limitation of photosynthesis by carbon metabolism1

Plant Physiol. (1986) 81, 1115-11220032-0889/86/81/11 15/08/$01.00/0

Limitation of Photosynthesis by Carbon Metabolism1I. EVIDENCE FOR EXCESS ELECTRON TRANSPORT CAPACITY IN LEAVES CARRYING OUTPHOTOSYNTHESIS IN SATURATING LIGHT AND CO2

Received for publication December 30, 1985 and in revised form March 15, 1986

MARK STITTInstitut fur Biochemie der Pflanze, Untere Karspile 2, 3400 Gottingen, Federal Republic ofGermany

ABSTRACT

It has been investigated how far electron transport or carbon metabo-lism limit the maximal rates of photosynthesis achieved by spinach leavesin saturating light and CO2. Leaf discs were illuminated with high lightuntil a steady state rate of 02 evolution was attained, and then subjectedto a 30 second interruption in low light, to generate an increased demandfor the products of electron transport. Upon returning to high light thereis a temporary enhancement of photosynthesis which lasts 15 to 30seconds, and can be up to 50% above the steady state rate of 02 evolution.This temporary enhancement is only found when saturating light inten-sities are used for the steady state illumination, is increased when lowlight rather than darkness is used during the interruption, and is maximalfollowing a 30 to 60 seconds interruption in low light. Decreasing thetemperature over the 10 to 30°C range led to the transient enhancementbecoming larger. The temporary enhancement is associated with anincreased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, andincreased levels of triose phosphate and ribulose 1,5-bisphosphate. Sinceelectron transport can occur at higher rates than in steady state condi-tions, and generate a higher energy status, it is concluded that leaveshave a surplus electron transport capacity in saturating light and CO.From the alterations of metabolites, it can be calculated that the enhanced02 evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that thecapacity for sucrose synthesis ultimately limits the maximal rates ofphotosynthesis, by restricting the rate at which inorganic phosphate canbe recycled to support electron transport and carbon fixation in thechloroplast.

When a plant leaf is photosynthesizing in low light or lowC02, the rate of photosynthesis can be increased by raising thelight intensity or the CO2 concentration. However, a point isreached where further increases of light or ofCO2 do not lead toany further increase in the rate of photosynthesis. At this point,it would appear that an internal ceiling has been reached whereat least one of the components of the photosynthetic apparatuscannot operate any faster. In principle, this ceiling could beimposed by the number or turnover rate of the photochemicalcenters, by the capacity for photosynthetic electron transport, orby the rate at which ATP and NADPH can be consumed in thereactions of carbon metabolism which convert CO2 and H20,ultimately, to carbohydrate end products. We do not have simplemethods to show to what extent these components are imposinga ceiling on the maximal rates of photosynthesis. The aim of thisand the following (17) paper is to describe simple procedures

' Supported by the Deutsche Forschungsgemeinschaft.

which reveal when carbon metabolism is restricting the rate atwhich the available light and CO2 can be utilized, to investigatein what conditions this may happen, and to ask which compo-nents of carbon metabolism may be responsible for this restric-tion.The approach described in this paper depends upon electron

transport and the various reactions of carbon metabolism re-sponding in a different way to a sudden decrease in the lightintensity. After darkening, electron transport is immediatelystopped, while stromal enzymes are only gradually inactivated(3, 8, 9), and the synthesis of sucrose in the cytosol continues for15 to 60 s (22). It will be shown here that this slower inhibitionof carbon metabolism can be exploited to generate an increased'demand' for the products of electron transport. This shouldallow a transient enhancement of the rate of 02 evolution im-mediately after returning a leaf to high light in conditions wherethe leaf possesses excess capacity for electron transport which isnot being utilized during steady state photosynthesis.

MATERIALS AND METHODSSpinach (Spinacia oleracea var Mazurka) was grown in hydro-

ponic culture (13) under a 9 h light/15 h dark cycle with a lightintensity of 340 ,E -m2 * s-' and a CO2 concentration in the lightof 380 ,l/L. The temperature was 22°C in the light and 16C inthe dark. Leaf discs were taken from fully expanded leaves of 5-to 7-week-old plants. Xanthium was grown under a 12 h light/12 h dark cycle, with a light intensity of 300gE m 2 -s-' and atemperature of 24°C in the light and 14C in the dark. The shadeleaf grew at a light intensity of about 100 AE m-2 s'. Ivy wasobtained from a plant growing in a shaded site in the botanicalgarden.02 evolution was measured in a Hansatech leaf disc 02 elec-

trode (4). Temperature was controlled by water flow through awater jacket. Light was provided by projectors, the intensitybeing varied with neutral filters. CO2 (about 5%) was suppliedfrom 200 ,ul of a 2 M KHCO3/K2CO3 mixture (pH 9.3) on felt inthe base of the electrode. Increasing the pH to 10.0 or decreasingthe concentration to 0.3 M did not decrease the rate of photosyn-thesis. Usually, photosynthesis of 3 to 5 leafdiscs (1 cm diameter)was measured. The leaf discs were placed on a fine sheet ofaluminum, which was punctured repeatedly with holes to allowgas exchange under the leaf material, but not punctured in thearea without plant material. The aluminum prevented lightpenetrating into the electrode chamber below the leaf, wherethermal expansion of the electrode walls and felt matting couldproduce artefacts with the light sources used in these experiments.After inclusion of the aluminum, the highest light intensitiesused did not produce significant alterations in 02 concentrationdue to thermal expansion when control experiments were carriedout using leaf material which had been killed by freezing in liquidN2 and rethawed.

