O2 Specificity of RubisCo

J. Plant Pbysiol.

Vol. 143. pp. 643-650 (1994)

Effect of Temperature on Partitioning of Photosynthetic Electron Flow Between CO 2 Assimilation and O2 Reduction and on the C0 2/0 2 Specificity of RubisCo JALEH GHASHGHAIE

and GABRIEL CORNIe

Lab. Ecologie Vegetale, B£t. 362, Univ. Paris-Sud, 91405-0rsay, France Received August 4, 1993 . Accepted December 6, 1993

Summary

Photosynthetic electron transport rate and partitioning of electrons between COz assimilation and Oz reduction were estimated in vivo at different temperatures using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence emission on intact leaves of both hairy-willow herb (Epilobium hirsutum L.) and French bean (Phaseolus vulgaris L.). In E. hirsutum leaves at low temperatures (below 15 0q, leaf net COz assimilation, A, was stimulated by normal Oz compared with air containing 2 % Oz, while at high temperatures the inhibition of A by normal Oz via stimulation of the oxygenase function of RubisCo masked the stimulatory effect of Oz on A. As a consequence of stimulation of A, the non-cyclic electron flow rate always remained higher in normal air than in air with 2 % Oz. In P. vulgaris, switching to non-photorespiratory conditions by increasing COz concentration stimulated A but did not change electron transport rates, indicating that only the partitioning of electrons between COz and Oz was changed. Two methods used to estimate the total photosynthetic electron transport rate gave similar results. The validity of the technique used was also tested by estimation of the COzlO z specificity factor of RubisCo, S. In both species, the estimated values of S agreed with that of the literature, obtained using spinach enzyme in vitro within a wide range of temperature (18 to 32 0q. However, in E. hirsutum S was considerably higher at low temperatures (below 18 0q. Overall, our results suggest that in both species, at temperatures above 18°C, carboxylation and photorespiration are the main processes consuming photosynthetic electrons, the processes of Oz reduction other than photorespiration being negligible.

Key words: Chlorophyll fluorescence, electron transport, Epilobium, Phaseolus, photorespiration, photosynthesis, quantum yield, temperature. Abbreviations: A = leaf net COz assimilation; C = intercellular COz concentration; F'm = maximum chlorophyll fluorescence measured using saturating pulse under actinic light; Fo = minimum chlorophyll fluorescence; .::IF = difference between steady-state chlorophyll fluorescence and F'm; Fv = difference between Pm and Fo; qP = photochemical quenching; 1A = linear electron flow allocated to COz assimilation; JL = linear electron flow allocated to Oz reduction and reassimilation of photorespired COz; J1 and Jz = total linear electron transport rates calculated using two methods; PPFD = incident photosynthetic photon flux density;
644

