505Plant, Cell and Environment (2001) 24, 505-515 intercellular partial pressure of CO 2 ; PPFD, photosynthetic photon flux density; R, dark respiration; R VPDB , 13 C/ 12 C ratio of standard VPDB; R s , 13 C/ 12 C ratio of sample; Rubisco, ribulose 1,5 bisphosphate carboxylase-oxygenase; RWC, leaf relative water content; VPD, vapour pressure deficit; d 13 C, carbon isotopic composition. INTRODUCTIONCarbon isotope discrimination during leaf CO 2 assimilation has been extensively studied and models have been developed (Farquhar, O'Leary & Berry 1982;Evans et al. 1986). The simple version of these models, which does not include the discrimination during respiration, has been validated for many species, suggesting that the discrimination during respiration is negligible and does not significantly modify the net discrimination during on-line measurements compared to the predicted values (for a recent review see Brugnoli & Farquhar 2000). Yet, the carbon isotope signature of plant dry matter integrates not only the discrimination during net CO 2 assimilation in the light (including CO 2 diffusion from the atmosphere to the chloroplasts, carboxylation, photorespiration and day respiration) but also the discrimination that could occur during the night-time respiration. Therefore, any fractionation during the night and/or the use of heavy or light substrates for dark respiration (releasing 13 C-enriched or 13 C-depleted CO 2 compared with leaf material) should change the isotopic signature of the remaining leaf material. Moreover, when non-photosynthesizing organs are taken into account, the release of 13 C-enriched or 13 C-depleted CO 2 will further contribute to changes in whole-plant carbon isotopic signature. Henderson, von Caemmerer & Farquhar (1992) observed in some C 4 species that the discrimination determined on leaf dry matter was significantly greater than that measured on-line. Using a modelling approach, they proposed that at least a part of this difference could be explained by the fractionation during dark respiration, releasing CO 2 enriched in 13 C relative to the plant material. We obtained similar results on Phaseolus vulgaris (unpublished results) and Nicotiana sylvestris (Duranceau, ABSTRACTThe variations of d 13 C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO 2 respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well-watered conditions, CO 2 respired in the dark was 13 C-enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant 13 C-enrichment by about 6‰ in respired CO 2 compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the 13 C-enrichment in respired CO 2 increased in dehydrated N. sylvestris and decrea...

The variations in δ 13 C in both leaf carbohydrates (starch and sucrose) and CO 2 respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in 13 C) with decreasing stomatal conductance and decreasing p i /p a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in 13 C). This may be explained by increased p i /p a but other possible explanations are also discussed. Interestingly, the variations in δ 13 C of CO 2 respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ 13 C of CO 2 respired in the dark and sucrose, respired CO 2 always being enriched in 13 C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in 13 C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing 13 C-enriched CO 2 ; and that (ii) this leads to 13 C depletion in the remaining leaf material.Abbreviations: A, leaf net CO 2 assimilation; a, fractionation against 13 C for CO 2 diffusion through air; b, net fractionation against 13 C during CO 2 fixation by Rubisco and PEPc; δ 13 C, carbon isotopic composition; ∆, discrimination against 13 C during CO 2 assimilation; d, the term including the fractionation due to CO 2 dissolution, liquid phase diffusion and also discrimination during both respiration and photorespiration; DW, leaf dry weight; dδ 13 C, the difference between CO 2 respired in the dark and plant material in their carbon isotope composition; d∆, variation in modelled discrimination at a given p i /p a relative to a reference value at p i /p a = 0·7; FW, leaf fresh weight; g c , leaf conductance to CO 2 diffusion; HPLC, high-performance liquid chromatography; LMA, leaf mass per area; p a , ambient partial pressure of CO 2 ; p i , intercellular partial pressure of CO 2 ; PEPc, phosphoenolpyruvate carboxylase; PPFD, photosynthetic photon flux density; R PDB , 13 C/ 12 C ratio of standard PDB; R S , 13 C/ 12 C ratio of sample; Rubisco, ribulose 1,5 bisphosphate carboxylase-oxygenase; RWC, leaf relative water content; SW, leaf saturated weight; VPD, vapour pressure deficit.δ 13 C of CO 2 respired in the dark in relation to δ 13 C of leaf carbohydrates in P. vulgaris 523

