Relationship betweenPhotosynthesis and Respiration · photosynthesis and dry weight ofliving material on the plant. Thisservedasabasis for developmentofthetheoretical concepts ofgrowth

Plant Physiol. (1983) 71, 574-5810032-0889/83/7 1/0574/08/$00.50/0

Relationship between Photosynthesis and RespirationTHE EFFECT OF CARBOHYDRATE STATUS ON THE RATE OF CO2 PRODUCTION BY RESPIRATIONIN DARKENED AND ILLUMINATED WHEAT LEAVES

Received for publication June 21, 1982 and in revised form October 25, 1982

JOAQUIN AZCON-BIETO AND C. BARRY OSMONDDepartment ofEnvironmental Biology, Research School of Biological Sciences, The Australian NationalUniversity, P.O. Box 475, Canberra City, A. C. T. 2601, Australia

ABSTRACT

The rate of dark CO2 efflux from mature wheat (Triium aestivum cvGabo) leaves at the end of the night is less than that found after a periodof photosynthesis. After photosynthesis, the dark CO2 efflux shows com-plex dependence on time and temperature. For about 30 minutes afterdarkening, CO2 efflux includes a large component which can be abolishedby transferring Muminated leaves to 3% 0 and 330 microbar CO2 beforedarkening. After 30 minutes of darkness, a relatively steady rate of CO2efflux was obtained. The temperature dependence of steady-state darkCO2 efflux at the end of the night differs from that after a period ofphotosynthesis. The higher rate of dark CO2 effiux followin photosyn-thesis is correlated with accumulated net CO2 assimilation and with anincrease in several carbohydrate fractions in the leaf. It is also correlatedwith an increase in the CO2 co_msation point in 21% 02, and an increasein the light compensation point. The interactions between CO2 efflux fromcarbohydrate oxidation and photorespiration are discussed. It is concludedthat the rate of CO2 efflux by respiration Is comparable in darkened andilluminated wheat leaves.

The rate of CO2 efflux by respiration from single leaves andwhole plants in the dark is linearly related to the rate of previousphotosynthesis when the latter is varied by changing the light levelor CO2 concentration (18, 21). McCree (21) fitted an empiricalequation in which the rate of dark CO2 efflux is proportional tophotosynthesis and dry weight of living material on the plant.This served as a basis for development of the theoretical conceptsof growth and maintenance respiration by Penning de Vries (24).Both of these components of respiration are thought to involve,principally, carbohydrate oxidation through glycolysis, the pen-tose phosphate pathway and the tricarboxylic acid cycle. Growthrespiration appears to be less sensitive to temperature than main-tenance respiration (22). Explanations of the complex interactionsbetween photosynthesis, temperature, and dark respiration areuncertain, although it is probable that the interaction is mediatedby carbohydrate level (6, 8).The extent to which tricarboxylic acid cycle respiration contin-

ues in the light in green leaves is uncertain. Graham (12) con-cluded that biochemical evidence suggests the tricarboxylic acidcycle continues to operate in illuminated leaves at about the samerate as it does in darkness. Physiological evidence is contradictory;some experiments are best explained in terms of significant CO2efflux in illuminated leaves from sources other than photorespir-ation (3, 12) whereas others suggest that these other sources arenegligible (7, 19, 23). The lack ofmethods for direct measurementofthe rate ofrespiration during photosynthesis greatly complicates

the resolution of this question. A new experimental approach isattempted in this paper.The experiments described here investigate the relationship

between photosynthesis in mature wheat leaves, its products (par-ticularly carbohydrates), temperature, and CO2 efflux by respira-tion in the light (Rd) in the dark (Rn) (see Ref. 3 for terminology).We conclude that the increase in dark CO2 efflux (Rn) after aperiod of photosynthesis is correlated with the amount of carbo-hydrate synthesized, and that the temperature dependence of darkCO2 efflux varies with leaf carbohydrate concentration. We alsoconclude that the rate ofrespiration in the light (Rd) is comparableto Rn and that it makes a significant contribution to total CO2efflux in illuminated wheat leaves.

