Light Energy Dissipation under Water Stress Conditions

Plant Physiol. (1990) 92, 1053-10610032-0889/90/92/1 053/09/$01 .00/0

Received for publication August 9, 1989and in revised form November 24, 1989

Light Energy Dissipation under Water Stress Conditions

Contribution of Reassimilation and Evidence for Additional Processes

Thomas Stuhifauth, Ralph Scheuermann, and Heinrich P. Fock*Fachbereich Biologie, Universitat Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern,

Federal Republic of Germany

ABSTRACT

Using 14CO2 gas exchange and metabolite analyses, stomatalas well as total intemal CO2 uptake and evolution were estimated.Pulse modulated fluorescence was measured during inductionand steady state of photosynthesis. Leaf water potential of Digi-talis lanata EHRH. plants decreased to -2.5 megapascals afterwithholding irrigation. By osmotic adjustment, leaves remainedturgid and fully exposed to irradiance even at severe water stress.Due to the stress-induced reduction of stomatal conductance, thestomatal CO2 exchange was drastically reduced, whereas thetotal CO2 uptake and evolution were less affected. Stomatalclosure induced an increase in the reassimilation of internallyevolved CO2. This 'CO2 recycling' consumes a significant amountof light energy in the form of ATP and reducing equivalents. As aconsequence, the metabolic demand for light energy is onlyreduced by about 40%, whereas net photosynthesis is diminishedby about 70% under severe stress conditions. By CO2 recycling,carbon flux, enzymatic substrate turnover and consumption oflight energy were maintained at high levels, which enabled theplant to recover rapidly after rewatering. In stressed D. lanataplants a variable fluorescence quenching mechanism, termed'coefficient of actinic light quenching,' was observed. Besideswater conservation, light energy dissipation is essential andinvolves regulated metabolic variations.

The restriction of water supply to the roots of a plantinduces water-conservation activities. By osmotic adjustmentsome plants are able to maintain water supply and turgidityfor a prolonged period. In Digitalis lanata the accumulationof abscisic acid triggers stomatal closure and reduces transpi-ration (29). Nevertheless, increases in quantum flux belowsaturation level do still, to some extent, induce stomatalopening and enhance gas exchange, even at severe stress levels(30).The attempt to conserve water by a reduction of stomatal

gas exchange involves the potential for further damage: lesslight energy can be utilized by CO2 assimilation, as transpi-ration and CO2 uptake are concomitantly decreased; thepotentially resulting overreduction ofthe photosynthetic elec-tron transport chain might cause photoinhibitory damage (5,6, 8). Thus, the potential of a plant to dissipate excess lightenergy may contribute to its ability to withstand water stress.The observation that the stomatal conductivity is relatively

more reduced than the activity of Calvin cycle enzymes, led

to the assumption that stomatal effects might dominate overmesophyll effects in the restriction of photosynthesis. Alter-natively, it has been postulated (23) that an internal CO2 cycleis activated, which consumes excess light energy and preventsthe leaf from photoinhibition by the refixation of internallyproduced CO2. This 'CO2 recycling' might explain why theenzymatic CO2-fixation activity of the Calvin cycle is lessreduced than stomatal aperture: additional enzymatic capac-ity is required for the refixation of photorespired CO2. Anincreased RuBP (Table I) oxygenation, dissipating light energyas ATP and reduction equivalents, can only be induced by analtered internal 02/CO2 ratio. However, the intercellular oxy-gen concentration is hardly affected by stomatal closure (16),and previous reports of the constancy of the intercellular CO2concentration (29, 30) contradict the assumption of photores-piratory enhancement. However, recent reports of a 'patchy'distribution of stomatal aperture over a single leaf ( 13, 21) ledto the conclusion that in many cases reports of constant C1are erroneous.The aim of our investigations was to characterize the proc-

esses triggered as a response to water stress in the C3 plant D.lanata. For that purpose, transpiration, leaf conductance andnet photosynthesis were measured in an open gas exchangesystem. We monitored the distribution of stomatal apertureover the leaf area by autoradiography (13). The CO2 fluxesover the stomata were estimated by the 12CO2/'4CO2-method(9). Reassimilation of CO2 can be calculated from gas ex-change data according to the equations ofGerbaud and Andre(16). The carbon flux through the glycolate pathway wasestimated directly by the newly developed analyses of thespecific radioactivities of phosphoglycolate and glycolate. Weattempted to clarify how the conservation of water is com-bined with the dissipation of light energy in the water stressedplant.To test if the rest of surplus light energy is dissipated also

by other processes besides CO2 recycling, we examined thelight reactions by measurements of Chl fluorescence. By thesaturation pulse method it is possible to estimate the redoxstate of the electron acceptor of PSII, the 'energization' of thethylakoid membrane due to the proton gradient and thequantum efficiency of photosynthetic electron transport (27).An irradiation of 250 ,umol photons m-2 s-' was used for thegrowth and measurement of D. lanata as we were interestedin the reaction of intact plants in a light range where qE is not