Leaf gas-exchange, carbon isotope discrimination (D) during photosynthesis, carbon isotope composition (d13 C) of leaf dry matter, leaf carbohydrates and ‰ d13 C of dark respiratory CO 2 were measured both in wild type (WT) and in a respiratory mutant of Nicotiana sylvestris Spegazz. plants. The mutation caused a dysfunction of complex I of the respiratory chain which has been described in detail by Gutierres et al. 1997, PNAS, 94, 3436. The aim of this work was to verify if this mutation has an influence on carbon isotope discrimination during photosynthesis and dark respiration. Another objective was to study the possible effect of respiratory fractionation on the isotopic composition of dry matter and on the discrimination measured on-line, in comparison with the expected D based on the model developed by Farquhar et al. 1982, AJPP, 9, 121. On-line D measured on leaves during photosynthesis was lower in the mutants (16.5‰ 0.9) than in the WT (20.1‰ 0.6), mainly due to lower conductance to CO 2 diffusion (both across stomatal pores and in the gaseous and liquid phases across the mesophyll) in the mutants. No statistically significant difference in the fractionation during dark respiration was observed between WT and mutant plants. However, respiratory CO 2 was enriched in 13 C compared to sucrose and glucose by about 2–3 and 2.5–4‰, respectively. The enrichment in 13 C (about 2‰) observed in leaf metabolites and leaf organic matter in the mutants compared to the WT can be explained by differences in .during photosynthesis. However, the fractionation in the whole-leaf organic matter of both WT and mutant plants was higher (more depleted in 13C) than expected based on the .values obtained with on-line measurements during photosynthesis. The observed discrimination during dark respiration, releasing 13 C-enriched CO 2 , may partly explain the higher fractionation in the remaining leaf organic matter compared to the overall discrimination during photosynthesis, as measured on-line.

The variations in δ 13 C in both leaf carbohydrates (starch and sucrose) and CO 2 respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in 13 C) with decreasing stomatal conductance and decreasing p i /p a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in 13 C). This may be explained by increased p i /p a but other possible explanations are also discussed. Interestingly, the variations in δ 13 C of CO 2 respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ 13C of CO 2 respired in the dark and sucrose, respired CO 2 always being enriched in 13 C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in 13 C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing 13 C-enriched CO 2 ; and that (ii) this leads to 13 C depletion in the remaining leaf material. Key-words: Phaseolus vulgaris;carbohydrates; carbon isotopes; dark respiration; discrimination; drought.Abbreviations: A, leaf net CO 2 assimilation; a, fractionation against 13 C for CO 2 diffusion through air; b, net fractionation against 13 C during CO 2 fixation by Rubisco and PEPc; δ 13 C, carbon isotopic composition; ∆, discrimination against 13 C during CO 2 assimilation; d, the term including the fractionation due to CO 2 dissolution, liquid phase diffusion and also discrimination during both respiration and photorespiration; DW, leaf dry weight; dδ 13 C, the difference between CO 2 respired in the dark and plant material in their carbon isotope composition; d∆, variation in modelled discrimination at a given p i /p a relative to a reference value at p i /p a = 0·7; FW, leaf fresh weight; g c , leaf conductance to CO 2 diffusion; HPLC, high-performance liquid chromatography; LMA, leaf mass per area; p a , ambient partial pressure of CO 2 ; p i , intercellular partial pressure of CO 2 ; PEPc, phosphoenolpyruvate carboxylase; PPFD, photosynthetic photon flux density; R PDB , 13 C/ 12 C ratio of standard PDB; R S , 13 C/ 12 C ratio of sample; Rubisco, ribulose 1,5 bisphosphate carboxylase-oxygenase; RWC, leaf relative water content; SW, leaf saturated weight; VPD, vapour pressure deficit. INTRODUCTIONPlants discriminate against 13 C during photosynthetic CO 2 fixation. The carbon isotope discrimination (∆) by plants involves both physical and biochemical processes. Farquhar, O'Leary & Berry (1982b) developed a theoretical model which predicts a linear relationship between ∆ and the ratio of intercellular to atmospheric partial pressures of CO 2 (p i /p a ) in C 3 plants:where a (= 4·4‰) is the fractionation due to diffusion of CO 2 from ambient air in...