on (

mm

ol m

-2 s-

1)

0 1000

(b)50

40

30

20

10

0

800 1000

Ci (ml l-1

)

CO

2 a

ssim

ilat

ion (

mm

ol m

-2 s-

1)

0 600200 400

(c)06

05

04

03

02

01

00200

sgat activity

(nmol min-1

mg-1

protein)

seri

ne

(mm

ol m

-2)

0 50 100 150

Figure 2 Eiexclect of high and low nitrogen supply on wild-type

barley (wt) and heterozygous mutants with reduced activitiesof SGAT (sgat) The plants were grown in a glasshouse with

high (5 mM NOiexcl

3 HN) or low (05 mM NOiexcl

3 LN) nitrogensupply (a) Relationship between the rate of CO2 assimilation

and photon poundux density (PFD) measured in 350 ml7 1 CO2

(n ˆ 3^4 lines sect se) (b) Relationship between the rate of CO2

assimilation and Ci measured at a PFD of 1213 mmolm7 2 s7 1

(n ˆ 3^4 lines sect se) (b) Serine contents in plants grown with

low (05 mM NOiexcl

3 ) nitrogen supply (n ˆ 4 leaves sect se)

wt

110

gdc

11

wt root

11

wt

1100

Figure 3 Western blot for H-protein of the GDC complex

H-protein was detected using an antiserum against H-protein

from wheat (provided by J Lorang and T Wolpert OregonState University USA) For leaves of the homozygous GDC

mutant of barley (gdc) and the roots of wild-type barley (wtroot) 1 mg fresh weight was loaded for leaves of wild-type

barley (wt) 01 mg fresh weight (110) and 001 mg fresh

weight (1100) were loaded

T-proteins of the GDC complex and SHMT is inducedby light (Raman amp Oliver 1997) Expression of the sub-units of the GDC complex has been studied in detailGDC is a mitochondrial multi-enzyme complex cata-lysing the conversion of glycine NAD and THF into CO2NH3 NADH and N5N10-methylene THF (Douce ampNeuburger 1999) The GDC complex is formed from P-H- T- and L-proteins with an approximate stoichiometryof 2 P-protein dimers27 H-protein monomers9 T-protein monomers1 L-protein dimer In wheat leaves theamounts of P- H- and T-proteins were highest in the leaftip and declined towards the base (Rogers et al 1991)showing that their expression is under developmentalcontrol Only the L-protein which is also a component ofthe pyruvate dehydrogenase complex was alreadypresent in the leaf base In pea the amounts of P- H-and T-proteins increased when etiolated seedlings weretransferred to the light (Turner et al 1993) During thedevelopment of pea leaves the expression of the genesencoding the P- H- and T-proteins of the GDC complexwas coordinated with the expression of the rubisco rbcS

genes (Vauclare et al 1996) However the mRNAsencoding the GDC proteins peaked before the GDCcomplex was formed while at this stage rubisco wasalready active This implies that post-transcriptionalregulation controls the formation of the GDC complexpossibly with a photorespiratory metabolite such asglycine acting as a signal In mutants with reducedactivities of GDC we have shown that the amount of P-protein was reduced in plants that had a content of H-protein that was lower than 60 of wild-type contentswhile the amounts of T- and L-proteins were normal(Blackwell et al 1990 Wingler et al 1997) This indicatesthat the mutation in this GDC mutant is probably in agene encoding H-protein and that the synthesis of P-protein is also regulated downwards when the formationof functional GDC complexes is limited by the avail-ability of H-protein After very long exposure H-proteinwas also visible in the leaves of the GDC mutant onWestern blots (centgure 3) Dilution of extracts from wild-type leaves revealed that the amount in the mutant wasabout 1 of that in the wild-type There was no diiexcler-ence in the content of H-protein in the roots of the GDCmutant compared with the wild-type These resultssuggest that in addition to the photorespiratory gene for

H-protein barley contains a second gene which isconstitutively expressed in roots and leaves but its func-tion is unknown

The other enzyme whose expression has been inten-sively analysed is NADH-dependent HPR Induction ofthe expression of the HPR gene in cucumber by lightinvolves a phytochrome-dependent component (Bertoniamp Becker 1993) In the dark expression of the HPR genecan be induced by cytokinin (Chen amp Leisner 1985Andersen et al 1996) This induction has been shown tobe at least in part due to transcriptional regulation Ithas also been suggested that photorespiratory metaboliteshave an eiexclect on the expression of the HPR gene Whenphotorespiration in cucumber plants was suppressed inhigh CO2 the HPR mRNA decreased (Bertoni amp Becker1996) There was however no eiexclect of high CO2 onHPR activity in pea (Thibaud et al 1995)

In tobacco we found that cytokinin can prevent thesenescence-dependent decline in HPR protein (Wingler et

al 1998) This was observed in naturally senescing plantswhich endogenously produced cytokinin by over-expression of isopentenyl transferase as well as in leafdiscs poundoating on cytokinin-containing solutions Glucoseon the other hand accelerated the decline in HPRprotein (centgure 4a) and it also overrode the eiexclect of cyto-kinin Thus the reduced expression of the HPR geneobserved in high CO2 (Bertoni amp Becker 1996) could bepart of the response of plants to sugar accumulationdescribed as sugar-mediated gene expression (Koch1996) In contrast the increase in sugar contents observedin drought-stressed barley (Wingler et al 1999b) did notlead to a decline in HPR protein Instead the amount ofHPR protein increased in drought-stressed leaves(centgure 4b) This was also the case in the SGAT and theGDC mutants suggesting that either a general drought-related signal or a metabolite formed in the photorespira-tory pathway before the GDC reaction (eg glycollate)could have acted as the signal

