Salvador Nogués

Metabolic Origin of Carbon Isotope Composition of Leaf Dark-Respired CO2 in French Bean

The carbon isotope composition (δ13C) 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 δ13C 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 δ13C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of δ13C 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 δ13C 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 physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.)

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (Nase) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and Nase activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by Nase activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with Nase inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψs). At the nodule level, drought had an inhibitory effect on Nase activity. This decrease in Nase activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N2 fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.

Ear of durum wheat under water stress: water relations and photosynthetic metabolism

The photosynthetic characteristics of the ear and flag leaf of well-watered (WW) and water-stressed (WS) durum wheat (Triticum turgidum L. var. durum) were studied in plants grown under greenhouse and Mediterranean field conditions. Gas exchange measurements simultaneously with modulated chlorophyll fluorescence were used to study the response of the ear and flag leaf to CO2 and O2 during photosynthesis. C4 metabolism was identified by assessing the sensitivity of photosynthetic rate and electron transport to oxygen. The presence of CAM metabolism was assessed by measuring daily patterns of stomatal conductance and net CO2 assimilation. In addition, the histological distribution of Rubisco protein in the ear parts was studied by immunocytochemical localisation. Relative water content (RWC) and osmotic adjustment (osmotic potential at full turgor) were also measured in these organs. Oxygen sensitivity of the assimilation rate and electron transport, the lack of Rubisco compartmentalisation in the mesophyll tissues and the gas-exchange pattern at night indicated that neither C4 nor CAM metabolism occurs in the ear of WW or WS plants. Nevertheless, photosynthetic activity of the flag leaf was more affected by WS conditions than that of the ear, under both growing conditions. The lower sensitivity under water stress of the ear than of the flag leaf was linked to higher RWC and osmotic adjustment in the ear bracts and awns. We demonstrate that the better performance of the ear under water stress (compared to the flag leaf) is not related to C4 or CAM photosynthesis. Rather, drought tolerance of the ear is explained by its higher RWC in drought. Osmotic adjustment and xeromorphic traits of ear parts may be responsible.

The Photosynthetic Role of Ears in C3 Cereals : Metabolism, Water Use Efficiency and Contribution to Grain Yield

This review concerns ear photosynthesis and its contribution to grain filling in C3 cereals. Ear photosynthesis is quantitatively important to grain filling, particularly in dry areas where source (i.e., assimilate) limitations can occur. Compared to the flag leaf, ear photosynthesis exhibits higher water stress tolerance. Several factors could be involved in the ears “drought tolerance.” First, although degree of C4 metabolism in ear parts has been reported, current evidence supports only typical C3 metabolism. Second, recycling of respired CO2 (i.e., refixation) could have considerable impact on final crop yield by preventing loss of CO2. Because refixation of CO2 is independent of atmospheric conditions, water use efficiency (measured as total ear photosynthesis divided by transpiration) could be higher in the ear than in the flag leaf. Moreover, ear parts (in particular awns) show higher relative water content and better osmotic adjustment under water stress compared to the flag leaf. This capacity, in addition to persistence of photosynthetic components under drought (delayed senescence), might help the ear to continue to fix CO2 late in the grain filling period.

Respiratory carbon metabolism following illumination in intact French bean leaves using 13C/12C isotope labeling

The origin of the carbon atoms in the CO2 respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using 13C/12C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO2 respired in the dark after a light period revealed that the CO2 was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO2 lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates.

In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus

*Nitrogen assimilation in leaves requires primary NH(2) acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO(2) evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. *In this study, we carried out isotopic labelling experiments with (13)CO(2) and (15)N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed (13)C- and (15)N-nuclear magnetic resonance analyses. *Our results indicated that the production of (13)C-glutamate and (13)C-glutamine under a (13)CO(2) atmosphere was very weak, whereas (13)C-glutamate and (13)C-glutamine appeared in both the subsequent dark period and the next light period under a (12)CO(2) atmosphere. Consistently, the analysis of heteronuclear ((13)C-(15)N) interactions within molecules indicated that most (15)N-glutamate and (15)N-glutamine molecules were not (13)C labelled after (13)C/(15)N double labelling. That is, recent carbon atoms (i.e. (13)C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by (15)N incorporation. *We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.

Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2

Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO2] conditions (700 versus 370 μmol mol−1) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO2. The ambient 13C/12C isotopic composition (δ13C) of air CO2 was changed from –10.2‰ in ambient [CO2] to –23.6‰ under elevated [CO2] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO2] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO2] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO2 enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ13C of ear total organic matter and respired CO2, soluble sugar δ13C revealed that a small amount of labelled C reached the ear. The 12CO2 labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber (Cucumis sativus L.) and tomato (Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate-glutathione (AsA-GSH) cycle enzymes and the production of H(2)O(2) in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA-GSH cycle enzymes followed by an increased H(2)O(2) accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.

Water and nitrogen conditions affect the relationships of Δ13C and Δ18O to gas exchange and growth in durum wheat

Whereas the effects of water and nitrogen (N) on plant Δ13C have been reported previously, these factors have scarcely been studied for Δ18O. Here the combined effect of different water and N regimes on Δ13C, Δ18O, gas exchange, water-use efficiency (WUE), and growth of four genotypes of durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husn.] cultured in pots was studied. Water and N supply significantly increased plant growth. However, a reduction in water supply did not lead to a significant decrease in gas exchange parameters, and consequently Δ13C was only slightly modified by water input. Conversely, N fertilizer significantly decreased Δ13C. On the other hand, water supply decreased Δ18O values, whereas N did not affect this parameter. Δ18O variation was mainly determined by the amount of transpired water throughout plant growth (Tcum), whereas Δ13C variation was explained in part by a combination of leaf N and stomatal conductance (gs). Even though the four genotypes showed significant differences in cumulative transpiration rates and biomass, this was not translated into significant differences in Δ18Os. However, genotypic differences in Δ13C were observed. Moreover, ∼80% of the variation in biomass across growing conditions and genotypes was explained by a combination of both isotopes, with Δ18O alone accounting for ∼50%. This illustrates the usefulness of combining Δ18O and Δ13C in order to assess differences in plant growth and total transpiration, and also to provide a time-integrated record of the photosynthetic and evaporative performance of the plant during the course of crop growth.

Photosynthetic down-regulation under elevated CO2 exposure can be prevented by nitrogen supply in nodulated alfalfa.

Increasing atmospheric CO₂ concentrations are expected to enhance plant photosynthesis and yield. Nevertheless, after long-term exposure, plants acclimate and show a reduction in photosynthetic activity (called down-regulation), which may cause a reduction in potential yield. Some authors suggest that down-regulation is related to nutrient availability, and more specifically, to an insufficient plant C sink strength caused by limited N supply. In this paper, we tested whether or not N availability prevents down-regulation of photosynthesis in nodulated alfalfa plants (Medicago sativa L.). To do so, we examined the effect of the addition of different levels of NH₄NO₃ (0, 10, and 15 mM) to 30-day-old nodulated alfalfa plants exposed to ambient (approximately 400 μmol mol⁻¹) or elevated CO₂ (700 μmol mol⁻¹) during a period of 1 month in growth chambers. After 2 weeks of exposure to elevated CO₂, no significant differences were observed in plant growth or photosynthesis rates. After 4 weeks of treatment, exclusively N₂ fixing alfalfa plants (0 mM NH₄NO₃) showed significant decreases in photosynthesis and Vc(max). Photosynthetic down-regulation of these plants was caused by the C/N imbalance as reflected by the carbohydrate and N data. On the other hand, plants supplied with 15 mM NH₄NO₃ grown under elevated CO₂ maintained high photosynthetic rates owing to their superior C/N adjustment. The intermediate N treatment, 10 mM NH₄NO₃, also showed photosynthetic down-regulation, but to a lesser degree than with 0 mM treatment. The present study clearly shows that external N supply can reduce or even avoid acclimation of photosynthesis to elevated CO₂ as a consequence of the increase in C sink strength associated with N availability.