1115

Plant Physiol. Vol. 81, 1986

Samples for metabolite analyses were killed by rapidly openingthe electrode, lifting the leaf pieces out on the aluminum foil,and tipping them into an adjacent beaker of liquid N2, whichwas illuminated by the same light intensity. There was no inter-vening dark period. To minimize alterations due to decrease ofC02, the electrode and liquid N2 were gassed by a stream of airwhich had been passed through a solution of 2 M K2C03/KHCO3pH 9.3. The leaf discs were extracted in 10% HC104 and analyzedfor Ru 1,5P22, PGA, triose-P, Frul,6P2, Fru6P, and Glc6P as in(20). The reliability of the extraction technique was checked byshowing that over 90% of representative amounts of each sub-strate added with the HC104 could be recovered. Adenine nu-cleotides were measured spectrophotometrically as in Lowry andPassoneau (14) using a Sigma 2FP 22 dual wavelength photom-eter, which allowed reliable measurements of under 50 pmolADP, and 25 pmol AMP. The high ATP/ADP ratios in leaves,and the very low AMP, mean that enzymic analysis gives moreaccurate results for the ADP and AMP level in leaves than canbe achieved by assay using luciferase even though the theoreticalsensitivity of the latter method is higher. In the luciferase assayADP and AMP are obtained by subtracting the large ATP signal.

RESULTSTransient Enhancement of Photosynthesis in Saturating Light

following a Short Period in Low Light. When a leaf is subjectedto a short period of darkness and then reilluminated there areoften marked oscillations in the rate of photosynthesis, and theinitial peak rates of 02 evolution and C02 fixation can exceedthe steady state rate of photosynthesis (18, 19, 25). Before study-ing why this stimulation occurs, it was investigated whether thesize of this transient enhancement of photosynthesis could beincreased. A few minutes in the dark leads to inactivation ofseveral enzymes in the Calvin cycle (3, 8, 9), as well as a depletionof stromal metabolites (22) which will restrict the rate of photo-synthesis achieved immediately after reillumination in high light.This suggested that the initial peak might be increased evenfurther by substituting low light for darkness and by shorteningthe duration of the interruption, so that the rate of electrontransport is temporarily decreased without a concomitant inac-tivation of the Calvin cycle. In these, as in all experiments in thisarticle, leaf discs were illuminated in the presence of saturatingC02 in a leaf disc 02 electrode (4).

In Figure IA, leaf discs photosynthesizing in high light weresubjected to a 60 s interruption in either complete darkness, ora range of low light intensities. A transient enhancement of therate of photosynthesis was always found immediately after re-turning to saturating light, but the stimulation (compared to therate in continuous high light) was larger following an interruptionwith low light (50%) than after darkness (16%). For furtherexperiments a light intensity of about 180 ,uE. m 2sI was se-lected for the low light interruption. In Figure lB the durationof the interruption in low light was varied before returning tohight light. Even 5 s in low light was enough to produce atransient enhancement after returning to saturating light, andthe response was maximal after 15 to 60 s in low light. Longerperiods in low light led to a decreased response. For subsequentexperiments a treatment of 30 s was used.The experiment of Figure 2 investigated whether the light

intensity used before and after the interruption influences themagnitude of the transient enhancement. The discs were illu-minated at one of 4 different light intensities between 500 and1500 uE m 2.s-' for 20 min until the steady state rate ofphotosynthesis was achieved, before lowering the light intensity

2Abbreviations: Ru 1 ,5P2, ribulose 1 ,5-bisphosphate; Fru 1,6P2, fructose1,6-bisphosphate; Fru6P, fructose 6 phosphate; Glc6P, glucose 6 phos-phate; PGA, glycerate-3-phosphate.

346/ 306,h296' '294 270 Enhaincedratecm

0 26 2 270/ pmol 02 mgChF1hE20

E 178 108 78 31 <10 Light intensity178- 108 78 31 <10 during nterupbonStandard light =1500 pEm-2.s-1 pE m2 s-

Control rote = 231 pmot 02 mg Chl-1 h-

10 20 30Time (min)

E_ / y_~~~~/

0 B 3250221

5sec/220 5sec. Light 1500 pEm~245E

20 / n / eueto18,.-.1020-

331

1410sec. L~~~~~ight 1500 pEmT2 s-1rnn. ~~~~Reduce 10 178 piEm-r62-s1

21 for varying times.10 20

Time (min)

FIG. 1. Transient enhancement of the rate of photosynthesis of spin-ach leaf discs photosynthesizing at saturating light and CO2 following a

brief interruption with low light. A, Dependence on the light intensityduring the interruption with low light. Leaf discs were illuminated with1500 ME * m-2 _ s-' until steady state rates of photosynthesis were achieved,and then were darkened or illuminated with low light (details shown infigure) for 60 s before returning to 1500 ME m2 s'. The 02 evolutionin steady state was 231 Amol 02-mg Chl-'-h-'; the enhanced ratesfollowing the dark or low light are shown in the legend. B, Dependenceon the duration of the period in low light (intensity 180 uE-m2.s-').The rate following the interruption with low light is shown on the figure.All experiments were carried out at 20C with saturating CO2 using three1 cm diameter leaf discs in a leaf 02 electrode (see "Materials andMethods.")

to 180 ,uE. m-2 _ s-' for 30 s. The light intensity was then returnedto the original level, and the magnitude of the transient enhance-ment measured. At the lowest light intensity used, which was

limiting, no transient enhancement was detected. The enhance-ment appeared with near-saturating or saturating light intensities.When the light intensity was further increased in the range wheresteady state rate of photosynthesis was already saturated, themagnitude of the enhancement increased even further.