MATERIALS AND METHODS

Plant Material. Triticum aestivum cv Gabo, Viciafaba, Eucalyp-tus grandis, and Lolium perenne plants were grown from seed in acabinet in 12-cm pots of soil. They were watered twice a day, andwere fertilized every other day with nitrate-type Hewitt solution.Total nitrate concentration was 12 mm. Quantum flux (400-700nm) was 500 to 600 pEm2.s-'. The day/night temperatureregime was 25/20°C with a daylength of 13 h. RH was between60 and 80%. Mature leaves of 30-d-old plants were selected at theend of the night period.Gas Exchange Techniques. CO2 and water exchanges were

measured in leaves using an open system gas analysis apparatus,utilizing an IR CO2 analyzer (model 865, Beckman Instruments)which was operated in both differential and absolute modes, anda dew point hygrometer (model 880; Cambridge Systems, Wal-tham, MA).One or two attached intact leaves were inserted in a well-

ventilated aluminum leaf chamber (boundary layer conductanceto diffusion of water vapor was 2.2 mol_m-2_s-'). Illuminationwas provided by a 2.5 kw water-cooled, high-pressure, xenon-arclamp (model XBF 2500, Osram), the UV and IR componentsbeing removed with a Schott KG 2B filter. Quantum flux (400-700 nm) incident on the leaf was 1000 ,uE_m 2_s- , and it wasmeasured with a quantum sensor (model LI-190 SR, LambdaInstruments). Leaf temperature, which was controlled by circulat-ing water through a jacket, was measured with two copper-con-stantan thermocouples (0.1 mm diameter) in contact with thelower surface.

Air with the desired partial pressure of CO2 was obtained byinjection of 5% CO2 in air into C02-free air through a stainlesssteel capillary tubing. A self-venting pressure regulator (modelMAR-1P; Clippard Minimatic, cincinnati OH) and a pressuregauge were used to control the injection rate. C02-free air withdifferent 02 concentrations was obtained by mixing compressedambient air with N2 from a cylinder, and then by passing the

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RESPIRATION AND PHOTOSYNTHESIS

resulting gas through two columns of soda lime (Carbosorb, self-indicating; BDH Chemicals Ltd, Poole, England). The 02 concen-tration was measured with an 02 electrode (model 5331, YSI). Thegas was then humidified in a gas washing bottle with a scintereddisc. The dew point of the gas was maintained by passing the gasthrough a glass condenser, the temperature of the latter beingcontrolled by circulating the water from a temperature-controlledwater bath. Air flow through the leaf chamber was monitoredwith a mass flowmeter (model AFSC-IOK; Hastings, Hampton,VA). Flowmeters with needle valves were used to distribute gasflow throughout the system. Copper tubing was used in the circuit.The outputs of all sensors were registered on a digital voltmeter,

and the outputs from the CO2 analyzer and dew point hygrometerwere continuously recorded. The outputs from the sensors allowedcalculation of the rate of net CO2 assimilation, stomatal conduct-ance, intercellular CO2 partial pressure, and dark CO2 efflux inair. All these parameters were calculated according to the equa-tions given in (30). Leaf area was measured with an electronicplanimeter (model LI-3050A, Lambda Instruments).Measurement of the CO2 and Light Compensation Points. The

CO2 compensation point, r, was either measured by using a closedsystem and allowing the leaf to equilibrate with its CO2 at-mosphere, or by interpolation of a curve of 'net CO2 assimilationversus intercellular CO2 partial pressure' to zero assimilation. Thegas exchange apparatus was modified by the inclusion of a closedsystem. A metal bellows pump (model MB-21E; Metal BellowsCorp., Sharon, MA) circulated the air through the system. Plugvalves (model B-4P4T; Nupro Co., Cleveland, OH) were used tomanually switch from open to closed system or vice versa. Bothmethods were compared in the same leaf of wheat at two 02concentrations (21% and 3%) yielding identical results (not shown).When r was measured in closed system, its value was taken

after 60 min in order to obtain steady-state values; then, the rateof dark CO2 efflux was measured 30 min after the light wasswitched off.The light compensation point was measured by interpolation of

a curve of 'net CO2 assimilation versus quantum flux' to zeroassimilation. Light intensity was changed by interposing copperscreens.