In contrast to their cytosolic counterparts the expres-sion of the plastidic isoforms of GS and GOGAT (GS-2and Fd-GOGAT) is not strongly regulated by nitrogensupply but is highly responsive to light (Hecht et al 1988Migge et al 1996 Migge amp Becker 1996) An involvementof photorespiratory signals in the regulation of theexpression of GS-2 has been suggested by Edwards amp

Photorespiration and stress protection AWingler and others 1521


wt

(b)(a)

gs sgat gdc

0day

H2O Glc Stl

13 0 13 0 13 0 13

Figure 4 Western blots for NADH-dependent HPR NADH-dependent HPR was detected using an antiserum against theNADH-dependent HPR of spinach (Kleczkowski et al 1990) (a) Eiexclect of glucose on the amount of NADH-dependent HPR

Discs of tobacco leaves were incubated for ten days poundoating on water 50 mM glucose (Glc) or 50 mM sorbitol (Stl) They were

kept at 23 8C and cycles of 16 h of light (20 mmol m7 2 s71) and 8 h of darkness A leaf area of 33 mm2 was loaded per lane(b) Eiexclect of drought stress on the amount of NADH-dependent HPR in wild-type barley (wt) and heterozygous mutants with

reduced activities of plastidic GS (gs) SGAT (sgat) or GDC (gdc) 0 and 13 days after withholding water The plants weregrown in cycles of 12 h light (460 mmol m7 2 s7 1) at 24 8C and 12 h darkness at 20 8C Eight micrograms of protein were loaded

per lane

Coruzzi (1989) because suppression of photorespirationin 2 CO2 led to a decrease in the GS-2 mRNA inpea Cock et al (1991) however were not able to detectany short-term eiexclect on the GS-2 mRNA whenPhaseolus vulgaris plants grown in high CO2 were trans-ferred into air as would have been expected if the expres-sion was under photorespiratory control Only in the longterm did plants growing in high CO2 show a lowerexpression of GS-2 than air-grown plants Growth ofArabidopsis or tobacco plants at a CO2 concentration of03 which is probably high enough to suppress photo-respiration did not aiexclect the amount of GS-2 mRNAcompared with plants grown in air (Beckmann et al 1997Migge et al 1997) Equally there was no eiexclect onFd-GOGAT mRNA in tobacco An involvement of photo-respiratory metabolites in the expression of GS-2 orFd-GOGAT is therefore questionable

4 PHOTORESPIRATION IN C4 PLANTS

In C4 plants CO2 is centrst centxed as bicarbonate by phos-phoenolpyruvate carboxylase (PEPC) in the mesophyllcells resulting in the formation of the C4 acids malate oraspartate After transport of these acids to the bundlesheath cells where rubisco is located CO2 is released inreactions catalysed by NADP-malic enzyme NAD-malicenzyme or phosphoenolpyrurate carboxylunase Becauseof the high resistance of the walls of bundle sheath cells tothe diiexclusion of CO2 the CO2-pumping mechanism of C4

plants results in a high CO2 concentration at the site ofrubisco and consequently in a strongly reduced rate ofRuBP oxygenation There are however indications thatphotorespiration also occurs in C4 plants It has beenshown that high concentrations of O2 can inhibit photo-synthesis and this inhibition becomes more severe whenthe external CO2 concentration is reduced belowambient The optimum partial pressure of O2 for photo-synthesis in C4 plants is however higher (5^10 kPa) (Daiet al 1993 1995 1996 Maroco et al 1997) than in C3

plants (about 2 kPa) due to a greater demand for ATPThe oxygen sensitivity of C4 photosynthesis is dependenton the plant species and on the leaf age Dai et al (1995)showed that photorespiration as indicated by the inhibi-tion of photosynthesis at supraoptimal O2 concentrationsis higher in young and senescing than in mature leaves ofmaize Within the NADP-malic enzyme and the NAD-malic enzyme subtypes of C4 plants the dicotyledonspecies tend to have higher rates of photorespiration thanthe monocotyledon species (Maroco et al 1997) This isprobably due to a higher degree of bundle sheath leaki-ness for CO2 in the dicotyledons For the NAD-malicenzyme type Amaranthus edulis the rate of photo-respiration was estimated by measuring NH3 productionin the presence of the GS inhibitor phosphinothricin inair compared with 07 CO2 (Lacuesta et al 1997) Itwas calculated that the rate of photorespiration accountedfor 6 of net photosynthesis

By isolating photorespiratory mutants of A edulisDever et al (1995) obtained further evidence that photo-respiration occurs to a signicentcant extent in this C4

species In a screen similar to that devised by Somervilleamp Ogren (1979) for the isolation of photorespiratorymutant of Arabidopsis it was possible to isolate A edulis

mutants of the C4 photosynthetic pathway as well asmutants lacking the ability to metabolize photo-respiratory glycine Similar to photorespiratory mutantsof C3 plants these glycine accumulators are only viable inhigh CO2 In air they did not only accumulate highconcentrations of glycine but also its precursor in photo-respiratory metabolism glyoxylate (Wingler et al 1999c)Furthermore the production of photorespiratory NH3which is formed in the GDC reaction was stronglyreduced in these mutants (Lacuesta et al 1997) On theother hand the rate of photorespiration calculated fromthe rate of NH3 production was increased to 48 of netphotosynthesis in an A edulis mutant lacking the C4

enzyme PEPC (Lacuesta et al 1997) probably becauseCO2 was directly assimilated by rubisco An increasedsensitivity to O2 (Maroco et al 1998a) and a lowerecurrenciency of electron transport for CO2 assimilation(Maroco et al 1998b) also indicated an increased rate ofphotorespiration in the PEPC mutant The resultsobtained for the A edulis mutants clearly demonstratethat even though C4 cycle activity leads to a reduction inthe rate of photorespiration this process is not completelyabolished in C4 plants