JALEH GmSHGHAIE and GABRIEL CORNIC

Introduction

As shown by works published in the past few years, the measurement of chlorophyll fluorescence, together with net CO2 uptake on intact leaves, makes it possible to estimate the photosynthetic electron transport rate in vivo (Weis and Berry, 1987; Gentyet al., 1989). It has also become possible, using the same technique, to estimate the partitioning of electrons between CO2 and O 2 reduction during photosynthesis of an intact C 3 leaf in normal air, and thus the rate of O 2 uptake (v o) by a leaf. This requires assumptions about the stoichiometry between the photosynthetic electron transport and the reduction of O 2 via the glycollate pathway (Peterson, 1989; Cornic and Briantais, 1991; Krall and Edwards, 1992). The use of the quantum yield of photosystem II (PSII) electron flow using the .:W/F'm ratio (~F is the difference between steady state, Fs, and maximum chlorophyll fluorescence, F'm, measured using saturating pulse under actinic light), as proposed by Genty et al. (1989), has proven very convenient since it does not require the measurement of Fo (the minimum chlorophyll fluorescence emission). In this method the relationship between the PSII quantum yield of electron transport (.:W/F'm) and the quantum yield of leaf gross CO2 uptake (~c, ~c is the net CO2 uptake (A) plus dark or light respiration divided by incident or absorbed photon flux density) measured under non-photorespiratory conditions is first established and represents a «calibration curve». In non-photorespiratory conditions the photosynthetic electrons are used mainly to reduce CO 2• This «calibration curve» allows to estimate, on similar leaves, the photosynthetic electron flow (i) by measuring the .:W/F'm parameter, (ii) the amount of light absorbed by the leaves and (iii) taking into account that 4 electrons are used for the photosynthetic assimilation of 1 molecule of CO2 • It is clear, as stressed by Peterson (1989) and Cornic and Briantais (1991), that the estimation of the electron flux allocated to CO2 assimilation and to O 2 reduction by a leaf in normal air, relies on the assumption that the way the photosynthetic electrons are used in the chloroplast does not change the quantum yield of electron transport. This has been recently demonstrated by Gentyet al. (1992). The validity of the techniques used to estimate the photosynthetic electron transport on leaves has been tested by estimating the relative specificity factor of RubisCo, S = (Ko Vel Kc Vo), where Vc and Vo are the maximum velocities for carboxylation and oxygenation and Kc and Ko are the Michaelis constants for CO2 and O 2 in vivo. S can be calculated from the following relation (Laing et al., 1974): (I) in which V o and v c are the rates of O 2 and CO2 reduction calculated as explained above and [C02] and [02] the concentrations, at a given temperature, of oxygen and carbon dioxide dissolved in the leaves, respectively. When the temperature of the leaves is around 25 °C, the calculated values of S are in good agreement with the values measured in vitro on purified RubisCo (Peterson, 1989; Cor-

nic and Briantais, 1991), strongly suggesting that O 2 uptake during photosynthesis at this temperature is mainly due to oxygenase activity of RubisCo. However, there is no data showing that this method can be used at different temperatures. Recently, Krall and Edwards (1992) estimated the electron transport rate of photosynthetic electrons by intact leaves using only the ~F/F'm parameter and an estimation of the light absorbed by PSI!. They demonstrated that this method gives, for C 4 plants (no photorespiration), estimates that were in good agreement with those given by the measurement of leaf net CO2 uptake. However, since this method relies only on the measurement of chlorophyll fluorescence emission under a modulated background of non-actinic light with a low intensity and since usually only the upper face of the leaf is illuminated, the PSII quantum yield that is measured represents only that of the first layer of photosynthesizing leaf cells. The aim of the present work was (i) to compare two approaches used by Cornic and Briantais (1991) and Krall and Edwards (1992) for in vivo determination of whole-chain electron transport within the leaves and allocation of these electrons to CO2 assimilation and O 2 reduction at different temperatures and (ii) to examine if the effect of temperature on the specificity factor of RubisCo estimated from simultaneous measurements of CO 2 exchange and fluorescence on intact leaves at different temperatures is compatible with the published values for the purified enzyme. The experiments were conducted with intact leaves of both hairy-willow herb and French bean. Material and Methods Plant material Epilobium hirsutum L. (hairy-willow herb) and Phaseolus vulga·

ris L. (French bean) were grown in a controlled-environment cham-

ber in pots containing an organic soil watered daily to field capacity; they were also watered with nutrient solution once a week. Photosynthetic photon flux density at the top of the plants was about 380J,Lmol·m- 2 ·s-t, photoperiod 16h, air temperature 24°C (day) and 20°C (night), and relative humidity about 70 %. The experiments were performed on the cotyledonary leaves of IS-day-old plants of French bean and on the 4th or 5th mature leaf counted from the top of I-month-old plants of hairy-willow herb.