Effect of a Period of Photosynthesis on the Rate of CO2 Effluxin the Dark. A pair of mature wheat leaves from the same plantwas enclosed in the photosynthetic chamber and the rate of darkCO2 efflux in ambient air was monitored for 2 h at the end of thenight and after a period of photosynthesis of 6.25 h at ambientCO2 and 02 levels. Leaf temperatures were 13.5, 20, 24, 27, and30°C in darkness. Leaf temperatures during the light period were2 to 40C higher than in the dark period. This experiment wasrepeated three times at every leaf temperature. A different plantwas used every time.

In a similar experiment, the rate of dark CO2 efflux wasmonitored for 1 h at the end of the night and after a period ofphotosynthesis of 6.25 h in which the 02 concentration in the airin the last 20 min was 3%. In this experiment, temperature waskept constant in the light and in the dark. In the experimentsperformed at 30°C, the 02 concentration in the dark period was21% or 3%. Three replicates were done at every 02 concentration,but no difference was found in the time course of dark CO2 effluxafter the light period (not shown). This experiment was alsoperformed at 200C in leaves selected from six plants, but the 02concentration in the dark period was 21%.The results of these experiments are shown in Figures 1 and 2.

The curves representing the time course of dark CO2 efflux areaverages of three or six individual curves. The statistical variationof the data was very small and it is not shown. The standard errorswere less than 5% of the absolute values and ranged between 0.01and 0.08 ,umol CO2*m2* s', the lower values being more commonspecially at lower temperatures.

In-5

E

x

G)

-

-

o

2 _ \ 30 C

27C

24C

0 II20 C

13.5 C

IIIU 30 60 90 120

Time in the dark (minutes)FIG. 1. Time course of dark CO2 efflux of mature wheat leaves after a

period of photosynthesis of 6.25 h at ambient CO2 and 02 levels ( ).(-- -), rate of dark CO2 efflux at the end of the preceding nightperiod.

Relationship between Dark CO2 Efflux and Leaf CarbohydrateStatus. Mature wheat leaves were allowed to photosynthesize forvariable periods of time up to 7 h, at ambient and high (800 ,ubar)external CO2 partial pressures; then, the rate of dark CO2 effluxwas measured 30 min after the termination of the photosyntheticperiod. Leaves were immediately killed in liquid N2 and storedfrozen for carbohydrate determination (see below). Leaf temper-ature was 21°C in darkness and 23.50 in the light.

Study of the Temperature Dependence of Dark CO2 Efflux. Therate of dark CO2 efflux of mature wheat leaves selected at the endof the night was measured at different temperatures up to 40°C.The first measurement was made at 11 C. Other leaves wereallowed to photosynthesize for 6.25 h at 22°C, at ambient CO2and 02 levels. Then, dark CO2 effux was measured at 20°C, 30min after the light was switched off; leaf temperature was steeplyraised to 42°C in some experiments or decreased to about 8°C inother experiments.

Carbohydrate Determination. Leaves killed in liquid N2 werefreeze-dried. Carbohydrates were extracted in boiling water for 15min and analyzed using an enzymic method. Free glucose plusfructose were measured from the leaf extract using a glucose-specific assay (Calbiochem-Behring Glucose s.v.r. No. 870104),after converting fructose to glucose with P-glucoisomerase (SigmaP-538 1). Glucose was converted to glucose 6-P by hexokinase, andthen oxidized by glucose 6-P dehydrogenase, reducing a molarequivalent of NADP. The change in A at 340 nm is proportional

Iri

575

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AZCON-BIETO AND OSMOND

%02 30C and increased within 30 min to the level of leaves kept in 21% 02

- 21 30C throughout the photosynthetic period. If the 02 concentration2 - 2during the measurement period in the dark was lowered to 3%,

the result was the same.3 -- >- _ These experiments suggest that the initially high rates of dark

CO2 efflux are in part due to O2-dependent processes in the light1 - - - - - - - period. Presumably, large pools of photorespiratory intermediates

continue to be decarboxylated in the dark, for about 30 min at

020°C and 30°C.The rate of dark CO2 efflux 30 min after the termination of the

20 C photosynthetic period increased in proportion with the total net/°°2 CO2 assimilation which had occurred during this period (Fig. 3).