5 THE FLEXIBILITY OF PHOTORESPIRATORY

METABOLISM

The photorespiratory carbon and nitrogen cycles arenot completely closed (Givan et al 1988) and photo-respiratory metabolites can be provided as substrates forother processes (Keys 1999) Glycine produced in thephotorespiratory pathway can for example be used forthe synthesis of glutathione (Noctor et al 1997 1998 1999)or be exported out of the leaves (Madore amp Grodzinski1984) Some poundexibility in photorespiratory nitrogenmetabolism is achieved by the use of alternative aminodonors In addition to glutamate and serine alanine andasparagine provide amino nitrogen for the synthesis ofglycine (Betsche 1983 Ta amp Joy 1986) This is due to thelack of specicentcity of the respective aminotransferasesGlutamateglyoxylate aminotransferase also uses alanine(Nakamura amp Tolbert 1983) and SGAT also catalysesasparagineglyoxylate and serinepyruvate aminotrans-ferase reactions (Murray et al 1987)

There are several possible pathways for the metabolismof glyoxylate (Igamberdiev 1989) For example glyoxylatecanbe reduced back to glycollateby an NADPH-dependentHPR or an NADPH-dependent glyoxylate reductase(Tolbert et al 1970 Kleczkowski et al 1986 1990) oroxidized to oxalate (Richardson amp Tolbert 1961 Halliwellamp Butt 1974) Glyoxylate can also be oxidativelydecarboxylated to formate (Zelitch 1972 Igamberdiev et

al 1999) This decarboxylation of glyoxylate is probably anon-enzymatic reaction involving the H2O2 formed inthe glycollate oxidase reaction (Halliwell amp Butt 1974Grodzinski 1978 Oliver 1979 Walton amp Butt 1981) SinceH2O2 is usually rapidly degraded by catalase the rate ofCO2 formation by glyoxylate decarboxylation is likely tobe low compared with the rate of CO2 formation by GDC(Oliver 1981 Walton 1982 Yokota et al 1985a) butassumes importance in mutants lacking SGAT (Murray etal 1987) SHMT (Somerville amp Ogren 1981) or in plantswith decreased catalase activities (Brisson et al 1998)

1522 AWingler and others Photoresp iration and stress protection


Formate can be activated by reacting withTHF in the C1-THF synthase pathway which provides one-carbon unitsfor the synthesis of purines thymidylate methionine andformylmethionyl-tRNA (Cossins amp Chen 1997) Theenzymes involved in the C1-THF synthase pathway inplants are a monofunctional N10-formyl-THF synthetase(Nour amp Rabinowitz 1991 1992) and a bifunctionalN5N10-methylene-THF dehydrogenaseN5N10-methenyl-THF cyclohydrolase (Kirk et al 1995) The reactionscatalysed by these enzymes result in the formation ofN5N10-methylene-THF from formate Since N5N10-methylene-THF is incorporated into serine in the SHMTreaction formate instead of the N5N10-methylene-THFproduced in the GDC reaction can be used as an alter-native substrate for the formation of serine (Giiexclord ampCossins 1982ab Prabhu et al 1996) Together with the C1-THF synthaseSHMT pathway the oxidative decarboxy-lation of glyoxylate to formate could therefore form aGDC-independent bypass to the normal photorespiratorypathway as shown for Euglena gracilis (Yokota et al 1985b)

By using homozygous GDC mutants of barley andA edulis we studied the signicentcance of such a bypass inhigher plants (Wingler et al 1999c) In contrast to wild-type plants the mutants showed a light-dependent accu-mulation of glyoxylate and formate which was suppressedin high (07) CO2 After growth in air the activity andamount of N10-formyl-THF synthetase were increased inthe mutants compared with the wild-types A similarincrease in N10-formyl-THF synthetase was induced whenleaves were incubated with glycine under illuminationbut not in the dark In addition the barley mutant wascapable of incorporating [14C]formate and [2-14C]glycol-late into serine Since the GDC activity in the mutant(1 of wild-type activity Wingler et al 1997) (centgure 3)was too low to support the rate of serine formation fromglycollate the formation of serine must have occurred viaa GDC-independent pathway Together these results indi-cate that the mutants are able to bypass the normalphotorespiratory pathway by oxidative decarboxylation ofglyoxylate and formation of serine from formate therebypartially compensating for the lack of GDC activity Theadvantage of this alternative photorespiratory pathway isthe absence of NH3 release In wild-type plants therelease of photorespiratory NH3 is however very low( 5 05 nmol m72 s71) (Mattsson et al 1997) because NH3

is ecurrenciently reassimilated by GS-2 In barley mutants withreduced activities of GS-2 on the other hand the alterna-tive pathway could be the mechanism by which the loss ofnitrogen as NH3 would be reduced (HIgraveusler et al 1996)