Rates ofphotosynthesis which exceed those occurring in steadystate can also be demonstrated by comparing the rate of photo-synthesis under conditions where two different light intensitiesare being rapidly alternated (cycled) with the rates achieved whenthese same two light intensities are given separately (Fig. 3).Cycling high and low light intensities allows the transient en-hancement of photosynthesis following a brief interruption inlow light to be repeated many times. In a typical experiment, a

leaf disc was illuminated at a selected upper light intensity untila steady state rate of photosynthesis was achieved, and the lightintensity then cycled at 10 s intervals between high light and a

standard low light intensity. After 6 min of cycling, the leaf was

1116 STITT

LIMITATION OF PHOTOSYNTHESIS. I

30

20

u

E

10

lU 20

Time (min)

FIG. 2. Relation between the transient enhancement of photosyn-thesis and the light intensity. Leaf discs were illuminated for 20 min untilsteady rates of photosynthesis were achieved, and the light intensity thendecreased to 180 ME m-2 - s-' before returning to the original light inten-sity. The experiment was carried out at 1500, 1100, 780, or 570 AE* 2-s_'. The photosynthesis rates in constant illumination, and immediatelyafter the interruption in low light, are shown on the figure (at 20°C).

Time (min)

FIG. 3. Comparison of rates of photosynthesis under constant illu-mination conditions, and during cycling at 10 s intervals between highlight and low light. Three spinach leaf discs were illuminated for 15 minat 570, 780, 1100, and 1500 qE-m-2-s-', until constant rates of photo-synthesis were reached. The light intensity was then alternated at 10 sintervals between the original high light intensity, and low light (180juE -

m 2.'). After 6 min the leaves were left in constant low light of 180,gE mM2-s-'. The temperature was 20'C.

further illuminated at the lower light intensity to measure thesteady state rate of photosynthesis in the low light. The rates ofphotosynthesis under the high and low light were then used toestimate the rate expected during the time when the light inten-sity is being cycled. In principle, this should be the average ofthe steady state rates at the high and low light intensities. Ac-tually, a lower rate might occur if the repeated alteration of lightintensity leads to an inbalance in metabolic conditions.

In Figure 3, a lower light intensity of 180 uE - m2. s-1 was usedin each case, and four different upper light intensities were used,between 570 AE m-2-s-', and 1500 ME-m-2_- . In Table I therates of 02 evolution are taken from Figure 3, and the expectedrate of 02 evolution during the period of cycled light is estimatedfor comparison. When light was cycled between 180 and 570

Table I. Enhanced Use ofSaturating Light when Supplied in Cycleswith Low Light

Calculated from Figure 3.

02 Evolution

Expected MeasuredLight Measured In in Activity as a

Intensity Constant cycling with cycling Percentage of180 ME. with 180 Expected

-2 I-ih18m s 2

E-m-2 s' qmol-mg' Chl-h'1500 236 215 166 1291100 226 169 161 105780 216 152 156 98570 162 120 129 93180 96

ME m2 * s', the measured rate of 02 evolution remained belowthat estimated by averaging the rates of02 evolution at these twolight intensities. However, at higher light intensities the rate of02 evolution when the light was being cycled was up to 30%above that predicted from the rates attained in constant light.This can also be seen from the slopes in Figure 3. The 02

evolution was hardly altered by increasing light from 780 to 1500uE*m-2 * s-' during constant illumination but there was a markedincrease if the slopes are compared during the time when thesetwo light intensities were being continually interrupted with lowlight. The overadditive 02 evolution in the presence of cycledlight resembles the transient enhancement following a singleinterruption with low light, in that neither occur when the lightintensity is limiting (Fig. 3, Table I). Overadditive 02 evolutionduring cycled light also was not found when cycles of over 1 minwere used, or when darkness was substituted for low light (notshown).The transient enhancement of the rate of photosynthesis fol-

lowing a short interruption in low light suggests that leaves mayindeed have unused electron transport capacity during photosyn-thesis in saturating light. The enhancement is not observed whenlimiting light intensities are used, as expected since electrontransport should be exerting at least a co-limitation on the rateof photosynthesis. To provide further evidence for these ideas,experiments were carried out in which the relation betweenelectron transport and carbon metabolism was modified byvarying the conditions or the plant material. The resulting alter-ation of the light saturation curve should be accompanied by ashift in the light intensities at which oscillatory phenomena ortransient enhancements of 02 evolution are observed, if theseoccur in conditions when reactions in carbon metabolism limitthe maximal rate ofphotosynthesis and there is a surplus capacityfor transport.

Relation between Light Saturation, Temperature and TransientEnhancement of Photosynthesis. There is a complex relationbetween temperature and light saturation curves in C3 plants. Ingeneral, high temperature leads to higher rates of photosynthesisat high light intensities, but the net rate of photosynthesis in lowlight may even be decreased at high temperature. Consequently,at higher temperature the light intensity needed to saturatephotosynthesis is raised (2). Figure 4 illustrates that spinach leafdiscs in an 02 electrode in saturating CO2 display this response.At the light intensities used, photosynthesis was still not fullysaturated at 30°C, and the intensity needed to saturate photosyn-thesis decreased progressively at 20, 15, and 10°C.The relation between light intensity and the transient enhance-

ment of photosynthesis after a brief period of low light wasstudied at each of these temperatures. This was done by meas-uring the rate following a single interruption for 30 s (Table II),and by comparing the rates in constant light with those when

Appearance of transient enhancementin saturating light

Enhanced 372/ 287 / 223 162

)02 evolutionpimoa mgCht1 h1hNSteawdy

state 236 226 216 162

pE m2 s1 1500 1100 780 570

1117


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0 500 1000 15~~~000C

E 200

E *

0

0

0 500- 1000 1500Light intensity (iE. mrr2 s1-)

FIG. 4. Light saturation curves for spinach leaf discs in saturatingCO2 at different temperatures.