2 21 Dark CO2 efflux was also positively correlated with specific leaf------ weight which greatly increased during the light period due to the

accumulation of products derived from photosynthesis, mostly1 /3 carbohydrates (not shown). Dark CO2 efflux was correlated with

several leaf carbohydrate fractions (Fig. 4). The relationship be-_ _ _I tween dark CO2 efflux and fructosans was not investigated here.

0 10 20 30 40 50 60 The temperature dependence of dark CO2 efflux at the end ofthe night period was compared with that of leaves after 6.25 h of

Time in the dark (minutes) photosynthesis in air. At the end of the night, dark CO2 effluxshowed an exponential relationship with temperature, with a

Time course of dark CO2 efflux of mature wheat leaves after a single apparent activation energy, Ea, of 12.9 kcal . mol' in thehotosynthesis of 6.25 h in which the 02 concentration in the air range from I I to 40°C (Fig. 5). However, CO2 efflux after a long20 min was 3% (-). The time course of dark CO2 efflux after period of photosynthesis presented a very different pattern inf 6.25 h of photosynthesis at ambient 02 concentration (see Fig. response to temperature. Its rate was higher at all temperatures,for comparison --- -). The 'end of night' level of dark CO2 but Ea declined in the range from 200C to 400C while it increased

[so shown (- ). in the range from 10°C to 20°C.

icose concentration in the range from 0 to 10 ng-mf' Relationship between Dark CO2 Effix, Carbohydrate Status,neasured with Varian 634 spectrophotometer. The assay da CO2 and Light Com ntion Points. Coincident with theermed wt n.634strophotomterb. a

increase in the rate of respiration following a period of photosyn-

rampled at tmper itatedbn abqot thesis, r in 21% 02 also increased in the same leaf (Table I; Fig.

ampe. inshedrwhen chatnge leabsor 6). Interestingly, the values for r showed the largest increases afterurred. Sucrose was hydrolyzed by incubating the leaf .- l but370C for 2 h in a water bath with invertase (Sigma I- treatments i which photorespmation would have been least but. N acetate buffer (pH 4.6). Inasmuch as invera also the rate of carbohydrate formation would have been maximalreported to hydrolyze some small fructosans (29), the (800

abar CO2f 2% O2). These correlations were confirmed in

sulting from the action of this enzyme are described as another set ofexpenments in which aleafCwas initially illuminatedtase fraction. This fraction was obtained by subtracting for 4 h in air containing 750 obar CO2, 21% 02 (ow photorespir-nt offree glucose plus fructose from total glucose assayed ation), then in air containing low CO2 prereses (slightly aboveincentration was obtained by incubating the leaf extract F) for a second period of 4 h (high photorespiration). Table IIFor 48 h in a water bath with 0.5% 'Clarase 900' (Miles shows agin that F was higher after hichperiod whichthe ratesries) in 0.1 N acetate buffer (pH 4.6). Clarase 900 is a of photorespiration were lower and the rates of carbohydrate)f several digestive enzymes which hydrolyze starch and formation were higher.) hexoses. Starch was obtained by subtracting the glucose Measurements of r from many experiments in which dark CO2

in glucose plus fructose and invertase fractions from total glucoseassayed in the Clarase digest.

RESULTS

Properties and Temperature Dependence of Dark CO2 Efflux.Dark CO2 efflux measured after a period of photosynthesis wasmuch higher than at the end of the preceding night period (Fig.1). This effect occurred at all temperatures studied. However, theincrease in total dark CO2 efflux due to the effect ofphotosyntheticactivity was relatively higher at lower temperatures (eg. 20°C).At higher temperatures (e.g. 300C), the rate of dark CO2 effluXreturned to the level at the end of the night within 2 h. At lowertemperatures, it took longer (e.g. 5 h at 200C). That is, the effectof the photosynthetic activity on dark CO2 efflux was moreaccentuated and lasted longer at lower temperatures.

It was commonly found that the rate of dark CO2 efflux washigher in the first 30 min after illumination and did not attain asteady slow rate of change until after about 60 min of darkness.When the 02 concentration of the atmosphere was lowered from21% to 3% during the last 20 min of the light period and the rateof dark CO2 efflux measured in 21% 02, a different pattern wasobtained (Fig. 2). The rate of dark CO2 efflux was initially low

c4AE

E

M.

x

aCMoIn

1.S

0

0 100 300 500 700

Integrated net CO2 assimilation (mmol rmf2)FIG. 3. Relationship between dark CO2 efflux and integrated net CO2

assimilation in mature wheat leaves. (0), Leaves selected at the end of thenight; (0), leaves photosynthesizing at ambient CO2 levels (A), leavesphotosynthesizing at 800,ubar CO2.