6 ENERGY REQUIREMENT

OF PHOTORESPIRATORY METABOLISM

Oxygenation of 1mol RuBP results in the formation of1mol glycerate-3-phosphate and 1mol glycollate-2-phos-phate which is converted into 05 mol glycerate-3-phos-phate in the photorespiratory pathway (centgure 1) Thisconversion requires 05 mol ATP for the phosphorylationof glycerate to glycerate-3-phosphate and 05 mol ATPand 1mol reduced Fd (Fdred equivalent to 05 molNADPH) for the reassimilation of NH3 in the GS-GOGAT cycle (table 1) Furthermore the formation ofRuBP from the 15 mol glycerate-3-phosphate formed peroxygenation requires 225 mol ATP and 15 mol NADPHIn total 325 mol ATP and 2 mol NADPH are consumedper oxygenation Since 05 mol CO2 is released per mol ofO2 centxed and assuming a photorespiratory CO2 release of25 of A (Sharkey 1988) the vo equals 05 when the vc is125 and when A is 1 In the presence of photorespirationthe consumption of ATP and reducing equivalents perCO2 centxed (5375 mol ATP and 35 mol NADPH) is clearlyhigher than in the absence of photorespiration (3 molATP and 2 mol NADPH) When photosynthetic CO2

assimilation is reduced because of stomatal closure theratio of vo to vc rises and the energy requirement for CO2

assimilation becomes even higher In addition the CO2

released during photorespiration can be partially reassi-milated which would result in an additional energy sink

For the alternative photorespiratory pathway involvingoxidative decarboxylation of glyoxylate and formation ofserine from formate the carbon balance is the same as inthe normal pathway In this pathway 05 mol ATP arerequired in the 10-formyl-THF synthetase reaction and05 mol NADPH in the 510-methylene-THF dehydro-genase reaction This requirement is equivalent to theconsumption of 05 mol ATP and 1mol Fdred for thereassimilation of NH3 in the normal photorespiratorypathway In contrast to the normal pathway the alter-native pathway does not include the GDC reaction whichcan provide the NADH for the reduction of hydroxy-pyruvate (Hanning amp Heldt 1993) so that additionalreducing equivalents are required in the HPR reaction

7 PROTECTION AGAINST STRESS

Because of the high energy requirement of photo-respiratory metabolism it has been suggested that photo-respiration is important for maintaining electron poundow toprevent photoinhibition under stress conditions (Osmond



Table 1 Energy requirement of photosynthesis and photoresp iration

(Oxygenation plus carboxylation for vc ˆ 125 vo ˆ 05 and A ˆ 1 125(3 ATP + 2 NADPH)+ 05 (325 ATP + 2 NADPH)ˆ 5375 ATP + 35 NADPH)

carboxylation oxygenation

phosphorylation of glycerate ouml 05 ATPreassimilation of NH3 ouml 05 ATP + 1 Fdred (05 NADPH)

Calvin cycle 3 ATP + 2 NADPH 225 ATP + 15 NADPHsum 3 ATP + 2 NADPH 325 ATP + 2 NADPH

1981 Wu et al 1991 Osmond amp Grace 1995 Kozaki ampTakeba 1996 Osmond et al 1997) In the main twodiiexclerent approaches have been employed to study thecontribution of photorespiration to the protection of thephotosynthetic apparatus (i) Photorespiration has beenreduced at the site of rubisco by reducing the O2 concen-tration to between 1 and 2 At these O2 concentra-tions the Mehler reaction is still active while due to itshigher Km the oxygenase reaction of rubisco is stronglyreduced (Osmond 1981) (ii) In other studies the role ofphotorespiration has been analysed in plants in whichphotorespiratory enzyme activities were reduced byfeeding inhibitors by mutagenesis or by antisense trans-formation Since photorespiration per se (ie oxygenationof RuBP and formation of glycollate-2-phosphate) is notdirectly aiexclected by reducing the activities of enzymes ofthe photorespiratory pathway it is not possible toconclude from such experiments whether or not thephotosynthetic apparatus of plants is protected by photo-respiration Instead an impairment of the poundux throughthe photorespiratory pathway can lead to photoinhibitionby inhibiting photosynthetic CO2 assimilation caused byan insucurrencient regeneration of RuBP or by an accumula-tion of toxic metabolites The best approach to study therole of photorespiration in photoprotection would be toalter the properties of rubisco (eg Bainbridge et al 1995Whitney et al 1999) with the aim of abolishing the oxyge-nase activity without inpounduencing the carboxylase activityHowever this goal has proved dicurrencult to achieve

In addition to the energy consumption byphotorespiration energy can also be dissipated byelectron transport to O2 in the Mehler-peroxidasepathway or as heat by non-photochemical quenchingNon-photochemical quenching is linked to the formationof zeaxanthin in the xanthophyll cycle and an increasedproton gradient across the thylakoid membrane (Rubanamp Horton 1995) Since the Mehler-peroxidase pathwayresults in an increased acidicentcation of the thylakoidlumen it can also lead to increased energy dissipation bynon-photochemical quenching (Schreiber amp Neubauer1990)

(a) Drought stress

In drought-stressed leaves CO2 assimilation isdecreased resulting in a reduced electron consumption byphotosynthesis Consequently the importance of mechan-isms protecting the photosynthetic apparatus is increasedUnder conditions of mild to moderate drought stress thedecline in photosynthesis mainly results from lower Ci

caused by stomatal closure (Kaiser 1987 Cornic ampBriantais 1991 Quick et al 1992 Lal et al 1996 Sanchez-Rodr|guez et al 1999) When the stomatal control ofphotosynthesis is overcome for example by removing thelower epidermis or by increasing the external CO2

concentration CO2 assimilation can be restoredsuggesting that at least during mild drought photo-synthesis is not inhibited due to damage to the photo-synthetic apparatus (Cornic 2000) Under theseconditions extractable activities or activation states ofphotosynthetic enzymes such as rubisco chloroplastfructose-16-bisphosphatase or NADP-dependent malatedehydrogenase do not typically decrease (Sharkey ampSeemann 1989 Lal et al 1996 Sanchez-Rodr|guez et al