Table II. Influence ofLight Intensity on the Magnitude oftheTransient Enhancement ofPhotosynthesis following a Short Period in

Low Light: Interaction with TemperatureLeaf discs were illuminated for 20 min at the temperature and light

intensity shown, and light then decreased for 30 s to 180 gE-m 2s',before returning to the original intensity. The rate after returning to highlight is related to the steady state rate to give the transient enhancement.

Transient Enhanced Rate (asPercentage of Rate in Constant

Light Illumination)

10°C 15°C 200C 30°CE.m-2 .-1

1500 137 147 142 1181100 123 125 126 103780 118 110 103 100570 106 100 100 100

Table III. Influence ofLight Intensity on the Enhanced Use oftheSatutrating Light during Cycles with Low Light: Interaction with

TemperatureFollowing 20 min at the temperature and high light intensity shown,

the light intensity was alternated at 10 s intervals with 180 jsE.m 2- I

for 10 min. The rate at 180 uE*m 2.s' was then measured. Enhance-ment was estimated as in Table 1.

Enhancement of Photosynthesis duringCycling of High Light with Low Light

Intensity of (180 uE. m-2.s1), Expressed asHigh Light Percentage of Expected Rate

10°C 15°C 200C 30°CiE. m~2 -I

1500 149 145 129 1031100 144 140 105 93780 125 120 98 89570 100 94 93 89

light is cycled at 10 s intervals (Table III). The difference betweenthe precise answers given by the two methods are probably dueto a leaf responding differently to a single transient and to arepeated rapid alteration of light intensity. However, both testsagree in showing that at 30°C there was hardly any enhancementof photosynthesis, compared to steady state rates, and then onlyat the highest light intensity used. As the temperature was loweredthe enhancement appeared at progressively lower light intensities.The extent of the enhancement also increased at lower temper-atures, in the sense that the rate achieved during the transientenhancement became larger relative to the steady state rate ofphotosynthesis.

In all these experiments the enhancement of photosynthesisappears at the point where the slope ofthe light saturation curvebegins to decrease. This is illustrated in more detail in Figure 5,where the light saturation curve for steady state photosynthesisat 1 5°C and 30°C (-) is compared with the rates of photosyn-thesis obtained transiently at a given light intensity immediatelyfollowing a 30 s interruption with light of 180 E. m-2s-'(- - - - ). These results are replotted from the experiments ofTableII. The dotted line gives a minimum estimate of the capacity forelectron transport capacity in given conditions, indicating howthe leaves have considerable unused electron transport capacityin saturating light at 1 5C, while at 30°C the electron transportapparently remains limiting for most of the conditions used inthese experiments.Comparison of Sun and Shade Leaves. Leaves which have

developed in different light intensities can vary in their mor-phology and physiology. Consequently, 'shade' leaves havehigher rates of photosynthesis at low light intensities, but pho-tosynthesis is saturated by lower light intensites than in 'sun'leaves. Figure 6 shows the differing steady state light saturationcurves (-) of Xanthium leaves which have developed in highand low light. The transiently enhanced rates of photosynthesisfollowing a 30 s interruption with low light are shown by thedotted lines. The enhancement of photosynthesis is only foundin saturating or near saturating light, and consequently appearsat lower light intensities in the shade leaf than in a sun leaf. Themagnitude of the enhancement is also larger in the shade leaf.Studies with Ivy (not shown), which is a plant adapted to lowlight conditions, revealed a transient enhancement of 02 evolu-tion of up to 100% at light intensities as low as 200 MtE.m 2s'at 20C. In Ivy leaves, such low light intensities were already

Light intensity (pE- mr2- sg1l)FIG. 5. Light saturation curves of steady state photosynthesis and the

transiently enhanced rates of photosynthesis at 15°C and 30C. Theresults are taken from Figure 3 and Table II.

1118 STITT


3001

z

E200

0E

a

c

10040

1000Light intensity ( pE m-2. 1)

2000

FIG. 6. Light saturation curves of steady state photosynthesis, and thetransient enhancement after a period of low light, for sun and shadeleaves from Xanthium. The leaves were from the same plant, one at thetop and one in shadow of the first leaf. Leaf discs were illuminated atsaturating CO2 at 20C in different light intensities until steady photosyn-thesis was achieved, before interrupting with 30 s at 200 HE* m-2 * s' lightto measure the following enhancement of photosynthesis.

Induction in spinach40 at different light intensities at 150 1100

, E -

-5E12020

C

0

0 10 20

Time in light (min)

FIG. 7. Induction of photosynthesis in spinach at different light inten-sities. Leaf discs were taken from plants which had been 18 and 20 h inthe dark, and were illuminated in saturating CO2 (see "Materials andMethods") at 15'C at 110. 5 70, or 1100 IAE * m-2.S- Iight.

close to saturating. Thus, leaves vary greatly in the light intensityat which C02-saturated photosynthesis becomes unable to utilizehigher light intensities, and this cannot be due to a saturation oftheir electron transport capacity as the rate of electron transportcan be significantly raised following a brief interruption in lowlight.

Limitation during Photosynthetic Induction. In spinach plantswhich have been in the dark for 20 h, the induction period lastsup to 20 min. This lengthy period of suboptimal photosynthesishas been attributed to the need to activate the enzymes involvedin carbon metabolism or to the need for adequate pools ofintermediates to be built up to allow adequate rates of catalysis(8). It would be predicted that less electron transport capacity isneeded to match the available activity of reactions in carbonmetabolism during the induction period than in steady statephotosynthesis. In agreement, the induction period can even beshortened at low light intensities (Fig. 7) as the full activity ofenzymes does not have to be achieved before they match themaximum rate of photosynthesis at a lower light intensity. Inthe experiment shown in Table IV, the enhancement of photo-

Table IV. Influence ofLight Intensity on the Transient EnhancementofPhotosynthesis during the Induction Period

Leafdiscs were taken from spinach plants which had been predarkenedfor 20 h and were illuminated at 1 5'C at the light intensity shown. After7 min, the light intensity was lowered to 180 MEW m-2 for 30 s, and thenreturned to the original intensity.