-

E

0

x

-6

4)

FIG. 2.period of pin the last:a period of1) is givenefflux is at

to the gluand was rwas perfofrom the 4ance occextract at5875) in Chas beensugars resthe invertthe amouiStarch co:at 370C fLaboratonmixture osucrose to

1.Ok1.0

*0 -

0.5F- .00

ons *- r - - -

576 Plant Physiol. Vol. 71, 1983

A

0 /




Carbohydrate concentration ( g glucose equiv m-2)

E

0

E

C

a-

50 100 150 200 250

Carbohydrate concentration (m mol Cmr2)FIG. 4. Relationship between dark CO2 efflux and several carbohydrate fractions in mature wheat leaves.

Leaf temperature (°C)

I

0

-1

50 40 30

310 320 330

20

340

10

350

0

360

47'

E2

E

1 x

U0)

00.3 --v.l.0a)

Inverse of absolute temperature x105

FIG. 5. Arrhenius plots for dark CO2 efflux of mature wheat leaves selected at the end of the git period (0) or at the end of a period ofphotosynthesis of 6.25 h at ambient CO2 and 02 pressures (A). Apparent activation energies (E.) are expressed in kcal- mol'. They can be convertedto Qlo values by using the formula 'log Qio = 2190.EJ/T T2,' where T,-T2 = 10 K.

effiux (R.) was varied by varying CO2 and 02 partial pressuresduring the period of photosynthesis are shown in Figure 7. Ex-trapolation of this relationship to zero R. presumably yields thephotorespiratory component of r in these mature wheat leaves.The correlation between an increase in r and R. following a

period of photosynthesis was also observed at temperatures otherthan 21PC (e.g. 15°C and 30°C) (data not shown) and in otherspecies (Table III).The increase in r following a period of photosynthesis was

reflected in a decrease in net rate of photosynthesis over a rangeof CO2 partial pressures and was not due to a change in the slopeof the curve of net CO2 assimilation versus intercellular CO2

partial pressure (Fig. 6). The displacement of this curve was 1.0± 0.2,umol C02-m 2-s , which is an average value obtained infour experiments including that shown in Figure 6. This valuecompared well with the increase in the rate of dark CO2 effluxobserved after a period of photosynthesis. The rate of CO2 effluxinto C02-free air in the light was also higher following a period ofphotosynthesis (Fig. 6).The light compensation point also increased in the same leaf

after a period of active photosynthesis. Figure 8 shows the corre-lation between the light compensation point and dark CO2 effluxwhich was varied by the period of prior photosynthesis underdifferent conditions of temperature and CO2 partial pressure.

A~~~~~~~

N.,

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AZC5N-BIETO AND OSMOND

Table I. r and Dark CO2 Efflux, R,, Measured at the End ofthe Night and after a Period of Photosynthesis of 5Hours in the Same Leafof Wheat.

r was measured in closed system (see "Material and Methods"). r and R. were measured in 21% 02. Leaftemperature was 21°C in the light and in the dark. Net CO2 assimilation rates were about 24 (A) and 30 (B) nmo1C02m .2.s'. The values shown are means ± SE of three to four independent experiments.

CO2 and O2 Levels during the Photosynthetic At the End of the Night After 5 h LightPeriod F F

Abar JIMol CO2. pbarJMol CO2~~ubar -2.s-I pbr -2 -Imsm *sA. 370 ubar C02, 21% 02 35 ± 1.5 0.65 ± 0.12 38 ± 0.5 0.94 ± 0.04B. 800 ,ubar CO2, 21% 02 or 800 ,ubar CO2, 2% 36.5 ± 2.5 0.62 ± 0.03 42 ± 1.5 1.18 ± 0.16

02

CA

E

E0E

0

-

z)z

8s

6L

Before

30After

40 50 60 70 80 90

Intercellular CO2 partial pressure (pbar)

CO2 Evolutionin CO2 - free air

FIG. 6. Curve of net CO2 assimilation versus intercellular CO2 partial pressure measured at the end of the night and after a period of photosynthesisof 3 h at 800 g2bar CO2 in the same leaf of wheat. Leaf temperature was 21 'C. Measurements proceeded from high to low CO2 partial pressures, asindicated by the arrows.