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Plant Physiol. Vol. 92,1990

expected to influence F,, during the induction of photosyn-thesis ( 12).

MATERIALS AND METHODS

Growth and Stress Application

Digitalis lanata EHRH., a cultivar ofthe Boehringer Mann-heim GmbH, was grown under defined conditions as previ-ously described (30). Water stress was induced slowly bywithholding irrigation from the 10-week-old plants in 5-Lpots. Leaf water potential of the youngest fully expanded leaffell from -0.7 to -2.5 MPa within about 18 d. At -2.5 MPairrigation was necessary to guarantee complete recovery ofthe plants.

Water Potential, Osmolality, and Conductivity

Leaf water potential was estimated in a pressure chamber.Osmolality of the leaf was measured with an osmometer(Halbmikroosmometer, Knauer, Oberursel, FRG). The spe-cific conductivity of the sap was estimated with an LF 521electrode (Wissenschaftliche Werkstatten, Weilheim, FRG).

Gas Exchange Measurements

Measurements were carried out with the attached leaf in anopen gas exchange system previously described (30). Theconditions were the same as during growth (350 ,uL CO2 L-',250 ,umol photons m-2 s-' and a RH ofabout 75%). Syntheticair, nitrogen and CO2 were mixed by mass flow controllers(Tylan series 260, Eching, FRG). Calculations of GP, and PR,were carried out according to Canvin and Fock (9) and RAand PRi according to Gerbaud and Andre (16). Calculationsof gross photosynthesis by "'CO2 application are slightly

Table I. Abbreviations Used in This Paper

RuBP, ribulose bisphosphateC,, intercellular C02 concentrationFo, instantaneous (dark) fluorescenceFm, maximal fluorescenceFp, peak fluorescenceFv, variable fluorescence at a given time(Fv)m, maximal variable fluorescence(Fv)0, variable fluorescence at full reduction of 0GP, gross C02 uptakeNP, net C02 uptakePR, photorespiration of C02qE, coefficient of nonphotochemical quenchingqo, coefficient of photochemical quenchingqc, coefficient of quenching by electron transport processesqL, coefficient of actinic light quenchingRA, reassimilation of C02rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase

Indices-c, total C02 flux, estimated by the specific radioactivity of me-

tabolitesi, total C02 flux, calculated from 14002 gas exchange measure-ments

-s, stomatal C02 flux, estimated according to Canvin and Fock1972 (9)

underestimated due to the about 5% slower uptake of the'4CO2 isotope ( 15).

Flux Studies

At steady-state gas exchange, the leaf was killed after 1 min"'CO2 assimilation (18.5 GBq/mol CO2) in a special cuvettefor rapid fixation (7). The extraction was carried out at -20°C,using the extraction procedure of Atkins and Canvin (3) andKrampitz and Fock (18). Glyoxylate and other substanceswith oxofunctions were transformed to their correspondingphenylhydrazones according to Baker and Tolbert (4). Com-bined ion-exchange chromatography (3) on Dowex H+ andDowex formate resulted in fractions containing glycolate withglycerate and phosphoglycolate with phosphoglycerate.The glycolate fraction was divided into two aliquots. With

the first aliquot, the total radioactivity and the mass of theglycolate fraction were estimated by scintillation counting andHPLC (1, 18) and with the second aliquot, the glycolate-oxidase reaction was carried out according to (4). After acombined ion-exchange chromatography which removed theglyoxylate-phenylhydrazone synthesized from glycolate, onlyglycerate remained in the fraction formerly containing alsoglycolate. The difference in radioactivity and mass betweenthe two aliquots enables the estimation of the specific activityof glycolate.