1999 Wingler et al 1999b) In the long term droughtstress has however been shown to result in lowerfructose-16-bisphosphatase activities in Casuarina

equisetifolia (Sanchez-Rodr|guez et al 1999) and severedrought stress led to a decline in the amounts ofsedoheptulose-17-bisphosphatase and NADP-dependentglyceraldehyde-3-phosphate dehydrogenase proteins inbarley (Wingler et al 1999b) The amounts of photo-respiratory enzyme proteins (proteins of the GDCcomplex GS-2 SGAT) were not aiexclected by droughtstress and the amount of NADH-dependent HPR evenincreased (centgure 4b) In combination the decline in Ci

and sustained activities of rubisco and photorespiratoryenzymes are likely to result in increased rates of photore-spirationoumlnot only relative to photosynthesis but also inabsolute terms Therefore photorespiration could serve asan important means to maintain electron poundow

Studies conducted with diiexclerent species under a varietyof conditions provide partly contradictory data on the roleof photorespiration during drought stress For exampleBiehler amp Fock (1996) showed that gross uptake of O2

increased with increasing water decentcit in wheat The abso-lute rate of photorespiration determined as the rate ofglycollate formation was however decreased at a waterpotential (Aacute) of 726 MPa This suggests that at this Aacuteelectron poundow to O2 was increased due to the Mehler reac-tion and not due to photorespiration Since the droughtstress in this study was quite severe reactions of the Calvincycle might have been inhibited which could result inreduced contents of RuBP (Sharkey amp Seemann 1989)and consequently in lower rates of RuBP oxygenationBrestic et al (1995) found that electron transport in theleaves of drought-stressed Phaseolus vulgaris plants wasreduced by switching from 21 to 2 oxygen demon-strating that photorespiration serves as an electron sink indehydrated leaves Incubation in 2 oxygen in the lighthowever did not aiexclect the parameter FvFm measuredafter 20 min dark adaptation Accordingly it can beconcluded that photorespiration is not essential forprotecting the photosynthetic apparatus against photo-inhibition under drought stress

In our studies we used heterozygous barley mutants ofchlorophyll pounduorescence which contained approximately50 of wild-type activities of the photorespiratoryenzymes GS-2 GDC and SGAT to study the role ofphotorespiration during drought stress (Wingler et al1999b) These mutants have normal rates of photo-synthesis in moderate light and in ambient CO2 In lowCO2 on the other hand photosynthesis is reduced in theGS and GDC mutants The rationale behind our studywas that if photorespiration is increased in dehydratedleaves photosynthesis should decline to a greater extentin the mutants than in the wild-type with increasingdrought stress and the control exerted by the photo-respiratory enzymes on photosynthesis should increase Inaddition it appeared possible that a further down-regulation of photosynthetic CO2 assimilation due torestrictions in the photorespiratory poundux could lead tophotoinhibition

In well-watered plants reduced activities of GS-2GDC or SGAT did not aiexclect photosynthesis Droughtstress was induced by withholding water over a period oftwo weeks and the decrease in leaf Aacute was monitored



daily With decreasing Aacute rates of CO2 assimilationdeclined almost linearly in the wild-type (centgure 5a) Inthe mutants with reduced activities of photorespiratoryenzymes this decline was accelerated resulting in lowerrates of CO2 assimilation at moderate drought stress Thecontrol exerted by photorespiratory enzymes on photo-synthesis was therefore increased in moderately drought-stressed leaves However under severe drought stress therates of CO2 assimilation were equally low in the wild-type and in the mutants The Ci-values determined by gasexchange measurements declined in all plants withincreasing drought stress until a Aacute of about 715 MPawas reached (centgure 5b) When desiccation became moresevere the relationship between Aacute and Ci broke downand the estimated Ci-values rose again This phenomenonie constant or even increased Ci-values calculated fromgas exchange data is often encountered in severelydrought-stressed leaves (Cornic et al 1989 Lal et al 1996Sanchez-Rodr|guez et al 1999) During severe droughtstress Ci-values are often overestimated due to thepatchiness of stomatal closure (Sharkey amp Seemann 1989)and altered lateral diiexclusion of CO2 (Lal et al 1996) vc ascalculated from the Ci data and the CO2 assimilationrates decreased with increasing drought stress (centgure 5c)As shown for the assimilation rates vc was higher indrought-stressed leaves of the wild-type than in leavesof the mutants The calculated values for vo rose until a Aacute of7 15 MPa was reached (centgure 5d ) As a consequence ofthe questionable Ci-values the relationship between Aacute

and vo broke down when drought stress became moresevere Together with the lower rates of photosynthesisthe calculated values for vo indicate that during moderatedrought stress when the calculation of Ci was probablystill valid photorespiration was increased This was alsoconcentrmed by an increase in glycine contents in drought-stressed leaves of the GDC mutant (Wingler et al 1999b)

The lower rates of photosynthesis in the heterozygousmutants were accompanied by decreased quantum ecurren-ciencies of photosystem (PS) II electron transport Thisdecreased electron consumption in photosynthesis andphotorespiration in the mutants did not lead to a declinein FvFm which would have indicated chronic photoinhi-bition Instead energy dissipation by non-photochemicalquenching increased In the SGAT and GDC mutantsthis was accompanied by a strong increase in the forma-tion of zeaxanthin As shown by Brestic et al (1995) andDemmig et al (1988) xanthophyll-cycle-dependent energydissipation seems to be an important mechanism forprotecting against the deleterious eiexclect of light indrought-stressed leaves