CO2 exchange Gas exchange was measured using an open system that has already been described (Cornie and Ghashghaie, 1991). The CO2 concentration was determined using an infra-red gas analyser (BINOS; Leybold Heraeus, Hanau, FRG) and the dew point of partial pressure of water with a dew-point Hygrometer (Elcowa electronique, system 1100 DP; Mulhouse, France). The leaf assimilation chamber was similar to that described by Dietz et al. (198S), which allows measurements on a small area of a leaf (about 3 cm2). The chamber volume was 6.5 mL, air flow in the system was usually 30 L . h -1 and leaf boundary-layer resistance for water vapour was 0.09 s . mm -1. Light was provided to the leaf from a tungsten lamp (with an infrared filter) through one of the branches of a fiber optic system (PAM 101 F; Walz, Effeltrich, FRG) equipped with a Walz chlorophyll fluorometer. This chamber allows measurements of leaf net CO2

In vivo estimation of COz/ O 2 specificity of RubisCo at varying temperature uptake and chlorophyll fluorescence emission on the same part of the leaf. The leaf gas exchange parameters were calculated according to Von Caemmerer and Farquhar (1981). Experiments were started after leaf net CO 2 uptake; leaf transpiration and chlorophyll fluorescence parameters reached a steady-state rate around 26°C in air containing 350 ilL' L -I CO2 and 21 % O 2 under limiting light of 450 and 240 Ilmol'm- 2 's- 1 for E. hirsutum and P. vulgaris, respectively. Leaf net CO 2 uptake on these plants was saturated at about 1200Ilmol'm- 2 's- 1 for (E. hirsutum) and 800Ilmol'm- 2 's- 1 for (P. vulgaris); Care was taken to maintain leaf-air vapour pressure difference constant (VPD around 0.9KPa) by reducing the humidity of the air in the chamber when decreasing the temperature.

Chlorophyll fluorescence Leaf fluorescence emission was measured with a PAM chlorophyll fluorometer (Walz). The fiber-optic system was essentially arranged as by Dietz et al. (1985). The frequency of modulated light was 1.6 kHz. Saturating pulses (1 s) of white light (12500 Ilmol· m- 2 's- l ) were provided by a KL 1500 Schott light source (Schott, Wiesbaden, FRG) activated by a PAM 103 trigger control unit at intervals of 300 s. Three fluorescence levels were measured: Fo, Fs and F'm, where Fo is the minimal fluorescence induced by far red light, Fs is the steady-state fluorescence reached upon continuous actinic illumination and F'm the maximal fluorescence induced by the 1 s saturating pulse under actinic light. From these measurements the ratio ~F/F'm calculated as (F'm-Fs)/F'm gives a relative measurement of the quantum yield of PSII electron flow (Genty et aI., 1989). The quantum efficiency of excitation energy capture by oxidized PSII reaction centers, Fv/F'm, was calculated as (F'm-Fo)1 F'm and the coefficient for photochemical quenching of fluorescence, qP, was calculated from ~F/Fv (Schreiber et aI., 1986).

Comparing two estimations 0/ the electron transport rate in the leaves The linear relationship between the quantum yield of PSII electron transport (~F/F'm) and the quantum yield of leaf gross photosynthesis (4)e, mol C0 2/moi photons) obtained under non-photorespiratory conditions (see Fig. 3) allows calculation of the total electron transport rate of leaves under photo respiratory conditions (e.g. in 21 % O 2). The measurement of MIF'm alone on similar leaves in normal air allows a measurement (using the relation between MIF'm and 4>e in non-photorespiratory conditions) of the quantum yield of photosynthetic electron transport (4)e X 4, mole-/mol photons incident), which when multiplied by the incident PPFD (expressed in Ilmol· m- 2 's- I), gives the corresponding value of whole-chain electron transport, J I (expressed in Ilmole- . m- 2 's- I ):

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a

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(3)

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645

Estimating the partitioning 0/the electron transport between CO2 assimilation 0A) and 02-dependent dissipative processes 0zJ The total rate of photosynthetic electron flow under photorespiratory conditions can be expressed as follows:

(4) where,

(5) In this expression Rci (day respiration) is the rate of CO2 evolution in the light from processes other than photorespiration and 0.5v o is the rate of CO 2 produced by the photorespiratory pathway (one CO2 evolved for every two oxygenations). Equation 5 represents a maximum estimation of Ve calculated assuming that all the CO2 produced is reassimilated. Rci is difficult to measure (e.g. Brooks and Farquhar, 1985; Sharkey, 1988) and is usually estimated by measuring the respiration rate in the dark, RD. From equations 4 and 5, it follows that:

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(6)

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For E. hirsutum, dark respiration (RD) was measured in the assimilation chamber after at least 30 min of leaf acclimation to the dark at 26°C; the leaf was then illuminated and Rci was estimated (assuming Rci = RD) at different temperatures using a QIO of 2.]A was first calculated and JL = 6v 0 was deduced from equation 6. For P. vulgaris, light respiration (RL = RD + 0.5v 0) was estimated from the A/C; (C; is the intercellular CO 2 concentration in the leaves) curves at different temperatures and then Ve was calculated from equation 5. JLwas calculated from equations 4 and 8 and JA deduced from equation 6. Better estimates of day respiration can be obtained by analyzing the A/C; curves under various light intensities by measuring r* (Brooks and Farquhar, 1985).

Estimating the CO2/0 2 specificity/actor 0/RubisCo Ve and v 0 were calculated as described above and were used to estimate the C0 2/0 2 specificity factor of RubisCo (Laing et aI., 1974) using equation 1. CO2 and O 2 concentrations in the cell were calculated taking into account C; and the actual O 2 concentration in the air using the Bunsen solubility coefficient at appropriate temperatures. Diffusional resistance for gases through an aqueous phase was not taken into account.

Results

Effect 0/leaftemperature on CO2 uptake and quantum yield o/PSII electron transport (ilE/Pm)

Leaf net CO2 uptake by leaves (A) and quantum yield of PSII electron transport (~F/F'm) of E. hirsutum were measured at normal CO2 both in 21 % and 2 % O 2 (Fig. 1 A and 1 B, closed and open symbols, respectively), whereas those of P. vulgaris were measured only in 21 % O 2 both at saturating and limiting CO 2 (Fig. 2, closed and open symbols, respectively).

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Fig. 1: Effect of temperature on: A) leaf net photosynthesis, B) quantum yield ofPSII electron transport (£\F/F'm), C) photochemical quenching coefficient of fluorescence (qP) and D) quantum efficiency of excitation energy capture by oxidized PSII reaction centers (Fv/F'm) of Epilobium hirsutum intact leaves both in normal air (closed symbols) and air with 2 % O 2 (open symbols). PPFD was 450Ilmol'm- 2 's- 1 and air-leaf vapour pressure difference (VPD) was maintained constant (around 0.9 KPa) during the experiments. Each point is the mean of 3 (for air with 2 % O 2) or 4 (for normal air) measurements performed independently on different leaves. The error bars are the SE.

measured at 350 ilL· L -1 CO2 varies a little with temperature and reached a maximum at about 29°C. Quantum yield of PSII electron transport decreases similarly with temperature in the two CO2 concentrations. Gentyet al. (1989) and Cornic and Briantais (1991) found a linear relation between the quantum yield of PSII electron transport and the quantum yield of gross net CO 2 uptake (cI> c) measured in non-photorespiratory conditions on leaves submitted to a variety of environmental conditions. Fig. 3 A and B show in E. hirsutum and P. vulgaris, respectively, that such a relation is also found when leaf temperature is changed. In the case of E. hirsutum gross photosynthesis was obtained by adding dark respiration to A, whereas, in the case of P. vulgaris light respiration (estimated from the Ale curves measured at the temperature indicated in Fig. 2) was added to A. A similar relation was also reported by Labate et al. (1990) for maize leaves; however, the relation they found for barley leaves (C 3 plant) at high CO2 (about 800 ilL· L -1) differed substantially from those shown in Fig. 3 and did not extrapolate close to the origin. For E. hirsutum leaves, the same linear relationship is observed in air with 2 % O 2 and normal CO 2 concentration (Fig. 3 A, closed symbols) and in air with 2 % O 2 and saturating CO2 (Fig. 3 A, open symbols). PPFD was 450 and 300Ilmol·m- 2 ·s-t, respectively. For P. vulgaris, the same relationship was obtained in air with 21 % O 2 at saturating CO2 (450 to 1200 ilL· L -1 depending on leaf