After enzymatic dephosphorylation ofthe phosphoglycolatefraction and subsequent ion-exchange chromatography, massand radioactivity of phosphoglycolate were estimated with theabove described procedure for glycolate.Carbon flux (CF) through the photorespiratory pathway

can be estimated by the formula:

CF = PG* G* (smol C m-2 s-')

PG G

(1)

where G represents the glycolate concentration, G* the gly-colate radioactivity, PG the phosphoglycolate concentration,and PG* the phosphoglycolate radioactivity.

This equation is derived from Reiner's general formula (25)under the conditions of steady-state photosynthesis (constantcarbon flux, unchanged pool sizes) and a linear increase inthe radioactivities and specific activities ofglycolate and phos-phoglycolate in the first minute of "'CO2 uptake.

Photorespiration ofCO2 (PR,) equals one-fourth the carbonflux through the glycolate pathway since one out of 4 carbonatoms in glycolate is evolved as CO2 (14).

PRC= 1/4 CF (2)

Explanation of the Terms

Net photosynthesis (NP) is the difference between stomatalCO2 uptake (GP,) and stomatal CO2 liberation (PR,):

NP = GP, - PR, (Fig. 1). (3)

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LIGHT ENERGY DISSIPATION UNDER WATER STRESS CONDITIONS

(two oxygenations result in one glycine decarboxylation).The synthesis of the reduction equivalents (RE) consumed

in the Calvin cycle and by photorespiration requires four lightquanta (LQ) each:

RE = 2 GP + 4 PR

LQ = 4 RE

(9)(10)

Autoradiography

Autoradiography was carried out with rapidly fixed leafmaterial according to the procedure described by Downton etal. (13).

Fluorescence Measurements

Fluorescence measurements were carried out with the at-tached uppermost fully expanded leaf in the gas exchangesystem (30). White light (Philips 15 V 150 W projector lamp)was used to deliver 250 gmol photons m-2 s-' actinic radia-tion. Fluorescence measuring beam (0.1 ,umol photons m-2s-') and saturation pulses (700 msec 4500 ,umol photons m-2s-') at a frequency of 0.1 Hz were transferred by the fiberopticsof the PAM fluorometer (Walz, Effeltrich, FRG). Simultane-ous gas exchange and fluorescence measurements were carriedout, exactly resembling the cultivation conditions (350 ppmC02, 75% RH at 25°C).

ICalvin cycle|

I ATP

1 RuMP L 1 RuBP

Figure 1. Scheme of leaf carbon fluxes. GP8, stomatal C02 uptake;PR,, stomatal C02 liberation; PR,, PR,, total photorespiration; GPi,GP,, gross C02 uptake; RA1, RAG, C02 reassimilation. Indices: s-estimated according to Canvin and Fock 1972 (9); i-estimatedaccording to Gerbaud and Andre 1987 (16) by 14C02 gas exchange;c-estimated by the analyses of metabolite specific activities.

Totally evolved CO2 (PRi or PR,) is partly liberated throughthe stomata (PR,) and partly reassimilated (RAi or RAc):

PR, = PR, + RAi

PRC. = PR, + RA.

(4)

(5)

Gross CO2 fixation (GPi or GPc) consists of stomatal CO2uptake (GP,) and internally reassimilated CO2 (RAi or RA,):

GP, = GP, + RAi

GPc = GPs + RAc

(6)

(7)

Calculation of RuBP and Quanta Consumptions

The stochiometries of the Calvin cycle and the glycolatepathway (14) enable the calculation of turnover rates, if theCO2 fluxes are known (Fig. 2).RuBP is a substrate used for any oxygenation and carbox-

ylation at rubisco, therefore

HJOH H I-Co2

2 TP . I- - 2 PGA

2 NADPH2 ATP

ATP/NADPH = 1,5

CO. + 2 NADPH + 3 ATP - HCOH + H11O + 2 NADP* + 3 ADPI

Glycolate pathway

2 On Os2 ATP

2 RuMP - 2 RuBP 2 Glyoxylate

HJOHj )

3 TP -1- 3 PGA 2GG l 2Fd

3 NADPH3 ATP [1 ATP C20]- J

Glycerate - Ser NH, Glu

I ATP

2 Fd = 1 NADPH ATP/NADPH = 1,75

J1O + 3 02 + 4 NADPH + 7 ATP-CO0 + 5 H,O + 4 NADP* + 7 ADPI

Figure 2. Stochiometries derived from the fixation of C02 in theCalvin cycle (above) and the evolution of C02 in the glycolate pathway(below). PGA, phosphoglyceric acid; RuMP, ribulose monophosphate;TP, triose phosphates.