02 Evolution Enhancement (asPercentage ofeight Before 30Aftere30 Rate Before

Intensity s in low s in low Interruption inlight light Low Light)

E m2 .s-I moIO2*mg'Chl*h1'1100 73 147 200780 70 101 144570 49 61 125

synthesis following a 15 s interruption with low light (180 AE-m2 s') was measured during the induction period of spinach

leaves (7 min after the start of illumination). The transientenhancement is larger during induction, and is present at lightintensities where no effect appears once steady state rates ofphotosynthesis have been achieved. When photosynthesis is lim-ited by carbon metabolism during the induction lag, surpluselectron capacity appears at lower light intensities than duringsteady state photosynthesis.

Metabolite Levels during Steady State and Enhanced Photo-synthesis. To provide additional evidence that the utilization ofthe available capacity for electron transport and photophosphor-ylation is restricted during sustained photosynthesis in saturatinglight, and to investigate what components of carbon metabolismmay contribute to this restriction, the levels of metabolites weremeasured during steady state photosynthesis and during thetransient enhancement of photosynthesis. The light intensity wasdecreased from 1500 to 180 gE m2.s-' for 30 s, and thenreturned to 1500 ME m-2 .s-'. The experiment was carried outat 15'C to maximize the magnitude of the temporary enhance-ment of photosynthesis. In this experiment, the enhanced ratewas about 40% above the steady state rate of 02 evolution (Fig.8A). This period of rapid photosynthesis was followed by aninhibition of photosynthesis, to below the steady state rate, beforethe photosynthetic rate oscillated back to the constant steadystate conditions.Lowering the light intensity produced a decrease of the ATP/

ADP ratio while PGA increased, and the other sugar phosphatesdeclined (Fig. 8B-F). Upon returning to high light, these metab-olites showed marked and rapid alterations, falling into threegroups. One group (ATP, Rul,5P2, triose-P, Fru6P) varied inparallel with changes in the rate of 02 evolution, while a secondgroup (PGA, ADP, AMP) varied inversely with the rate ofphotosynthesis. Others (Fru 1,6P2, Glc6P) showed a less clearresponse. Fru 1,6P2 did not vary greatly in this experiment,although in others it showed changes similar to triose-P. Glc6Prose gradually following the return to high light. The sum of theesterified phosphate decreased during 30 s in low light and roseagain gradually after returning to high light, suggesting that freePi increases by about 100 nmol -mg Chl' during the interruptionin low light and decreases again gradually after returning to highlight.These measurements ofmetabolites represent the overall levels

in the leaf. Studies of metabolite distribution in leaves (15) (RGerhardt, unpublished data) and protoplasts in saturating lightand CO2 (10, 20-22) have shown that all of the Rul,5P2, mostof the PGA and Fru 1 ,6P2, and about half the Fru6P are locatedin the stroma. Most of the Glc6P and triose-P are in the cytosol.Since the ATP/ADP ratio is far higher in the cytosol than thestroma (21), most of the ADP and AMP are located in the

Xanthium - light saturationSun leaf

Shade leafSun leaf

Shade leaf

0. .-.- -.-

1119


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Time (min)

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2 3

Time (min)

stroma, while ATP is distributed equally between the stroma andthe cytosol.

Despite the size of these changes in metabolite levels, they are

small in relation to the fluxes through these pools during pho-tosynthesis. For reference, the use rate of these metabolites isestimated (Table V) during conversion of CO2 to sucrose. Com-parison ofthese use rates with the fluctuation of metabolites (Fig.8) shows that the PGA accumulated during 30 s in low light (70nmol * mg-' Chl) is equivalent to 0.93 s of steady state photosyn-thetic fluxes or, alternatively could support a 40% enhancementof photosynthesis for 2.3 s. The fluctuations of ATP and RuBPare even smaller than those of PGA. Consequently, the 40%increased rate of 02 evolution for 15 to 20 s cannot be due toconsumption of preexisting pools of these intermediates whichhave accumulated during the period in low light. The fluxesinvolved in sucrose synthesis are lower, so that the observed

FIG. 8. Alterations of metabolites dur-ing an enhancement of photosynthesis insaturating light, following a short periodin low light. Leaf discs (5) from plantswhich had been preilluminated (400 ,gE.m2.s') for 6 h were illuminated (1500gE._M2.s-') for 10 min, before loweringthe light to 180 IAE-M2_s-' for 30 s, andthen returning to 1500 gE-m2 s'.Other conditions were saturating CO2(see "Materials and Methods") and 15°C.Each time point was a separate incuba-tion of 5 discs, and was stopped by open-ing the electrode and transferring thediscs into liquid N2 within 2 s in theprevailing light intensity. A, 02 evolu-tion; B, adenine nucleotides; C, PGA andtriose-P; D, Rul,5P2 and Fru1,6P2; E,Glc6P and Fru6P; F, summed P-ester.

Time (min)

changes of Glc6P represent about 8 s of steady state photosyn-thesis, as do the estimated changes of free Pi.