(b) Salt stress

Under conditions of high salinity plants encountersimilar problems to those during drought stress Initiallyphotosynthesis is inhibited by closure of the stomata andthe resulting decrease in Ci while non-stomatal eiexclectsbecome limiting when the chloride concentrationincreases further (Walker et al 1981 Downton et al 1990

Photoresp iration and stressprotection AWingler and others 1525


250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30

leaf water potential (MPa)

Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10

Figure 5 (a) A (b) Ci (c) vc and (d ) vo with developing drought stress in wild-type barley (closed circles) and heterozygous

mutants with reduced activities of plastidic GS (open squares) SGAT (open triangles) or GDC (inverted triangles) The plantswere grown in cycles of 12 h light (460 mmol m7 2 s7 1) at 24 8C and 12 h darkness at 20 8C The data were calculated according to

Von Caemmerer amp Farquhar (1981) assuming a day respiration of 045 mmol m7 2 s7 1 and a CO2 compensation point of304 ml l7 1 (HIgraveusler et al 1996)

Fedina et al 1994) As for drought-stressed leaves severesalt stress can result in non-uniform closure of the stomataand patchiness of photosynthetic CO2 assimilation(Downton et al 1990) Several lines of evidence suggestthat stomatal closure in moderately salt-stressed leavesleads to enhanced rates of photorespiration The followingparameters all indicative of higher rates of photo-respiration have been shown to increase the CO2

compensation point (Fedina et al 1993 1994) the light todark ratio of CO2 production (Rajmane amp Karagde1986) the stimulation of photosynthesis by lowering theO2 concentration (Walker et al 1981 Fedina et al 1994)the activity of glycollate oxidase (Rajmane amp Karadge1986 Fedina et al 1993 1994) and the formation of photo-respiratory metabolites such as glycine serine and glycol-late (Downton 1977 Rajmane amp Karadge 1986 DiMartino et al 1999) The maintenance of considerablerates of electron transport in CO2-free air also indicates asignicentcant occurrence of photorespiration in salt-stressedleaves (Di Martino et al 1999) In addition to sustainedrates of electron transport due to photorespiration theformation of zeaxanthin during salt stress probably alsomitigates against photoinhibitory damage although thisprotection by zeaxanthin is not complete in high light(Sharma amp Hall 1992)

(c) Light stress in alpine plants

Plants are particularly prone to photoinhibition whenthey are exposed to high light in combination withdrought or low temperatures conditions mainly encoun-tered by plants growing at high altitudes These plantspecies are often highly resistant to photoinhibition andemploy diiexclerent protective strategies For Chenopodium

bonus- henricus grown in the Alps Heber et al (1996) foundthat photorespiration is essential for maintaining electronpoundow in high light whereas the Mehler-peroxidasepathway is insucurrencient to prevent photoinactivationSignicentcant rates of electron poundow were maintained whenphotosynthesis was inhibited by cutting the petioles orsubmerging the leaves in water On the other hand whenphotosynthesis and photorespiration were inhibited byfeeding HCN or glyceraldehyde the sensitivity of PS II tosunlight increased At very low temperatures photo-respiratory metabolism is greatly reduced and cannotmake a signicentcant contribution to photoprotection Forthe alpine plant Geum montanum it was suggested that atlow temperatures cyclic electron transport around PS Iresults in a decrease in pH in the thylakoid lumen andprobably in combination with zeaxanthin formation toincreased energy dissipation at the site of PS II (Manuelet al 1999) A strong dependence on the operation of thexanthophyll cycle in high light and at low temperatureshas also been proposed for Soldanella alpina (Streb et al1998) Ranunculus glacialis on the other hand was lessdependent on the formation of zeaxanthin while feedingof the GS inhibitor phosphinothricin at low temperaturesresulted in photoinhibition This points to a signicentcantpoundux through the photorespiratory pathway even at lowtemperatures As photorespiration was however notblocked at the site of RuBP oxygenation it cannot beestablished from this experiment that photorespirationitself has a protective function Instead accumulation ofcompounds such as NH3 or glyoxylate or a depletion of

Calvin cycle intermediates could have led to secondaryeiexclects by for example reducing CO2 assimilation Inaddition to the mechanisms discussed above an increasedimportance of the antioxidant system comprising ascor-bate a-tocopherol and glutathione has been proposed toprotect alpine plants against photo-oxidation (Heber et al

1996 Streb et al 1997 1998) Since the glycine requiredfor the formation of glutathione is mainly provided byphotorespiration photorespiration could play an addi-tional role in the protection of alpine plants

8 CONCLUSION

Photorespiratory metabolism is not only a wastefulprocess inevitably resulting from the kinetic properties ofrubisco but precisely because of this inecurrenciency is alsoinvolved in stress protection In addition photorespira-tory metabolism can generate metabolites such asglycine serine or one-carbon units for other processes inplants Abolishing photorespiration by engineeringrubisco may therefore not necessarily lead to improvedplant performance especially under unfavourable growthconditions

Our own research described in this paper was supported by theBiotechnology and Biological Sciences Research Council UK(research grants PG50555 and PO3666) We also thank VickiAnn for excellent technical assistance

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Heber U 1999 Protection against photoinhibition in thealpine plant Geum montanum Oecologia 119 149^158

Maroco J P Ku M S B amp Edwards G E 1997 Oxygensensitivity of C4 photosynthesis evidence from gas exchange

and chlorophyll pounduorescence analyses with diiexclerent C4

subtypes Plant Cell Environ 20 1525^1533Maroco J P Ku M S B Lea P J Dever L V Leegood