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Fluorescence Parameter Calculations and

qQ = (F)s-Fluorescence parameters (F),, Fp, Fm, (Fv)m, (Fv)s, F, qQ,

and qp-) were determined according to Schreiber et al. (27)and Stuhlfauth et al. (30). The PAM modulation fluorometerenables the estimation of instantaneous (F,), maximal (Fm),peak (Fp), variable (Fv), and variable fluorescence at fullreduction ofQ (Fv),, including maximal variable fluorescence(Fv )m (Fig. 3).The constancy of F,, within single measurements was con-

firmed by switching off the actinic light after different timeintervals within the induction kinetics.

According to Schreiber et al. (27), two components offluorescence quenching can be continuously recorded:

(Fv)m - (Fr).qE = (Fv)m

(12)

with

F, = (1 - qF) * (1 - qQ) * (Fv)m (13)

Nonphotochemical quenching (qE) is correlated with theATP-generating proton gradient over the thylakoid mem-brane (19), whereas qQ is an indicator for the relative oxidationof the primary electron acceptor Q of photosystem 11 (27).By analogy with the above equations, the definition of two

further quenching coefficients is possible:

q1 =(Fv)m -Fp(Fv)m

(14)

(1 1) and

=Fp-Fv

Fp

Figure 3. Time course of a representative fluorescence inductioncurve and derived parameters (leaf water potential -1.15 MPa). A,Recorder tracing; Fm, maximal fluorescence; F0, instantaneous fluo-rescence; (Fv)m, maximal variable fluorescence; Fp, peak fluores-cence; F,, variable fluorescence (time dependent); (F,,)8, variablefluorescence at full reduction of Q (time dependent). B, Time courseof photochemical (q,) and nonphotochemical (qE) fluorescencequenching and the fluorescence quenching coefficient qc during in-duction (left) and steady state (right) of photosynthesis. (Due to itsdefinition, qL represents a single value [0.51] and cannot be shownas a time-dependent curve).

(15)

with

F, = (1 - qL) (1 - qc) * (Fv)m (16)

At high actinic light intensities, the peak maximum ofvariable fluorescence, Fp, can equal (Fv)m. Therefore, Fp re-flects the amount of Q-reduction which can be reached byactinic light (26). When Fp is induced, (F0), is approximatelyidentical with (Fv)m (Fig. 3). The nonphotochemical quench-ing, qE, is still inactive during that period (26) as the protongradient is just starting to build up. Due to Equation 12 andEquation 14, qL approximates qQ at the time ofFp estimation,and variations of qL are dominated by changes in the redoxstate of Q. Therefore, qL may be taken as an indicator for therelative oxidation of Q induced by actinic light.The diminution of variable fluorescence, Fv, after the peak

maximum Fp, induced by the combined effects of electrontransport to NADP+ via PSI (photochemical quenching) andelectron-transport dependent processes (nonphotochemicalquenching) are summarized in qc.

RESULTS AND DISCUSSION

Down to a leaf water potential of -2.5 MPa, water-stressedDigitalis lanata plants remained capable of recovering com-pletely within 2 d after rewatering. Osmotic adjustment, low-ering the osmotic potential of the leaf sap in accordance withthe water potential, accounted for an almost constant turgorpotential over the whole stress range (Fig. 4A).The measurement of leaf sap conductivity indicates that

osmotic adaptation was only to a very limited extent broughtabout by ionic substances (Fig. 4B). Thus, neutral com-pounds, most probably synthesized from starch (J Beckedahl,unpublished results), were mainly responsible for the increasein osmolality.Due to this osmotic adaptation, even lower rosette leaves

ofseverely stressed plants did not loose turgidity and remainedconstantly exposed to the light.

Stomatal gas exchange was restricted under stress condi-

I

1 056 STUHLFAUTH ET AL.

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30

10

201

X 10

-1.0 -2.0a [MPaJ

Figure 4. Parameters of leaf water status versus leaf water potential('). A, Turgor potential (4/p) and osmotic potential (4,r) versus leafwater potential (4l). D. lanata plants were slowly dehydrated bywithholding watering. Points represent the mean of 5 replicates. Pointsize a ± SE. B, Specific conductivity (K) versus leaf water potential(4/). Points represent the mean of five replicates. Point size > ± SE.

15

v::.