DISCUSSION

Surplus Capacity for Electron Transport and Photophosphor-ylation. These results provide evidence that the maximal rate ofphotosynthesis in saturating light and CO2 does not reflect aceiling set by the capacity for electron transport and photophos-phorylation. When leafdiscs photosynthesizing in saturating lightare subjected to a short interruption in low light, there is atransient enhancement ofthe rate of02 evolution after returningto high light. This enhancement can be up to 50% above thesteady state rate of photosynthesis, lasts 15 to 20 s and is maximalafter 15 to 30 s interruption in low light rather than darkness.As the components of the electron transport chain ( 12) and thepools of ATP and NADPH in the stroma (5, 16, 20) turn over

1120 STITT

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Table V. Use Rates ofMetabolites dutring Steady State PhotosynthesisThe use rate of selected metabolites in the Calvin cycle is estimated

for a CO2 fixation rate of 136 tmol -mg-' Chl -h-'No. of Molecules Estimated Use

Metabolite Metabolized per RateCO2 Fixed

nmol-mg' Chl-s-'ATP 3 114RubP 1 38PGA 2 75pja 1/3 12.5Glc6PIa 1/6 5

a Turnover estimated for sucrose synthesis in cytosol, if 80% of pho-tosynthate is converted to sucrose.

in less than a second, this enhanced rate of 02 evolution overmany seconds implies that the flux through the electron transportchain, and the production and consumption of ATP andNADPH, are all proceeding more rapidly than during steadystate photosynthesis.The measurements of metabolite levels provide a second line

of evidence that electron transport capacity is not being fullyutilized during steady state photosynthesis in saturating light andCO2. During the period of enhanced photosynthesis, electrontransport is capable of generating 2-fold higher ATP/ADP quo-tients in the leaf than are found during steady state photosyn-thesis. The 3- to 4-fold decrease of the PGA/triose-P ratio alsoprovides evidence for an enhanced performance of electrontransport because the reactions involved in PGA reduction areclose to equilibrium (5) and this ratio can be taken as an indicatorfor the combined (ATP/ADP- Pi) (NADPH/NADP) quotients.The transient enhancement of 02 evolution is not observed in

a limiting steady state light intensity, appears as the light ap-proaches saturating intensities, and becomes even larger as thelight intensity is raised in the range where steady state photosyn-thesis is already light-saturated. When the conditions or plantmaterial were varied, the shift in the light-saturation curve ofphotosynthesis was always accompanied by a shift in the lightintensity at which the transient enhancement appears. Thus, therate of electron transport may be primarily determined by theavailable light input at limiting light intensities, although evenhere it would be premature to rule out a co-limitation by carbonmetabolism in some conditions. However, in saturating light thereactions in carbon metabolism impose a considerable restrictionon the fluxes through electron transport, and the regeneration ofATP and NADPH is depressed below their potential maximum.This can be directly observed as the rate of02 evolution decreasesfollowing the transient enhancement of photosynthesis. Thisinhibition of 02 evolution is accompanied by a decrease of theATP/ADP ratio, and an accumulation of PGA (Fig. 8). Clearly,something is preventing the thylakoid processes from operatingat their full capacity to generate ATP and NADPH. Studies oflight scattering in leaves from spinach and other species also ledto the conclusion that electron transport may not play a directrole in limiting photosynthesis in optimal conditions, as thetransthylakoid pH gradient remained high during photosynthesisin saturating light and CO2 (6). The question arises, what isresponsible for this restriction of electron transport?

Surplus Capacity of the Calvin Cycle. These results also suggestthat the available capacity for Calvin cycle turnover and carbox-ylation is not being fully utilized during steady state photosyn-thesis. The fluctuations ofthe PGA pool are too small to accountfor more than a fraction of the increment in 02 evolution duringthe period of enhanced photosynthesis, implying that Ru 1 ,5P2 isbeing carboxylated faster during the transient enhancement than

during steady state photosynthesis. In agreement, parallel meas-urements of 02 evolution and CO2 uptake have shown that bothchange in parallel during oscillations in the rate ofphotosynthesisof spinach leaf discs (25). The increased rate of carboxylationcannot be due to consumption ofaccumulated Ru 1 ,5P2, becauseRu 1,5P2 even increases during the period ofenhanced photosyn-thesis. Thus, the regeneration of Ru 1 ,5P2 from triose-P must alsobe occurring at higher rates during the transient enhancementthan during steady state photosynthesis. These increased fluxesaround the Calvin cycle do not stimulate electron transport andphotophosphorylation by consuming more ATP and NADPH.Rather, the use of the Calvin cycle capacity in steady stateconditions may even be restricted by the supply of energy fromelectron transport, as shown by the accumulation of PGA andthe low ATP/ADP ratio. During the transient enhancement ofphotosynthesis, the increased rate of electron transport andhigher ATP/ADP ratio may stimulate the Calvin cycle by allow-ing a temporary redistribution of carbon and phosphate out ofPGA into other stromal intermediates lying between triose-P andRu 1,5P2. This will increase the concentration of substrates andlower the concentration of inhibitors. For example, light activa-tion ofthe stromal Fru 1 ,6P2 requires Fru 1 ,6P2 (3), while catalysisby phosphoribulokinase is inhibited by PGA and ADP (9).Similarly, these changes could stimulate activity of Ru 1,5P2carboxylase, as an increase of Ru 1 ,5P2 in the range of 80 to 130,umol-mg-' Chl should allow increased rates of carboxylation(see Ref. 24 for discussion), while PGA is known to inhibitRul,5P2 carboxylase (1).

