Summary The first broad species survey of diurnal variation in carbon (C) isotope signatures of leaf dark‐respired CO2 (δ13Cres) is presented here and functional differences and diurnal dynamics are linked to fractionation in different respiratory pathways, based on 13C‐labelling experiments. δ13Cres was analysed with a rapid in‐tube incubation technique in 16 species. A large diurnal increase in δ13Cres (4–8‰) occurred in evergreen, slow‐growing and aromatic species and correlated significantly with cumulative photosynthesis, whereas no variation occurred in herbaceous, fast‐growing plants or temperate trees. The diurnal increase in δ13Cres declined almost proportionally to reductions in cumulative light and was reduced in growing compared with mature leaves. Pyruvate positional labelling provided direct evidence that functional groups differ in C allocation between respiratory pathways owing to different metabolic demands for growth, maintenance and secondary metabolism. Diurnal increase in C flux through pyruvate dehydrogenase (for investment in, for example, isoprene or aromatic compounds) combined with consistently low Krebs cycle activity resulted in pronounced increase in δ13Cres in evergreen and aromatic species. By contrast, fast growing herbs with high respiratory demand exhibited no diurnal changes since C was fully respired. Hence, diurnal δ13Cres pattern may provide information for C allocation in plants.

The study of the fate of assimilated carbon in respiratory fluxes in the field is needed to resolve the residence and transfer times of carbon in the atmosphere-plant-soil system in forest ecosystems, but it requires high frequency measurements of the isotopic composition of evolved CO2. We developed a closed transparent chamber to label the whole crown of a tree and a labelling system capable of delivering a 3-h pulse of 99% 13CO2 in the field. The isotopic compositions of trunk and soil CO2 effluxes were recorded continuously on two labelled and one control trees by a tuneable diode laser absorption spectrometer during a 2-month chase period following the late summer labelling. The lag times for trunk CO2 effluxes are consistent with a phloem sap velocity of about 1 m h(-1). The isotopic composition (delta13C) of CO2 efflux from the trunk was maximal 2-3 days after labelling and declined thereafter following two exponential decays with a half-life of 2-8 days for the first and a half-life of 15-16 days for the second. The isotopic composition of the soil CO2 efflux was maximal 3-4 days after labelling and the decline was also well fitted with a sum of two exponential functions with a half-life of 3-5 days for the first exponential and a half-life of 16-18 days for the second. The amount of label recovered in CO2 efflux was around 10-15% of the assimilated 13CO2 for soil and 5-13% for trunks. As labelling occurred late in the growing season, substantial allocation to storage is expected.

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast-mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 micromol m(-2) s(-1) photosynthetic active radiation (PAR; 80) and 350 micromol m(-2) s(-1) PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 micromol m(-2) s(-1) PAR. The data demonstrate that chloroplast-mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.

Abstract. Soil CO 2 efflux is the main source of CO 2 from forest ecosystems and it is tightly coupled to the transfer of recent photosynthetic assimilates belowground and their metabolism in roots, mycorrhiza and rhizosphere microorganisms feeding on root-derived exudates. The objective of our study was to assess patterns of belowground carbon allocation among tree species and along seasons. Pure 13 CO 2 pulse labelling of the entire crown of three different tree species (beech, oak and pine) was carried out at distinct phenological stages. Excess 13 C in soil CO 2 efflux was tracked using tuneable diode laser absorption spectrometry to determine time lags between the start of the labelling and the appearance of 13 C in soil CO 2 efflux and the amount of 13 C allocated to soil CO 2 efflux. Isotope composition (δ 13 C) of CO 2 respired by fine roots and soil microbes was measured at several occasions after labelling, together with δ 13 C of bulk root tissue and microbial carbon. Time lags ranged from 0.5 to 1.3 days in beech and oak and were longer in pine (1.6-2.7 days during the active growing season, more than 4 days during the resting season), and the transfer of C to the microbial biomass was as fast as to the fine roots. The amount of 13 C allocated to soil CO 2 efflux was estimated from a compartmentCorrespondence to: D. Epron (daniel.epron@scbiol.uhp-nancy.fr) model. It varied between 1 and 21 % of the amount of 13 CO 2 taken up by the crown, depending on the species and the season. While rainfall exclusion that moderately decreased soil water content did not affect the pattern of carbon allocation to soil CO 2 efflux in beech, seasonal patterns of carbon allocation belowground differed markedly between species, with pronounced seasonal variations in pine and beech. In beech, it may reflect competition with the strength of other sinks (aboveground growth in late spring and storage in late summer) that were not observed in oak. We report a fast transfer of recent photosynthates to the mycorhizosphere and we conclude that the patterns of carbon allocation belowground are species specific and change seasonally according to the phenology of the species.

The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

Soil CO₂ efflux is the main source of CO₂ from forest ecosystems and it is tightly coupled to the transfer of recent photosynthetic assimilates belowground and their metabolism in roots, mycorrhiza and rhizosphere microorganisms feeding on root-derived exudates. The objectives of our study were to assess patterns of belowground carbon allocation among tree species and along seasons. Pure ¹³CO₂ pulse labelling of the entire crown of three different tree species (beech, oak and pine) was carried out at distinct phenological stages. Excess ¹³C in soil CO₂ efflux was tracked using tunable diode laser absorption spectrometry to determine time lags between the start of the labelling and the appearance of ¹³C in soil CO₂ efflux and the amount of ¹³C allocated to soil CO₂ efflux. Isotope composition (δ¹³C) of CO₂ respired by fine roots and soil microbes was measured at several occasions after labelling, together with δ¹³C of bulk root tissue and microbial carbon. Time lags ranged from 0.5 to 1.3 days in beech and oak and were longer in pine (1.6–2.7 days during the active growing season, more than 4 days during the resting season), and the transfer of C to the microbial biomass was as fast as to the fine roots. The amount of ¹³C allocated to soil CO₂ efflux was estimated from a compartment model. Seasonal patterns of carbon allocation to soil CO₂ efflux differed markedly between species, with pronounced seasonal variations in pine and beech. In beech, it may reflect competition with other sinks (aboveground growth in late spring and storage in late summer) that were not observed in oak

The observed decrease in respiration during rosette leaf maturation of Nicotiana sylvestris wild-type (WT) plants was shown to be because of a decline in the cytochrome oxidase (COX) pathway activity, measured by 18 O/ 16 O oxygen discrimination, while the alternative oxidase pathway (AOX) remained stable. This suggests a higher contribution of the COX pathway to growth respiration than to maintenance respiration. Mitochondrial superoxide dismutase (MnSOD) activity paralleled the decrease in COX activity with leaf age, whereas chloroplastic FeSOD activity increased. Age-dependent respiratory changes were much less apparent in the Cytoplasmic Male Sterile II (CMSII) mitochondrial mutant devoid of respiratory complex I and previously shown to possess increased AOX content and enhanced respiration but lower photosynthesis in mature leaves. Respiration declined less rapidly with leaf age in CMSII than in the WT, and was significantly higher in the mutant when compared with the WT in mature leaves only. In contrast, photosynthesis was lower in the mutant than in the WT at all leaf stages. The higher respiration of mature CMSII leaves was supported exclusively by enhanced COX activity, in association with an increased mitochondrial MnSOD activity. Steady-state levels of AOX1 transcripts increased in maturing WT leaves, and the CMSII mutant had higher amounts of coxI, AOX1 and MnSOD transcripts than the WT. Enhanced activity of the proton-pumping COX route in the mutant can be viewed as a compensation for the lack of the first coupling site of the respiratory chain. However, this is not quite sufficient to ensure normal growth rates in the mutant.