R C Furbank R T amp Edwards G E 1998a Oxygenrequirement and inhibition of C4 photosynthesis An analysis

of C4 plants decentcient in the C3 and C4 cycles Plant Physiol

116 823^832Maroco J P Ku M S B Furbank R T Lea P J

Leegood R C amp Edwards G E 1998b CO2 and O2 depen-dence of PS II activity in C4 plants having genetically

produced decentciencies in the C3 or C4 cycle Photosynth Res 5891^101

Mattsson M HIgraveusler R E Leegood R C Lea P J amp

Schjoerring J K 1997 Leaf-atmosphere NH3 exchange inbarley mutants with reduced activities of glutamine synthe-

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Migge A amp Becker T W 1996 In tobacco leaves the genesencoding the nitrate reducing or the ammonium-assimilating

enzymes are regulated diiexclerently by external nitrogen-sources Plant Physiol Biochem 34 665^671

Migge A Meya G Carrayol E Hirel B amp Becker T W1996 Regulation of the subunit composition of tomato plas-

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Planta 200 213^220Migge A Carrayol E Kunz C Hirel B Fock H amp

Becker T 1997 The expression of the tobacco genes encodingplastidic glutamine synthetase or ferredoxin-dependent gluta-

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Migge A Kahmann U Fock H P amp Becker T W 1999Prolonged exposure of tobacco to a low oxygen atmosphere to

suppress photorespiration decreases net photosynthesis and

results in changes in plant morphology and chloroplast struc-ture Photosynthetica 36 107^116

Murray A J S Blackwell R D Joy K W amp Lea P J 1987Photorespiratory N donors aminotransferase specicentcity and

photosynthesis in a mutant of barley decentcient in serineglyoxylate aminotransferase activity Planta 172 106^113

NakamuraY amp Tolbert N E 1983 Serineglyoxylate alanine-

glyoxylate and glutamateglyoxylate aminotransferasereactions in peroxisomes from spinach leaves J Biol Chem

258 7631^7638Noctor G amp Foyer C H 1998 Ascorbate and glutathione

keeping active oxygen under control A Rev Plant Physiol

Plant Mol Biol 49 249^279Noctor G Arisi A-C M Jouanin L Valadier M-H Roux

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Noctor G Arisi A-C M Jouanin L Kunert K J

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Photorespiratory glycine enhances glutathione accumulation

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Osmond C B Badger M Maxwell K BjIcircrkman O amp

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Arch Biochem Biophys 150 698^707



Photorespiration metabolic pathways

and their role in stress protection

Astrid Wingler1 Peter J Lea2 W Paul Quick3 and Richard C Leegood3

1Department of Biology University College London Gower Street London WC1E 6BT UK2Department of Biological Sciences University of Lancaster Lancaster LA1 4YQ UK

3Robert Hill Institute and Department of Animal and Plant Sciences University of Shecurreneld Shecurreneld S10 2TN UK

Photorespiration results from the oxygenase reaction catalysed by ribulose-15-bisphosphate carboxylaseoxygenase In this reaction glycollate-2-phosphate is produced and subsequently metabolized in thephotorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate During this meta-bolic process CO2 and NH3 are produced and ATP and reducing equivalents are consumed thusmaking photorespiration a wasteful process However precisely because of this inecurrenciency photorespira-tion could serve as an energy sink preventing the overreduction of the photosynthetic electron transportchain and photoinhibition especially under stress conditions that lead to reduced rates of photosyntheticCO2 assimilation Furthermore photorespiration provides metabolites for other metabolic processes egglycine for the synthesis of glutathione which is also involved in stress protection In this review wedescribe the use of photorespiratory mutants to study the control and regulation of photorespiratory path-ways In addition we discuss the possible role of photorespiration under stress conditions such asdrought high salt concentrations and high light intensities encountered by alpine plants

Keywords drought stress glutamate synthase glutamine synthetase glycine decarboxylasehydroxypyruvate reductase serineglyoxylate aminotransferase

1 THE ORIGINS OF PHOTORESPIRATION

Photorespiration is a consequence of the oxygenation ofribulose-15-bisphosphate (RuBP) catalysed by RuBPcarboxylaseoxygenase (rubisco) The ratio of thecarboxylation rate (vc) to the oxygenation rate (vo) isdependent on the CO2 and O2 concentrations theMichaelis constants for these gases (Kc and Ko) and themaximal velocities (Vc and Vo) (Farquhar et al 1980)vcvo ˆVcKoVoKc ([CO2O2]) with the term VcKoVoKc

decentning the specicentcity factor of rubiscoUsing rubisco kinetics it is possible to calculate the

ratio of vc to vo and thereby estimate the rate of photo-respiration at diiexclerent CO2 concentrations (Sharkey1988) In order to calculate the rate of photorespiratoryCO2 release it has to be taken into account that one CO2

is released for every two oxygenation reactions (centgure 1)Sharkey (1988) estimated that under ambient conditionsthe rate of photorespiratory CO2 release is about 25 ofthe rate of net CO2 assimilation (A) With increasingtemperatures the specicentcity of rubisco for CO2 decreases(Brooks amp Farquhar 1985) and the solubility of CO2

decreases relative to that of O2 resulting in enhancedrates of photorespiration at high temperatures Due to thehigh rates of photorespiratory CO2 release photo-respiration is a wasteful process imposing a strong carbondrain on plants When the rate of photorespirationbecomes too high for example when the O2 concentra-tion is increased above the O2 compensation point for a

given CO2 concentration (Tolbert et al 1995) photo-respiration leads to a depletion of carbohydrates and toaccelerated senescence On the other hand long-termgrowth in low O2 (2 kPa) to suppress photorespirationappears to be detrimental to plants and results indecreased rates of photosynthetic CO2 assimilation(measured in air) poor plant growth and alterations in thechloroplast structure (Migge et al 1999) It has beensuggested that photorespiration is important for energydissipation to prevent photoinhibition (Osmond 1981Osmond amp Grace 1995 Osmond et al 1997 Kozaki ampTakeba 1996 Wu et al 1991) In addition photorespirationcan generate metabolites such as serine and glycine whichcan be exported out of the leaf (Madore amp Grodzinksi1984) or used in other metabolic pathways for exampleprovision of glycine for the synthesis of glutathione(Noctor et al 1997 1998 1999) Since glutathione is acomponent of the antioxidative system in plants (Noctor ampFoyer 1998) photorespiration may provide additionalprotection against oxidative damage in high light bysupplying glycine Thus photorespiration in addition tobeing wasteful may also be a useful process in plants