N5t

(MPaJ

Figure 5. Gross C02 uptake, estimated by analyses of metabolitespecific activities (GP,) or by '4CO2 gas exchange measurements(GPi), stomatal C02 uptake (GPS) and net C02 uptake (NP) versus

leaf water potential (4/). Points represent the mean of five replicates.Point size : ± SE.

tions (Fig. 5). Net photosynthesis (NP) and stomatal CO2uptake (GP,) were reduced by about 70% in comparison withunstressed controls. In comparison with stomatal CO2 uptake,gross CO2 uptake (GPi, GPC) was less reduced. The ratiobetween gross CO2 uptake and net photosynthesis was notfixed but dependent on the stomatal aperture. Calculationsfrom metabolite analyses (GP,) and "'CO2 gas exchange (GP,)produced similar results. Obviously, CO2 uptake through thestomata contributed less to gross CO2 fixation under stressconditions than under well-watered conditions. These find-ings for CO2 uptake can be explained by the results for CO2evolution (Fig. 6). Whereas stomatal CO2 liberation was re-duced by about 50%, the total photorespiratory CO2 evolution(PR,, PRC.) remained almost unchanged. Less internally pro-duced CO2 was liberated via the stomata under stress condi-tions. Consequently, reassimilation (RAC, RAj) of photores-pired CO2 was significantly increased when the stomata closed(Fig. 7). Fitting the data into the model of Farquhar et al. ( 14)resulted in an increase in the ratio of oxygenation rate tocarboxylation rate. This required a decreased CO2 concentra-tion at rubisco, as the 02 concentration within a leaf is hardlyaffected by stomatal closure (16). Such a drop in the CO2concentration contradicts the far-reaching constancy of C,previously reported under water deficiency conditions (30);however, Laisk (21) pointed out that C, may be overestimatedifthe stomatal apertures ofa leafare not uniform ('patchiness';11, 13). The autoradiography of leaves after 14C02 assimila-tion clearly indicates that this is the case for D. lanata (Fig.8). The conventional calculation resulted in an overestimationof C, at lowered leaf water potential (21). In contrast to thepreviously reported 'patchy' distribution of starch in Helian-thus, starch is homogenously distributed in Digitalis (30).As indicated by autoradiography, patches ('areoles'; 1)

were small (about 2 mm2), so that the area tested during gasexchange and fluorescence studies allowed a representativeaveraging.

14CO2 uptake remained possible at -2.0 MPa (Fig. 8),

6

C~'

-N

2

-1.0 -2.0[MPaJ

Figure 6. Total photorespiration, estimated by analyses of metabolitespecific activities (PR,) or by 14C02 gas exchange measurements(PRj), and stomatal C02 liberation (PR,) versus leaf water potential(4/). Points represent the mean of five replicates. Point size : ± SE.

A

pw

"P00, , , . , . ., . . . . . .. II..........

B

PRj@ Cp :PRO O_

i ~ ~ _

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4

RA A0~~~~a,2

RAi

-1.0 -2.0I [MPaJ

Figure 7. Reassimilation of photorespiratory C02, estimated by anal-yses of metabolite specific activities (RA,) or by 14C02 gas exchangemeasurements (RA,), versus leaf water potential (). Points representthe mean of five replicates. Point size > ± SE.

-210 MPa 3. MPG

Figure 8. Autoradiography of leaves rapidly fixed after 14C02 assim-ilation. Dark areas represent patches (areoles) of high C02 uptake. Incontrast to leaves at -2.0 MPA, white spots without C02 exchangedo occur at -3.0 MPa.

indicating that stomata were not completely closed even atsevere water stress.Due to photorespiration and reassimilation, the turnover

of RuBP was only reduced by about 40% (Fig. 9A), whereasstomatal CO2 uptake was diminished by about 70% at -2.5MPa (Fig. 5). The calculated value for the turnover of RuBPwas consistent with a 42% diminution of in vivo rubiscoactivity under the same stress conditions in D. lanata (SBarzen, unpublished results). Similar results have been ob-tained with Phaseolus vulgaris by Sharkey and Seemann (28).Thus, the reassimilation supports a relatively high enzymaticturnover under water stress conditions.The consumption of light energy, as calculated from the

turnover of reduction equivalents in the Calvin cycle and theglycolate pathway, was only reduced by about one-third atsevere stress (Fig. 9B), whereas net photosynthesis was dimin-ished by about 70%.Depending on water potential, the reassimilation of pho-