Table II. r and R. ofa Mature Wheat LeafMeasured after TwoConsecutive Periods ofPhotosynthesis of4 Hours at High and Low C02

Partial Pressures02 concentration was 21%. The rates of net CO2 assimilation during the

firt (A) and second (B) periods were about 23 and 2 jAmol C02m2s1,respectively. Two independent experiments were performed. The rest ofconditions were the same as in Table I.

C02Partial Pressure during the r RPhotosynthetic Period

,Abar pAmol CO2 m-2.S1A. 750 ubar CO2 43 ± 1 1.48 ± 0.02B. 50-75 ,barCO2 37 ± 2 0.80 ± 0.05

DISCUSSION

Prperties and Temerature D epedence of Dark CO2 Eflux.Dark C02 efflux (R.) of mature wheat leaves increased consider-ably after a long period of photosynthesis, as did that of tomatoleaves (18). At least two groups of substrates contributed to theCO2 efflux. Because 15% to 20% of the CO2 evolved in the first 30min ofd ss was abolished ifleaves were kept in low 02 duringthe latter part of the photosynthetic period, we conclude that thisCO2 are from photorespiratory substrates. In this sense, thelevels of glycine measured in wheat leaves during the ligt period,1.5 to 2 mmol-m2 (M. Berger, personal communication), are high

enough to sustain glycine decarboxylation in the dark for about30 min at the rates observed in our experiments. This observationcontrasts with the idea that the photorespiratory postilluminationburst in leaves is restricted to the first 2 to 5 min in darkness, ascommonly found in many species including wheat (1 1). However,this discrepancy may be explained by the short length of thepreceding light period utilized in previous studies (often only afew min) compared to the present experiments. The remainingCO2 efflux, was closely correlated with several carbohydrate frac-tions. This CO2 presumably arose from tricarboxylic acid cycleand pentose phosphate pathway oxidation of carbohydrate de-rived substrates. A similar correlation between dark CO2 effluxand carbohydrates has also been found in leaves of Cucumis sativa(8).The linear relationship between the rate of photosynthesis and

the subsequent rate of dark CO2 effluX (see also Ref. 18) can beexplained in terms of quantitative changes in carbohydrates, com-mon metabolites to both processes.The enhancement of leaf respiration by carbohydrates cannot

be primarily related to growth requirements since mature leaveswere used. Alternatively, excess respiration may be used forsynthesis of compounds (e.g. amino acids) in the leaf which canbe utilized for growth in other parts ofthe plant and for providingenergy for transport of assimilates (15). However, when the rateof sugar export from the leaf is reduced by e.g. lowering sinkdemand, carbohydrates accumulate in this organ and the rate of





Table III. r and R. ofMature Leaves of Several Plant Species Measured at the End of the Night and after aPeriod of Photosynthesis of5 Hours

A is the rate of net CO2 assimilation. The rest of the conditions were the same as in Table I.

CO2 Pressures during At the End of the Night After 5 h LightSpecies the Photosynthetic A

Period r RFr R,aiMOl C02m2 pLMOI C02m2 JIMOl C02 M2

Abar tiAbar phAbar s0,Eucalyptus gran- 340 13 40.5 0.82 45 1.07

dis 800 17 39 1.20 45 1.50Viciafaba 800 21 38 0.77 41 1.05Lolium perenne 800 22 34 0.65 38 0.87

L-

-0

-._L-

Q0

0.6

c

C0

a-

E0U1

U1

40H 0

01-11

**/0

0

0.0 00 0

_ @000 *.10 *

* * 00in- 0 0

00

0

y=30+9.4 xr = 0.83

30-

0 0.5 1.0 1.5

Dark CO2 efflux, Rn (pmol m2 S1)FIG. 7. Relationship between the CO2 compensation point and dark

CO2 efflux of mature wheat leaves at 21 'C.

