SUMMARYExperimental studies on CO 2 assimilation of mesophytic C3 plants in relation to relative water content (RWC) are discussed. Decreasing RWC slows the actual rate of photosynthetic CO 2 assimilation (A) and decreases the potential rate (A pot ). Generally, as RWC falls from c . 100 to c. 75%, the stomatal conductance (g s ) decreases, and with it A. However, there are two general types of relation of A pot to RWC, which are called Type 1 and Type 2. Type 1 has two main phases. As RWC decreases from 100 to c. 75%, A pot is unaffected, but decreasing stomatal conductance (g s ) results in smaller A, and lower CO 2 concentration inside the leaf (C i ) and in the chloroplast (C c ), the latter falling possibly to the compensation point. Down-regulation of electron transport occurs by energy quenching mechanisms, and changes in carbohydrate and nitrogen metabolism are considered acclimatory, caused by low C i and reversible by elevated CO 2 . Below 75% RWC, there is metabolic inhibition of A pot , inhibition of A then being partly (but progressively less) reversible by elevated CO 2 ; g s regulates A progressively less, and C i and CO 2 compensation point, Γ Γ Γ Γ rise. It is suggested that this is the true stress phase, where the decrease in A pot is caused by decreased ATP synthesis and a consequent decreased synthesis of RuBP. In the Type 2 response, A pot decreases progressively at RWC 100 to 75%, with A being progressively less restored to the unstressed value by elevated CO 2 . Decreased g s leads to a lower C i and C c but they probably do not reach compensation point: g s becomes progressively less important and metabolic limitations more important as RWC falls. The primary effect of low RWC on A pot is most probably caused by limited RuBP synthesis, as a result of decreased ATP synthesis, either through inhibition of Coupling Factor activity or amount due to increased ion concentration. Carbohydrate synthesis and accumulation decrease. Type 2 response is considered equivalent to Type 1 at RWC below c. 75%, with A pot inhibited by limited ATP and RuBP synthesis, respiratory metabolism dominates and C i and Γ Γ Γ Γ rise. The importance of inhibited ATP synthesis as a primary cause of decreasing A pot is discussed. Factors determining the Type 1 and Type 2 responses are unknown. Electron transport is maintained (but down-regulated) in Types 1 and 2 over a wide range of RWC, and a large reduced/oxidized adenylate ratio results. Metabolic imbalance results in amino acid accumulation and decreased and altered protein synthesis. These conditions profoundly affect cell functions and ultimately cause cell death. Type 1 and 2 responses may reflect differences in g s and in sensitivity of metabolism to decreasing RWC.

Drought and salinity are two widespread environmental conditions leading to low water availability for plants. Low water availability is considered the main environmental factor limiting photosynthesis and, consequently, plant growth and yield worldwide. There has been a long-standing controversy as to whether drought and salt stresses mainly limit photosynthesis through diffusive resistances or by metabolic impairment. Reviewing in vitro and in vivo measurements, it is concluded that salt and drought stress predominantly affect diffusion of CO(2) in the leaves through a decrease of stomatal and mesophyll conductances, but not the biochemical capacity to assimilate CO(2), at mild to rather severe stress levels. The general failure of metabolism observed at more severe stress suggests the occurrence of secondary oxidative stresses, particularly under high-light conditions. Estimates of photosynthetic limitations based on the photosynthetic response to intercellular CO(2) may lead to artefactual conclusions, even if patchy stomatal closure and the relative increase of cuticular conductance are taken into account, as decreasing mesophyll conductance can cause the CO(2) concentration in chloroplasts of stressed leaves to be considerably lower than the intercellular CO(2) concentration. Measurements based on the photosynthetic response to chloroplast CO(2) often confirm that the photosynthetic capacity is preserved but photosynthesis is limited by diffusive resistances in drought and salt-stressed leaves.