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In vivo estimation of CO/O 2 specificity of RubisCo at varying temperature

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Quantum Yield -1 (mol CO 2 , mol photon ) Fig.3: Relationship between the ratio ~F/F'm and the quantum yield of CO 2 uptake by E. hirsutum (A) and P. vulgaris (B) intact leaves at varying temperature. The measurements were performed under non-photorespiratory conditions. For E. hirsutum (A) the measurements were conducted at either normal CO2 and 2 % O 2 and a PPFD of 450Ilmol ' m- 2's- 1 (closed symbols) or in air with 1000I1L'L-' CO 2 and 2% O 2 and a PPFD of 300l1mol·m-2·s-' (open symbols). Different symbols correspond to the measurements of aifferent leaves. For P. vulgaris (B), PPFD was 240 I1mol'm-2's-1 and measurements were performed at either normal CO2 and 2 % O 2 (open symbols) or normal O 2 and saturating CO2 (closed symbols). temperature) as well as in air with 2 % O 2 and normal CO 2 under a PPFD of 240 Ilmol· m -2 . S -I.

Estimating the total electron transport rate and the partitioning ofelectrom between CO 2 assimilation and O 2dependent dissipative processes The non-cyclic electron transport rate under photorespiratory conditions estimated using the «calibration curves» in Fig. 3 is shown in Fig. 4 (closed circles; A: E. hirsutum; B: P. vulgaris). The estimates obtained using the method of Krall and Edwards (1992) are also shown (Fig. 4, dashed lines) and are always higher than those calculated using the «calibration curve» (Genty et al., 1989) for both species. The latter estimates 1) are used to calculate the allocation of the electrons to Oz-dependent dissipative processes (Fig. 4, closed squares) using equations 6 and 8. As expected from the .::IF"/Pm measurements presented in Fig. 1 B, the total electron flow measured in 21 % O 2 was always higher than that measured in 2 % O 2 in E. hirsutum

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Effect ofleaf temperature on CO/02 specificity factor (S) of RubisCo estimated in vivo The temperature response curves of S, estimated as described in Material and Methods, are presented in Fig. S for both species. The open circles show the data obtained using spinach RubisCo in vitro by Jordan and Ogren (1984). It is striking to see that the values obtained with P. vulgaris (Fig. S B, closed circles) fit with those obtained in vitro by Jordan and Ogren, although they are somewhat higher. The data obtained with E. hirsutum (Fig. S A, closed circles) show similar variations above 18 °C, although they are slightly higher than the in vitro ones obtained on spinach enzyme, but diverge substantially from in vitro values below 18

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Discussion

Effect ofO2 and temperature on leafphotosynthesis and partitioning ofnon-cyclic electron flow between CO2 assimilation and O2 reduction In C3 plants, at normal CO2 concentration, leaf photosynthetic CO2 assimilation can be inhibited or stimulated by O 2 depending on leaf temperature. In E. hirsutum, the inhibition of leaf photosynthesis by O 2 at high temperature is shown in Fig. 1 A. This effect of O 2 on A decreases with decreasing leaf temperature as a consequence of the temperature dependency of both the kinetic properties of RubisCo and the solubility ratio of 0/C02 (see Ku and Edwards, 1977 a, b). For temperatures below 15°C, leaf CO2 uptake is stimulated by O 2 (Fig. 1 A). Stimulation of leaf photosynthesis by O 2 has been reported at saturating light and high CO 2 concentrations Golliffe and Tregunna, 1968, 1973; ViiI et aI.,

