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It has been 75 yr since leaf respiratory metabolism in the light (day respiration) was identified as a low-flux metabolic pathway that accompanies photosynthesis. In principle, it provides carbon backbones for nitrogen assimilation and evolves CO2 and thus impacts on plant carbon and nitrogen balances. However, for a long time, uncertainties have remained as to whether techniques used to measure day respiratory efflux were valid and whether day respiration responded to environmental gaseous conditions. In the past few years, significant advances have beenmade using carbon isotopes, ‘omics’ analyses and surveys of respiration rates in mesocosms or ecosystems. There is substantial evidence that day respiration should be viewed as a highly dynamic metabolic pathway that interacts with photosynthesis and photorespiration and responds to atmospheric CO2 mole fraction. The view of leaf day respiration as a constant and/or negligible parameter of net carbon exchange isnow outdated and it should now be regarded as a central actor of plant carbon-use efficiency.

Day respiration of illuminated C₃ leaves is not well understood and particularly, the metabolic origin of the day respiratory CO₂ production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using ¹²C/¹³C stable isotope techniques on illuminated leaves fed with ¹³C-enriched glucose or pyruvate. The ¹³CO₂ production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the ¹³C-enriched compounds. Using different positional ¹³C-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 CO₂ in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with ¹³C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf ¹³C 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.

Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-L-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants.

Day respiration is the process by which nonphotorespiratory CO₂ is produced by illuminated leaves. The biological function of day respiratory metabolism is a major conundrum of plant photosynthesis research: because the rate of CO₂ evolution is partly inhibited in the light, it is viewed as either detrimental to plant carbon balance or necessary for photosynthesis operation (e.g., in providing cytoplasmic ATP for sucrose synthesis). Systematic variations in the rate of day respiration under contrasting environmental conditions have been used to elucidate the metabolic rationale of respiration in the light. Using isotopic techniques, we show that both glycolysis and the tricarboxylic acid cycle activities are inversely related to the ambient CO₂/O₂ ratio: day respiratory metabolism is enhanced under high photorespiratory (low CO₂) conditions. Such a relationship also correlates with the dihydroxyacetone phosphate/Glc-6-P ratio, suggesting that photosynthetic products exert a control on day respiration. Thus, day respiration is normally inhibited by phosphoryl (ATP/ADP) and reductive (NADH/NAD) poise but is up-regulated by photorespiration. Such an effect may be related to the need for NH₂ transfers during the recovery of photorespiratory cycle intermediates.

The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO2 showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO2 levels. The inhibition of photosynthesis was alleviated by high CO2 or low O2. The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO2 evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously.

The carbon isotope composition (δ 13C) of CO2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The δ 13C of CO2 linearly decreased when temperature increased, from -19‰ at 10°C to -24‰ at 35°C. It also progressively decreased from -21‰ to -30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO2 was enriched in 13C compared with carbohydrates, the most 13C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO2 was depleted in 13C even when compared with the composition of total organic matter. In the two types of experiment, the variations of δ 13C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of δ 13C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13C-enriched CO2 and β-oxidation of fatty acids producing 13C-depleted CO2 when compared with total organic matter (-27.5‰). These results are consistent with the assumption that the δ 13C of dark respired CO2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.

Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8. Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD. We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.

Carbon isotope discrimination during photosynthetic CO2 assimilation has been extensively studied and rigorous models have been developed, while the fractionations during photorespiratory and dark respiratory processes have been less well investigated. Whilst models of discrimination have included specific factors for fractionation during respiration (e) and photorespiration (f), these effects have been considered to be very small, i.e. not significantly modifying the net discrimination expressed in organic material. On this paper we consider the fractionation effects associated with specific reactions set against the overall discrimination which occurs during source-product transformations. We review the studies which have recently shown that discrimination occurs during respiration at night in intact C3 leaves, leading to the production of CO2 enriched in 13C (i.e., e = −6‰ ), and modifying the signature of the remaining plant material. Under photorespiratory conditions (i.e. increased oxygen concentration and high temperature), the photorespiratory fractionation factor may be high (with f around +10‰ ), and significantly alters the observed net photosynthetic discrimination measured during gas exchange. Fractionation factors for both respiration and photorespiration have been shown to be variable among species and with environmental conditions, and we suggest that the term `apparent fractionation' be used to describe the net effect for each process. In this paper we review the fractionations during photorespiration and dark respiration and the metabolic origin of the CO2 released during these processes, and we discuss the ecological implications of such fractionations.

Lateral diffusion of CO2 was investigated in photosynthesizing leaves with different anatomy by gas exchange and chlorophyll a fluorescence imaging using grease to block stomata. When one-half of the leaf surface of the heterobaric species Helianthus annus was covered by 4-mm-diameter patches of grease, the response of net CO2 assimilation rate (A) to intercellular CO2 concentration ($C_{\\text{i}}$) indicated that higher ambient CO2 concentrations ($C_{\\text{a}}$) caused only limited lateral diffusion into the greased areas. When single 4-mm patches were applied to leaves of heterobaric Phaseolus vulgaris and homobaric Commelina communis, chlorophyll a fluorescence images showed dramatic declines in the quantum efficiency of photosystem II electron transport (measured as $F_{\\text{q}}{}^{\\prime}/F_{\\text{m}}{}^{\\prime}$) across the patch, demonstrating that lateral CO2 diffusion could not support A. The $F_{\\text{q}}{}^{\\prime}/F_{\\text{m}}{}^{\\prime}$ values were used to compute images of $C_{\\text{i}}$ across patches, and their dependence on $C_{\\text{a}}$ was assessed. At high $C_{\\text{a}}$, the patch effect was less in C. communis than P. vulgaris. A finite-volume porous-medium model for assimilation rate and lateral CO2 diffusion was developed to analyze the patch images. The model estimated that the effective lateral CO2 diffusion coefficients inside C. communis and P. vulgaris leaves were 22% and 12% of that for free air, respectively. We conclude that, in the light, lateral CO2 diffusion cannot support appreciable photosynthesis over distances of more than approximately 0.3 mm in normal leaves, irrespective of the presence or absence of bundle sheath extensions, because of the CO2 assimilation by cells along the diffusion pathway.