respiration increases (2, 14). This suggests that carbohydrate maybe wastefully oxidized in some conditions in the absence of anyapparent major requirement.The rate of respiration extrapolated to a positive value at zero

carbohydrate (Fig. 4), which may represent maintenance respira-tion (24). Respiration at the end of the night, when carbohydratecontent of wheat leaves was very low, may be principally associ-ated with maintenance processes. As other authors have found (6,22), it increased exponentially with temperature. However, whencarbohydrates accumulated in the leaf as a result of the photosyn-thetic activity, the rate ofdark CO2 efflux increased, and the shapeof its temperature dependence changed dramatically, showingdifferent activation energies (Ea) above and below 20°C. It isunlikely that this break in the Arrhenius plot for dark CO2 effluxat high leaf sugar level could be attributed to membrane phasetransitions (26) because mitochondrial respiration is presumablyinvolved in both instances. The mechanism underlying this re-

sponse may involve the effect of substrate concentration on thetemperature dependence of enzymatic reactions. The apparent Eaofenzymatic reaction decreases at low substrates availability sincethe Km of enzymes for their substrates generally increases withtemperature (10). Therefore, a fixed substrate concentration couldbe saturating or limiting depending on temperature, and Ea should

CN-

IEUJ

c._

.5_anC0.aI.)

c

0

-I

20

15p

10k-

5

0° 0.5 1.0 1.5

Dark CO2efflux, Rn (pmol m-2s-1)FIG. 8. Relationship between the light compensation point and dark

CO2 efflux in mature wheat leaves. (0), Leaf temperature, 20°C; externalCO2 partial pressure, 330 ,ubar. (0), 30°C, 330 ,ubar CO2. (A), 20°C, 640Obar CO2. (A), 30°C, 640,ubar CO2.

be consequently affected. Considered as a multienzyme system,respiration could be saturated by substrates at low temperatureafter a period of photosynthesis, and its Ea should be very high.However, E. would decline at higher temperatures as soon assubstrates are present at concentrations close or below the Km ofkey enzymes.These explanations of the interaction between carbohydrate

levels and temperature on respiratory CO2 efflux assume thatthere is a direct regulation of respiration by substrate availability.The data suggest that glycolysis and mitochondrial reactions inleaves are not necessarily limited by the energy charge in a verynarrow range, at least when substrate levels are low (cf. Beevers,4). A similar conclusion was reached by Sagho and Pradet (27)who have shown that 02 uptake of maize root tips varied widelyin response to sugars while the energy charge remained constant.However, these results do not exclude the possibility that respira-tion is regulated in a complex way. As will be reported later, thewheat leaves studied here show a large cyanide resistant compo-nent of respiration when carbohydrate levels are high (J. Azc6n-Bieto, H. Lambers, and D. A. Day, unpublished). The Arrheniusplot of the cyanide-resistant respiration of wheat coleoptile mito-chondria also shows a discontinuity at 17.5°C (20).

Relationship between Dark CO2 Effux and the CO2 and Light

y=13.4xr=0.99

0

0

0

cf%r-

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AZCON-BIETO AND OSMOND

Compensation Points. r of mature leaves of wheat and other C3species varied during the photoperiod, its value being low at theend of the night period, but increasing during the day period.Similar changes of r after periods of light or darkness have beenreported in leaves of wheat (23) and Rumex acetosa (16). Incontrast to these results, r did not vary after prolonged exposureto darkness, leading to starvation in leaves of Nicotiana taba-cum (13).To investigate the nature of the changes in r in wheat leaves,

we varied the CO2 and 02 partial pressures in the atmosphereduring the photosynthetic period to produce different rates ofphotorespiration and photosynthetic carbohydrate formation. Weconcluded that variations in r during the photoperiod were prin-cipally related to processes other than photorespiration, and pre-sumably were associated with respiration, because r and R.increased maximally after periods in which the gas compositionof the air favored high rates of photosynthetic carbohydrateformation and minimal rates of photorespiration (low 02 and highCO2 pressures). Conversely, r and Rn were lower following aperiod in which the rate of photorespiration was maximal and therate of carbohydrate synthesis was reduced (low CO2 and ambient02 pressures). This conclusion is further supported by the strongcorrelation found between r and R. (Fig. 7). From this relation-ship, r has a positive value when R. is zero, which presumablyrepresents the photorespiratory component. Assuming an averagevalue of 1 ,umol C02 m2.s-1 (at 21VC) for Rn, we estimate thatRd, the CO2 efflux due to respiration, contributes about 25% ofthe CO2 efflux measured at r. This value for wheat leaves issimilar to that found in L. perenne (3). However, the contributionof Rd to r is variable, and it is correlated with the carbohydratelevel. This conclusion is consistent with the fact that externallyadded sugars increase the CO2 compensation point and the rate ofrespiration of leaves (28, 29, and unpublished results).The light compensation point of mature wheat leaves also