-10 -2 0

Figure 9. Metabolic parameters calculated from gas exchange andcarbon flux data. A, Turnover of ribulosebisphospate, estimated byanalyses of metabolite specific activities (RuBP,) or by 14002 gasexchange measurements (RuBP1), versus leaf water potential (@f).Points represent the mean of five replicates. Point size 3 ± SE. B,Relative utilization of light quanta by metabolic processes (Calvincycle and photorespiration), estimated by analyses of metabolitespecific activities (LQ,) or by 14002 gas exchange measurements(LQi), versus leaf water potential (@4. Light quanta consumption ofunstressed controls is taken as 100%. Points represent the mean offive replicates. Point size > ± SE. C, Relative participation of C02recycling (reassimilation and photorespiration) on total metabolic lightenergy consumption by Calvin cycle and photorespiration, estimatedby analyses of metabolite specific activities (@) or by 14CO2 gasexchange measurements (0), versus leaf water potential (4t). Pointsrepresent the mean of five replicates. Point size : ± SE.


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torespiratory CO2 accounts for 25 to about 50% of the meta-bolic quanta consumption (Fig. 9C). Therefore, photorespir-ation and reassimilation contribute significantly to the energybalance of a plant.The importance of reassimilation and photorespiration for

water stress tolerance could be demonstrated by gas-exchangemeasurements in air with reduced oxygen content. In air with2.5% 02, photorespiration and reassimilation were sup-pressed. When leaves ofstressed D. lanata plants were exposedto low oxygen conditions, net photosynthesis (Fig. lOA) andtranspiration (Fig. lOB) were enhanced in comparison tocontrol plants in 21% oxygen. If photorespiration and CO2recycling are suppressed, the plants open their stomata anduse external CO2 to consume light energy by net CO2 fixation.This stomatal opening leads to an increased loss of water bytranspiration.From that it may be speculated that the efficient dissipation

of light energy is more important than the conservation ofwater. The reassimilation ofinternally evolved CO2 is a mech-anism that dissipiates light energy at low water costs and thusmay contribute to the stress tolerance of C3 plants.Peak fluorescence (Fe), which is induced by the onset of

actinic light (250 ,umol photons m-2 s-'), decreases with

12

vlz-,IAN,e"aSt

5!

8

4

ca,1.2

0-

0.4

-1.0 -2.0

increasing water deficiency (Fig. 1 1). Such a decrease of Fphas also been reported for several other water stressed plants(17, 30), but it is not typical, e.g. for bean or sunflower undersimilar conditions (R Scheuermann et al., in preparation). Inthose stress-sensitive plants, where osmotic adaptation inresponse to water deficiency does hardly occur, Fp remainsalmost constant.

In contrast to Fp, constant (Fo) and maximal fluorescence(Fm), are not influenced by water stress treatment (Fig. 11).Fo remains unchanged, even during the fluorescence induc-tion, as indicated by switching off the actinic light afterdifferent time intervals within the induction kinetics. In ac-cordance with the results of Demmig-Adams et al. (12), F0was not influenced by qE at nonsaturating levels of actiniclight. Therefore, it may be concluded that mobilization of thelight harvesting complex does not occur as a response to waterstress in D. lanata. The constancy of Fm correlates quite wellwith the unchanged Chl a and b contents of leaves (30).The fluorescence quenching coefficients qE, qQ, and qc are

only slightly affected by lowering the leafwater potential (Fig.12). As pointed out by the unchanged qQ coefficient (Fig.12A) the redox status of Q, the primary electron acceptor ofPSII, remains unaffected. The relatively small increase innonphotochemical quenching, qE, (Fig. 1 2A) indicates a slightincrease in the transmembrane proton gradient of the thyla-koids and a concomitantly reduced ATP turnover under stressconditions (30).With respect to the constancy of qc during water stress

treatment (Fig. 12B) one may assume that Fp and steady stateF, are similarly altered.

In contrast to the other quenching coefficients, qL is quiteremarkably increased by water stress (Fig. 12B). As comparedwith well watered controls, this enhancement of fluorescencequenching can be attributed to a 30% increase of Q-oxidation

qJ

C..

LL.

IMPa]

Figure 10. Effect of 2.5 and 21% 02 on net C02 uptake andtranspiration. A, Net C02 uptake (NP) in air with either 21% (-) or2.5% (0) oxygen versus leaf water potential (46). Points represent themean of five replicates. Point size : ± SE. B, Transpiration (T) in airwith either 21% (0) or 2.5% (0) oxygen versus leaf water potential(4 Points represent the mean of five replicates. Point size : ± SE.