1977; Cornic and Louason, 1980; Sharkey, 1985; Sage and Sharkey, 1987). Nevertheless, this stimulatory effect of O 2 on A still remains difficult to understand. It is interesting to note that ~/F'm (quantum yield of PSII electron transport, Fig. 1B) and thus J1 (total electron transport rate), which is calculated from ~/F'm values and the relationship Fig. 3, are higher in 21 % O 2 compared with 2 % O 2 (Fig. 4 A, closed and open circles, respectively). This increase in the rate of total electron transport brought about by O 2 is likely to be related to the higher values of A observed in the same O 2 conditions below 15°C. It is likely that above 15 °C the inhibition of A by atmospheric O 2 via stimulation of the oxygenase function of RubisCo predominates, masking any effects due to the stimulation of whole-chain electron transport by O 2 • Lower .::lF/F'm at low O 2 concentration can be due to either lower values of qP or to lower values of Fv/F'm (or both). Clearly, between 20 and 30°C lower values of Fvl F'm in 2% O 2 (Fig. 1D) explain the low values of .::lF/F'm in that condition. Weis and Berry (1989) obtained similar results for cotton leaves and suggested that the drop in the quantum yield of PSII electron transport (and thus of the total electron transport) in 2 % O 2 can be explained by the lower ATP demand of the PCR cycle (3 ATP for regeneration of 1 RuBP) relative to the PCO cycle (3.5 ATP per RuBP). Thus, at low O 2 concentration (low rate of photorespiration) non-radiative dissipation of energy would increase (decrease of Fv/F'm) because of a higher .::lpH. However, the fact that this inhibitory effect of 2 % O 2 on leaf whole-chain electron transport rate could be observed on maize and Digitaria sanguinalis leaves (two C4 plants) at different CO2 concentrations and temperatures (Ghashghaie and Cornic, unpublished) suggest that another mechanism could be involved in that phenomenon. Moreover, if Krall and Edwards (1990), Gentyet al. (1990) and Cornic and Ghashghaie (1991) found a decrease in electron transport rate in wheat, ivy and French bean, respectively, Sharkey et al. (1988) on bean (at limiting light), Genty et al. (1990) on barley and Oberhuber and Edwards (1993) on Flaveria pringlei found no changes in electron transport rate when decreasing the O 2 concentration. For temperatures ranging from 6 to 20°C the difference between qP measured both in 21 % and in 2 % O 2 concentrations increases (Fig. 1 C) while that between Fv/F'm in the same O 2 conditions decreases; at around 6°C Fv/F'm is the same in 21 % and 2 % O 2 , respectively (Fig. 1 D). At low temperatures, minimum values of Fv/F'm seem to be reached, indicating that the lower values of ~/F'm in 2 % O 2 can be explained mainly by lower values of qP in the same condition. At low temperature, QA (the primary electron acceptor of PSII) is less reduced in 21 % O 2, suggesting that part of the photosynthetic metabolism in this condition is still sensitive to O 2•

Comparison of two methods for estimating the total electron transport rate The total electron transport rate estimated using the method of Krall and Edwards (1992), in which no account of leaf net CO2 uptake is taken, are close but always higher

In vivo estimation of CO/O 2 specificity of RubisCo at varying temperature than those obtained using the «calibration curve» {Fig. 4, dashed lines and circles, respectively}. This can be explained by the fact that the chlorophyll fluorescence emission is measured under a modulated background of non-actinic light with low intensity and that only the upper face of the leaf is illuminated (see Introduction). Thus, the measured ~F/F'm represents only the quantum yield of PSII electron transport of the upper cell layers, which as a mean, receives more light than the leaf as a whole. Light absorption by a leaf, taken as a constant by Krall and Edwards (1992), may also change depending on environmental conditions and the plant that is used (Brugnoli and Bjorkman, 1992). The difference between the two estimates are expected to decrease when PPFD approaches its saturating level (measurements here were done under limiting light). The measured leaf CO 2 uptake in non-photorespiratory conditions, which we used to calibrate the M/F'm measurements, gives the mean response of all leaf cell layers and thus a better estimate (though much more time consuming) of non-cyclic electron transport.