We have used a range of barley mutants with reducedphotorespiratory enzyme activities to study the followingaspects of photorespiratory metabolism (i) the controlexerted by photorespiratory enzymes on photosyntheticpoundux (ii) the eiexclect of photorespiratory metabolites onphotosynthetic metabolism (iii) regulation of the expres-sion of photorespiratory enzymes (iv) the occurrence ofalternative photorespiratory pathways and (v) the signif-icance of photorespiration under stress conditions

Phil Trans R Soc Lond B (2000) 355 1517^1529 1517 copy 2000 The Royal Society

doi 101098rstb20000712

Author for correspondence (rleegoodshecurreneldacuk)










malatemalate

2-OG 2-OG

Glu Glu

Gln

Glu



NADH


2 glycine serine

2 glycine serine

OAA

NAD malate

NADH

CO2

NH3

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glycerate glycerate

PEROXISOME


3 glycerate-3-P

Calvincycle

ADP ATP

2Pi

H+

OH-

2O2

NH3

2O2

2H2O2




























(a)30

20

10

0

2000

PFD (mmol m-2

s-1

)

CO

2 a

ssim

ilati

on (

mm

ol m

-2 s-

1)

0 1000

(b)50

40

30

20

10

0

800 1000

Ci (ml l-1

)

CO

2 a

ssim

ilat

ion (

mm

ol m

-2 s-

1)

0 600200 400

(c)06

05

04

03

02

01

00200

sgat activity

(nmol min-1

mg-1

protein)

seri

ne

(mm

ol m

-2)

0 50 100 150



high (5 mM NOiexcl







low (05 mM NOiexcl


wt

110

gdc

11

wt root

11

wt

1100
















wt

(b)(a)

gs sgat gdc

0day

H2O Glc Stl

13 0 13 0 13 0 13





per lane











METABOLISM



























(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

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0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526




















malatemalate

2-OG 2-OG

Glu Glu

Gln

Glu



NADH


2 glycine serine

2 glycine serine

OAA

NAD malate

NADH

CO2

NH3

OAA

glycerate glycerate

PEROXISOME


3 glycerate-3-P

Calvincycle

ADP ATP

2Pi

H+

OH-

2O2

NH3

2O2

2H2O2




























(a)30

20

10

0

2000

PFD (mmol m-2

s-1

)

CO

2 a

ssim

ilati

on (

mm

ol m

-2 s-

1)

0 1000

(b)50

40

30

20

10

0

800 1000

Ci (ml l-1

)

CO

2 a

ssim

ilat

ion (

mm

ol m

-2 s-

1)

0 600200 400

(c)06

05

04

03

02

01

00200

sgat activity

(nmol min-1

mg-1

protein)

seri

ne

(mm

ol m

-2)

0 50 100 150



high (5 mM NOiexcl







low (05 mM NOiexcl


wt

110

gdc

11

wt root

11

wt

1100
















wt

(b)(a)

gs sgat gdc

0day

H2O Glc Stl

13 0 13 0 13 0 13





per lane











METABOLISM



























(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526

































(a)30

20

10

0

2000

PFD (mmol m-2

s-1

)

CO

2 a

ssim

ilati

on (

mm

ol m

-2 s-

1)

0 1000

(b)50

40

30

20

10

0

800 1000

Ci (ml l-1

)

CO

2 a

ssim

ilat

ion (

mm

ol m

-2 s-

1)

0 600200 400

(c)06

05

04

03

02

01

00200

sgat activity

(nmol min-1

mg-1

protein)

seri

ne

(mm

ol m

-2)

0 50 100 150



high (5 mM NOiexcl







low (05 mM NOiexcl


wt

110

gdc

11

wt root

11

wt

1100
















wt

(b)(a)

gs sgat gdc

0day

H2O Glc Stl

13 0 13 0 13 0 13





per lane











METABOLISM



























(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526




















wt

(b)(a)

gs sgat gdc

0day

H2O Glc Stl

13 0 13 0 13 0 13





per lane











METABOLISM



























(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526





















METABOLISM



























(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526
































(a) Drought stress
















(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526














(b) Salt stress




250

(b)

(a)

200

150

100

12

16

8

4

0

50

0-30


Ci (m

l l-

1)

CO

2 a

ssim

ilation (

mm

ol m

-2 s-

1)

0 -20-10

16

(d)

(c)

12

8

4

16

12

20

8

4

0

0-30


vo (

mm

ol m

-2 s-

1)

vc

(mm

ol m

-2 s-

1)

0 -20-10











8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526


















8 CONCLUSION



REFERENCES



Planta 198 1^5


237^244




























































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526



















































Phytol 116 499^503

















217^224























505^511






Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526














Nature 275 741^743







653^659







































































J Exp Bot 46 1351^1362



119^121

































833^836









Planta 169 117^122































207 518^526









































833^836









Planta 169 117^122































207 518^526











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