- 1.0 -2.0P IMPal

Figure 11. Fluorescence characteristics versus leaf water potential(4fl. Maximal fluorescence (Fm), peak fluorescence (Fp) and darkfluorescence (Fo) in relative units. Fluorescence measurements werecarried out at 350 ppm C02, 75% RH at 250C in an open gasexchange system. Dark fluorescence was estimated at 0.1, variablefluorescence at 250 and 0-saturation at 4500 ,qmol photons m-2 s-',respectively. Points represent the mean of five replicates. Point size: ± SE.

A

. . . . . . , . . , . I . . . . .

B

F.

G _ * Z Z

F* U

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1.0

0.5

10

0.5

-10[MPa]

-20

Figure 12. Steady state values of the fluorescence quenching coef-ficients versus leaf water potential (i). A, Steady state values of thecoefficient of photochemical quenching (q0) and nonphotochemicalquenching (qE) versus leaf water potential. Points represent the meanof five replicates. Point size , ± SE. B, Steady state values of thefluorescence quenching coefficient qc and qL values versus leaf waterpotential. Points represent the mean of five replicates. Point size >± SE.

in response to activation of PSII by actinic light. In waterstressed D. lanata qL seems to be a relevant quenching param-eter, whereas in bean and sunflower it remains constant. (RScheuermann, unpublished data).At severe water deficiency (-2.5 MPa leaf water potential),

stomatal closure restricts transpiration and C02-fixation in D.lanata (29, 30). Under such conditions the demand for pho-tochemically synthesized reduction equivalents in the photo-synthetic carbon oxidation and reduction cycles is lowered byabout one-third (Fig. 9). The 30% increase in qL-quenchingmay thus help to balance the difference between offered lightenergy and its metabolic consumption.The structural basis for qL quenching has to be further

elucidated. The hypothesis that this type of quenching isbasically connected to osmotic conditions, is supported bythe fact that qL increases with osmotic and leaf water poten-tials, and is most efficient at the growth irradiation level,whereas an increase in light intensity from 250 to 850 ,molphotons m-2 s-' leads to an increase of q;, irrespective of leaf

water status (data not shown). Principally, the low reductionstatus of Q, correlated with qL-type Chl fluorescence quench-ing, can be achieved by pseudocyclic electron transport. Thereduction of H202 in the chloroplast causes a diminution ofFp and simultaneously stimulates qQ and qE, when the Calvincycle is suppressed (22). The assumption of pseudocyclicelectron flow is supported by the increase in glutathionereductase activity in water stressed D. lanata plants (S Barzen,unpublished data), as this enzyme contributes to H202 scav-enging in the chloroplast (2). The role of 02 as an effectiveacceptor of photosynthetic energy has been pointed out influorescence studies by Comic et al. (10). An alternativeexplanation for qL quenching may be given by an electroncycle (20, 22) operating either around PSII, or within PSII,by charge recombination, and thus lower Q-reduction.Our results confirm and extend the view that there is no

specific site of inhibition of photosynthesis under water stressconditions, but that a complex regulation is necessary to adaptlight and dark reactions to the environmental conditions (24).It may be speculated that qL quenching indicates the operationof an additional protective mechanism against water stressinduced photoinhibition that enables D. lanata to tolerateeven very low leaf water potentials.

D. lanata is adapted to dry habitats in southeastern Europe(29) and can endure periods of dryness and high irradiationin the first, vegetative year ofdevelopment. The maintainanceof high metabolic activities and capacities protects the plantfrom photoinhibition and enables rapid recovery after thestress period.

ACKNOWLEDGMENTS

We thank Dr. E. Weis for his helpful contributions during our

recent discussions and Dr. T. Buckhout for correcting the manuscript.

LITERATURE CITED

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2. Asada K, Takahashi M (1987) Production and scavenging ofactive oxygen in photosynthesis. In DJ Kyle, CB Osmond, CJArntzen, eds, Photoinhibition. Elsevier Science Publishers,Amsterdam, pp 227-288

3. Atkins CA, Canvin DT (1971) Photosynthesis and CO2 evolutionby leaf discs: gas exchange, extraction, and ion-exchange frac-tionation of '4C-labelled photosynthetic products. Can J Bot49:1225-1234

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Light Energy Dissipation under Water Stress Conditions