increased during the day, being correlated with the rate of respi-ration. This relationship extrapolated to the origin suggesting thatrespiration is a major component of the light compensation point.The rate of respiration in the light (Rd) can be estimated from

the displacement on the curve of net CO2 assimilation versusintercellular CO2 partial pressure by varying the rate ofrespirationin the dark (Rn) through changes in the leaf carbohydrate concen-tration. It can be concluded that the rates of Rd and Rn arecomparable in wheat leaves.

Peisker and Apel (23) also analyzed the responses of r, its 02dependence, and respiration in the dark in wheat leaves after adark period and after an extended light period (18 h) at high CO2concentrations. They found that respiration increased followingthe extended period of photosynthesis, that the 02 dependency ofthe CO2 compensation point increased, but that the latter increasedby only 30%o of the value expected on the basis of their model.Our data confirm their observations, but the different analysis ofour data does not support Peisker and Apel's (23) conclusion thatrespiration in the light is inhibited by 70%0. Whether the expecta-tions of their model, or technical discrepancies, are relevant to ourdifferent conclusions remains to be resolved.Graham (12) reviewed the literature and concluded that glycol-

ysis and tricarboxylic acid cycle can operate in illuminated greencells although some modifications probably occur in relation tothe dark pattern. This is suggested by the increase in the malate/aspartate ratio and the different labeling patterns after adminis-tration of radioactive carbon compounds (e.g. CO2, tricarboxylicacid cycle intermediates, amino acids, sugars) into citrate andother tricarboxylic acid cycle intermediates and related com-pounds, such as glutamate, glutamine, etc. (5, 12). The evidenceis consistent with the suggestion that glycolysis and tricarboxylicacid cycle are modified in the light to allow a continuous anapler-otic carbon flow for supplying a-oxoacids which the chloroplast

is unable to make (17). These compounds can be used for a varietyof synthetic reactions including amino acid and lipid formation.Important features of this anaplerotic flow are the probable op-eration of P-enolpyruvate carboxylase in the cytosol and malicenzyme and the mitochondrion to replenish carbon loss from thetricarboxylic acid cycle (1, 9). It is not known if the tricarboxylicacid cycle operates beyond succinate oxidation, and the operationof the mitochondrial electron chain in the light is a more uncertainaspect of the problem. If, however, respiration in leaves in thelight is cyanide insensitive, control ofelectron transport via energycharge is likely to be less effective. The CO2 arising from theabove mentioned reactions (e.g. 1 mol CO2 released/mol of glu-tamine formed) could well be responsible for most of the rate ofrespiration in illuminated leaves observed in our experiments.Non-green cells in the leaf also contribute to Rd, but it is notknown whether photosynthesis exerts the same influence on theirrespiratory metabolism as in green cells.The effect of the photosynthetic activity on the rate of respira-

tion in the light may be mediated by the supply of P-enolpyruvatefrom recently synthesized triose-P or from sugars. The latteralternative seems more unlikely inasmuch as the exogenous glu-cose is not metabolized through glycolysis in illuminated leavesincluding wheat (5, 12). High CO2 concentration enhances thecarbon traffic through the tricarboxylic acid cycle and relatedcompounds, presumably by increasing the supply of substrates forP-enolpyruvate carboxylase (25). This may help to explain whysome authors have failed to find significant CO2 efflux by respi-ration in illuminated leaves into C02-free air conditions (19). Thisalso suggests that respiration in daytime (Rd) may be underesti-mated at the CO2 compensation concentration.

Acknowledgments-We are very grateful to Dr. S. C. Wong for his help indesigning equipment and to Drs. G. D. Farquhar, D. A. Day, and H. Lambers foruseful comments. We also thank Mr. M. Berger for making available unpublisheddata.

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