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The carbon isotope composition (␦ 13 C) of CO 2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The ␦ 13 C of CO 2 linearly decreased when temperature increased, from Ϫ19‰ at 10°C to Ϫ24‰ at 35°C. It also progressively decreased from Ϫ21‰ to Ϫ30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO 2 was enriched in 13 C compared with carbohydrates, the most 13 C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO 2 was depleted in 13 C even when compared with the composition of total organic matter. In the two types of experiment, the variations of ␦ 13 C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of ␦ 13 C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13 C-enriched CO 2 and ␤-oxidation of fatty acids producing 13 C-depleted CO 2 when compared with total organic matter (Ϫ27.5‰). These results are consistent with the assumption that the ␦ 13 C of dark respired CO 2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.Photosynthetic CO 2 assimilation of C 3 plants discriminates against 13 CO 2 so that organic matter is, on average, 20‰ depleted in 13 C compared with atmospheric carbon dioxide (for recent review, see Brugnoli and Farquhar, 2000). Respiratory carbon fluxes in light (i.e. photorespiration and "day" respiration) are often assumed to be negligible or weakly fractionating processes. However, the carbon isotope signature of organic matter may be modified by nighttime respiration depending on the ␦ 13 C of the evolved CO 2 because respiratory carbon lost by many plants has been shown to be within 30% to 60% of the carbon fixed through photosynthesis (Evans, 1993; Amthor, 2000).In vitro studies using protoplasts have shown that respired CO 2 isotope composition is identical to that of the Suc supplied to the culture medium, indicating that no fractionation occurs during respiration in the dark (Lin and Ehleringer, 1997). A similar result was also obtained in long-term experiments with animals, where the isotope composition of CO 2 expired by mice (Mus musculus) reflected that of the diet (Perkins and Speakman, 2001). In contrast, it has been shown previously that CO 2 produced by respiration in the dark is 6‰ 13 C enriched when compared with Suc in intact French bean (Phaseolus vulgaris) leaves (Duranceau et al., 1999). Similar results were also obtained in Nicotiana sylvestris and sunflower (Helianthus annuus), although CO 2 was less 13 C enriched with ␦ 13 C values of 4‰ and 3‰, respectively (Ghashghaie et al., 2001). Moreover, it has been demonstrated that the ␦ 13 C value of CO 2 evolved in the dar...

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Day respiration of illuminated C 3 leaves is not well understood and particularly, the metabolic origin of the day respiratory CO 2 production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using 12 C/ 13 C stable isotope techniques on illuminated leaves fed with 13 C-enriched glucose or pyruvate. The 13 CO 2 production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the 13 C-enriched compounds. Using different positional 13 C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely metabolized to liberate CO 2 in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with 13 C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf 13 C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration.Illuminated leaves simultaneously assimilate CO 2 through the photosynthetic carbon reduction cycle and lose CO 2 through photorespiration and day respiration. In darkness, leaves no longer assimilate CO 2 via the photosynthetic carbon reduction cycle but produce CO 2 through dark respiration. Although dark respiration is known to involve glycolysis and CO 2 production through pyruvate dehydrogenation and the degradative Krebs cycle (Trethewey and ap Rees, 1994;Plaxton, 1996), the carbon metabolism that is responsible for the CO 2 respiratory release in the light is almost unknown. This is so because the day respiratory CO 2 flux is very low and masked by the photosynthetic carbon fixation and the photorespiratory CO 2 production in the light, and is thus difficult to study.Nevertheless, it has been repeatedly shown, using either the Laisk's (Laisk, 1977) or Kok's method (Kok, 1948), that the rate of day respiration (R d ) is less than that of dark respiration (R n ; for review, see Atkin et al., 2000) so that light is known to inhibit respiration, with a R d /R n value (usually denoted as m) ranging from 30% to 100% (for a recent study, see Peisker and Apel, 2001). Pioneering gas exchange measurements on mustard suggested that some enzymatic activities are inhibited in the light so that substrates accumulate (Cornic, 1973), explaining the respiratory burst when leaves are darkened: the light enhanced dark respiration. More recently, it has been shown in the unicellular alga Selenastrum minutum that pyruvate kinase (Lin et al., 1989) is inhibited by light. It is also the case of the pyruvate dehydrogenase complex that is partly inactivated by (reversible) phosphorylation in ...

Using a combination of gas-exchange and chlorophyll fluorescence measurements, low apparent COyOj specificity factors (1300 mol mol"') were estimated for the leaves of two deciduous tree species {Fagus sylvatica and Castanea sativa). These low values contrasted with those estimated for two herbaceous species and were ascribed to a drop in the CO2 mole fraction between the intercellular airspace (Q) and the catalytic site of Rubisco {C^) due to internal resistances to CO^ transfer. Q. was calculated assuming a specificity of Rubisco value of 2560 mol moP'. The drop between C, and Q. was used to calculate the internal conductance for CO2 (g,). A good correlation between mean values of net CO2 assimilation rate (A) and gi was observed within a set of data obtained using 13 woody plant species, including our own data. We report that the relative limitation of A, which can be ascribed to internal resistances to CO2 transfer, was 24-30%. High internal resistances to CO2 transfer may explain the low apparent maximal rates of carboxylation and electron transport of some woody plant species calculated from A/Cj curves.