Estimation of CO/02 specifu:ity factor ofRubisCo from gas exchange and fluorescence measurements The rate of electron flow and its partitioning between CO2 assimilation and O 2 reduction calculated using the method of Cornic and Briantais (1991) was used to estimate the CO/02 specificity factor of RubisCo (S). The calculated values for P. vulgaris and E. hirsutum (from 18 to 32°C) are in general agreement with the results obtained by Jordan and Ogren (1984) using spinach enzyme in vitro (Fig. 5B, open symbols). For E. hirsutum the calculated values of S deviate from in vitro data at temperatures lower than 18°C. In the S calculation three main hypotheses were considered:

i) No resistance to CO2 diffusion in aqueous phase was taken

649

Thus, the use of C j rather than C e, 6 electrons!O 2 fixed and dark respiration can not explain the considerably higher values of S in E. hirsutum at low temperatures.

Conclusion (1) The two methods used to estimate the photosynthetic electron transport rate, the first based on the «calibration» of the M/F'm signal with the oII e under non-photorespiratory conditions (Genty et al., 1989) and the second based only on the measurement of M/F'm parameter and the light absorbed by the leaf (Krall and Edwards, 1992), gave similar estimates. The slightly higher values obtained by the second method may be due to an overestimation of the light absorbed by the leaves. (2) The estimation of CO/0 2 specificity factor of RubisCo on intact leaves is in general agreement with the in vitro results on spinach enzyme Gordan and Ogren, 1984) for temperatures above 18°C. On E. hirsutum, the estimated values of S diverge from in vitro ones only at low temperatures. The fact that the values of S calculated in vivo are in good agreement with in vitro data within a wide range of temperature (18 to 32°C) suggests that in both species carboxylation and photorespiration are the main processes consuming linear photosynthetic electrons and that the processes of O2 reduction other than photorespiration are negligible in this range of temperature. The deviation of S from in vitro data below 18°C, which is observed quite clearly in E. hirsutum, may indicate that another O 2 reduction process different from oxygenation of RuBP takes place as temperature decreases. Acknowledgements

We would like to thank Dr. B. Genty and Dr. D. Epron for helpful discussions.

into account (C; was used to calculate S). When a 10% de-

crease in concentration from C to C e (C0 2 concentration in the chloroplast) is assumed (Brooks and Farquhar, 1985; Krall and Edwards, 1992) the calculated S (data not shown) is slightly increased for all ranges of temperature used here. ii) The stoichiometry of 6 electrons per O 2 fixed was used here

assuming reassimilation by the Calvin cycle ofall the cO2 pro· duced (see equation 8). It is clear from equations 1 and 5 that

if the CO2 produced during photorespiration is not entirely reassimilated, the calculated S will decrease since Ve will be smaller. The rate of reassimilation of CO 2 evolved by the photo respiratory pathway may change with temperature. When a minimum of 4 electrons per O 2 fixed is used (assuming no CO 2 reassimilation) calculated S is decreased (data not shown) but remains higher than in vitro data at low temperatures. iii) Rv was used as an estimate ofRd to calculate Vc. When R.! values estimated from the linear relationship between day respiration and dark respiration obtained on two C 3 plants (see Brooks and Farquhar, 1985) are used, the estimated values of S (Fig. 5 A, dotted line) are similar to in vitro values above 16°C, but diverge again from these at low temperatures (S estimated that way is about 330 at 8°C).

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