Possible Limitation by Pi Availability. During photosynthesis,triose-P are produced from CO2 and Pi in the chloroplast, whileconversion of triose-P to end products like starch and sucroserecycles Pi so that photophosphorylation and photosynthesis cancontinue. Many features of the results presented here could beunderstood if sucrose synthesis were limiting the rate of photo-synthesis, and the stromal Pi concentration were so low duringphotosynthesis in saturating light and CO2 that it restricts therate of photophosphorylation. This would explain why electrontransport operates at below maximum capacity during steadystate photosynthesis, even though there are high levels of PGA,low ATP/ADP ratios (Fig. 8), and large transthylakoid pH gra-dients (6).The transient enhancement ofphotosynthesis, increased ATP/

ADP ratios and lowered PGA/triose-P ratios following a briefinterruption in low light can also be explained if Pi availabilitywere to restrict the steady state rate of photosynthesis. Starchsynthesis is rapidly inhibited when the light intensity is decreased,but the synthesis of sucrose is only gradually inhibited (22).Consequently, the pools of phosphorylated intermediates in-volved in the synthesis of sucrose in the cytosol (e.g. Glc6P)decrease and free Pi increases. Since Pi and metabolites like PGAor triose-P are rapidly exchanged via the phosphate translocator(10), the pools of esterified metabolites in the stroma are alsodecreased (22). The estimated increment of 100 nmol Pi/mg Chl(Fig. 8F) would represent an increase of more than 2 mm in theaverage Pi concentration in the cytosol and stroma. Upon re-turning to high light, this increment of Pi could support steadystate photosynthesis for about 8 s (see above) or, alternatively,could support a 40% stimulation of steady state photosynthesisfor about 20 s (Fig. 8A). As Pi is reincorporated, the free Piconcentration will decrease again, and this could contribute tothe subsequent restriction of photosynthesis. The alterations ofesterified phosphate do not exactly match the rates of 02 evolu-tion in the later oscillations (Fig. 8, A and F). This may be dueto changes in the stroma being masked in measurements whichhave been made on unfractionated leaf material, but couldindicate that additional regulatory mechanisms have been trig-gered during these rapid changes in the rate of photosynthesis.

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Two other lines of evidence also suggest that availability of Pican restrict photosynthesis in leaves under conditions of saturat-ing light and CO2. In a following article (17) it will be shown thatspinach leaves show 02 insensitive photosynthesis in saturatinglight at l5°C with 500 to 550 ,ul/L CO2 and that this is accom-panied by an accumulation of PGA, and a decreased ATP/ADPratio, as expected (16) if Pi availability is restricting the rate ofphotosynthesis. In independent studies, Walker and Sivak (25)have also shown that oscillations in the rate of photosynthesisonly appear in conditions when light and CO2 approach saturat-ing levels, but that these oscillations can be induced under lowerlight intensities if mannose is supplied to leaves to sequesterphosphate, and can be reduced or abolished if phosphate is fed.

Studies which have been presented elsewhere (23) reveal howa Pi limitation could develop in vivo. It is striking that themaximal capacity for sucrose synthesis in spinach leaves resem-bles the maximal rates of photosynthesis in saturating light andCO2. Using extraction methods which require 5 s and assayingin optimal conditions yields activities at 20°C of about 40 and108 umol hexose.mg-'Chl-h-' for sucrose P synthase and thecytosolic fructose-1,6-bisphosphatase, respectively (I Wilke, un-published data; 23). The activity of sucrose P synthase resemblesthe maximal rate of photosynthesis, and it is unlikely that morethan 30% of the Frul,6Pase activity is attained in the presenceof Fru2,6P2 and AMP in vivo (23). Moreover, these activities willonly be attained when the enzymes are activated by high concen-trations of phosphorylated intermediates, and the inhhibitor Piis low (7, 23). This means there is a potential conflict betweenthe requirements for rapid sucrose synthesis and for rapid pho-tophosphorylation, as an increasing triose-P/Pi ratio in the cy-tosol decreases the rate at which Pi can return to the chloroplastvia the phosphate translocator (I 1). Provided the capacity forsucrose synthesis is well in excess of the rate of photosynthesisthen moderate alterations of phosphorylated intermediates andPi should allow sucrose synthesis to be activated without therecycling of Pi to the stroma becoming limiting. However, thenearer the rate of photosynthesis approaches the maximal capac-ity for sucrose synthesis, the higher the ratio of triose-P/Pi whichwill be required in the cytosol to allow matching rates of sucrosesynthesis, and the greater the possibility that recycling of Pi tothe stroma starts to limit the rate of photosynthesis.

CONCLUSIONS

In these experiments, a combination of nondestructive tech-niques and biochemical analysis has been used to show that aleaf photosynthesizing in saturating light and CO2 can haveexcess capacity for electron transport, which is not utilizedbecause reactions in carbon metabolism restrict the rate of pho-tosynthesis. In the case which was examined in detail, there wasalso surplus capacity for turnover of the Calvin cycle, and thelimitation may reside in the ability to recycle Pi during sucrosesynthesis in the cytosol. To demonstrate this limitation, the leafmaterial was moved from the conditions in which it grew intothe conditions in which higher light and CO2 were present. It isnot surprising that an internal ceiling is met following impositionof far higher photosynthetic rates, and it is unlikely that thisparticular limitation is of major importance in the conditions inwhich the spinach originally grew. Moreover, a different choiceof the conditions in which the plants grew and in which theexperiments were carried out would probably have produced adifferent answer to the question of what limits photosynthesis.This does not mean that a concept of limiting factors in photo-synthesis is without meaning. Rather, it emphasizes the need forprocedures such as those described here or by Walker andcoworkers (18, 19) which allow internal limitations to be identi-fied, albeit first in extreme conditions when such limitations canbe more easily defined. Such procedures will provide a way of

investigating how plants vary in their capacity for carbon metab-olism and how this capacity varies in response to changingconditions, and will also make it possible to study aspects ofcarbon metabolism in leaves under conditions where it is knownthat these reactions contribute to determining how rapidly theleaf is photosynthesizing.

Acknowledgments-I am grateful to T. Sharkey, H. W. Heldt, D. A. Walker, U.Schreiber and U. Heber for many discussions.

LITERATURE CITED

1. BADGER M, G LORIMER 1981 The interaction of sugar phosphates with thecatalytic site of ribulose-1,5-bisphosphate carboxylase. Biochemistry 20:22 19-2225.

2. BERRY J, 0 BJORKMAN 1984 Photosynthetic response and adaptation totemperature in higher plants. Annu Rev Plant Physiol 31: 491-543

3. BUCHANAN BB 1980 Role of light in the regulation of chloroplast enzymes.Annu Rev Plant Physiol 31: 341-374

4. DELIEU T, DA WALKER 1981 Polarographic measurement of photosyntheticmeasurement of photosynthetic 02 evolution by leaf discs. New Phytol 89:165-178

5. DiETz K-J, U HEBER 1984 Rate limiting factors in leaf photosynthesis. I.Carbon fluxes in the Calvin cycle. Biochim Biophys Acta 767: 432-443

6. DIETz K-J, S NIEMANIS, U HEBER 1984 Rate limiting factors in leaf photosyn-thesis. Biochim Biophys Acta 767: 444-450

7. DOEHLERT DC, SC HUBER 1984 Phosphate inhibition of spinach leaf sucrosephosphate synthase as affected by glucose 6 phosphate and phosphoglucoseisomerase. Plant Physiol 76: 250-253

8. EDWARDS GE, DA WALKER 1983 C3, C4; Mechanisms, and Cellular andEnvironmental Control of Photosynthesis. Blackwell Science Publications,London

9. FLUGGE Ul, M STITT, M FREISL, HW HELDT 1982 On the participation ofphosphoribulokinase in the light regulation of CO2 fixation. Plant Physiol69: 263-267

10. HAMPP R, M GOLLER, M ZIEGLER 1982 Adenylate levels, energy charge andphosphorylation potential during dark-light and light-dark transitions inchloroplasts, mitochondria and cytosol of mesophyll protoplasts from Avenasaliva L. Plant Physiol 69: 448-455

I 1. HEBER U, HW HELDT 1981 The chloroplast envelope: structure, function androle in leaf metabolism. Annu Rev Plant Physiol 32: 139-168

12. JUNGE W 1977 Physical aspects of light harvesting, electron transport andelectrochemical potential generation in photosynthesis ofgreen plants. In: ATrebst, M Arnon, eds, Encyclopedia ofPlant Physiology, NS, Vol 5. Springer,Berlin, pp 59-93

13. LILLEY R McC, DA WALKER 1974 The reduction of 3-phosphoglycerate byreconstituted chloroplasts and by chloroplast extracts. Biochim Biophys Acta368: 269-278

14. LOWRY OH, JU PASSONEAU 1972 A Flexible System of Enzymatic Analysis.Academic Press, New York

15. SANTARIUS KA, U HEBER 1965 Changes in the intracellular levels of ATP,ADP, AMP and Pi and regulatory functions of the adenylate system in leafcells during photosynthesis. Biochim Biophys Acta 102: 39-54

16. SHARKEY TD 1985 Photosynthesis in intact leaves of C3 plants: physics,physiology and rate limitations. Bot Rev 51: 53-105

17. SHARKEY TD, M STITT, R GERHARDT, D HEINEKE, K RASCHKE, HW HELDTLimitation of photosynthesis by carbon metabolism. II. 02 insensitive pho-tosynthesis. Plant Physiol 81: 1123-1129

18. SIVAK MN, DA WALKER 1984 What can be learned about the regulation ofphotosynthesis from multiple measurements: state ofthe art and perspectives.In: B Jeffcoat, AF Hawkins, AD Stead, eds, British Plant Growth RegulatorGroup, Monograph 12. Parchments Ltd, Oxford, pp 3-17

19. SIVAK MN, DA WALKER 1985 Chlorophyll a fluorescence: can it shed light onfundamental questions in photosynthetic carbon dioxide fixation. Plant CellEnviron 8: 439-448

20. STITT M, W WIRTZ, HW HELDT 1980 Metabolite levels in the chloroplast andextrachloroplast compartments of spinach protoplasts. Biochim BiophysActa 593: 85-102

21. STITT M, R McC LILLEY, HW HELDT 1982 Adenine nucleotide levels in thecytosol, mitochondria and chloroplasts of wheat leaf protoplasts. PlantPhysiol 70: 971-977

22. STITT M, W WIRTZ, HW HELDT 1983 Regulation of sucrose synthesis bycytoplasmic fructosebisphosphatase and sucrose phosphate synthase duringphotosynthesis in varying light and carbon dioxide. Plant Physiol 72: 767-774

23. STITT M, HW HELDT 1985 Control of photosynthetic sucrose synthesis byfructose 2,6 bisphosphate. VI. Regulation of the cytosolic fructose- 1,6-bisphosphatase in spinach leaves by an interaction between metabolic inter-mediates and fructose 2,6 bisphosphate. Plant Physiol 79: 599-608

24. VON CAEMMERER S, GD FARQUHAR 1986 Kinetics and activation of Rubiscoand some preliminary modelling of RuBP pool sizes. In J Viil, A Laisk, eds,Kinetics of Photosynthetic Carbon Metabolism in C3 Plants. ScientificCouncil on Photosynthesis, Acadamy of Science, USSR, Tallinn. In press

25. WALKER DA, MN SIVAK 1985 In vivo chlorophyll a fluorescence transientsassociated with changes in the CO2 content of the gas phase. In RL Heath, JPreiss, Regulation of Carbon Partitioning in Photosynthetic Tissue. Ameri-can Society of Plant Physiologists, Washington, DC, pp 93-208

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