To investigate the lignification process and its physiological significance under drought-stressed conditions, the changes in enzymes responsible for lignification and the related physiological parameters were determined in white clover (Trifolium repens L.) leaves during 28 d of water deficit treatment. Water deficit gradually decreased leaf water potential (Ψ_w) to −2.33 MPa at day 28. For the first 14 d of water deficit, ascorbate peroxidase and phenylalanine ammonia-lyase were highly activated. Neither a change in the parameters symptomatic of oxidative stress nor growth inhibition was observed. The reduction of leaf biomass occurred from 21 d of water deficit treatment when Ψ_w was −2.27 MPa or less, and was concomitant with the increase of lipid peroxidation and lignin content. As Ψ_w decreased below −1.67 MPa from 14 d of water deficit, the enhanced activation of guaiacol peroxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, and benzidine peroxidase was involved in lignification rather than in protection of plant tissues against the oxidative damage. The data indicate that a high activation of lignifying enzymes during terminal stress may be a drought stress-induced injurious symptom, which leads to reduced forage growth and digestibility.

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 (N_ase) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N_ase 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 N_ase 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 N_ase 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 (Ψ_s). At the nodule level, drought had an inhibitory effect on N_ase activity. This decrease in N_ase 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.

Despite its worldwide economic importance for food (oil, meal) and non-food (green energy and chemistry) uses, oilseed rape has a low nitrogen (N) use efficiency (NUE), mainly due to the low N remobilization efficiency (NRE) observed during the vegetative phase when sequential leaf senescence occurs. Assuming that improvement of NRE is the main lever for NUE optimization, unravelling the cellular mechanisms responsible for the recycling of proteins (the main N source in leaf) during sequential senescence is a prerequisite for identifying the physiological and molecular determinants that are associated with high NRE. The development of a relevant molecular indicator (SAG12/Cab) of leaf senescence progression in combination with a (15)N-labelling method were used to decipher the N remobilization associated with sequential senescence and to determine modulation of this process by abiotic factors especially N deficiency. Interestingly, in young leaves, N starvation delayed senescence and induced BnD22, a water-soluble chlorophyll-binding protein that acts against oxidative alterations of chlorophylls and exhibits a protease inhibitor activity. Through its dual function, BnD22 may help to sustain sink growth of stressed plants and contribute to a better utilization of N recycled from senescent leaves, a physiological trait that could improve NUE. Proteomics approaches have revealed that proteolysis involves chloroplastic FtsH protease in the early stages of senescence, aspartic protease during the course of leaf senescence, and the proteasome β1 subunit, mitochondria processing protease and SAG12 (cysteine protease) during the later senescence phases. Overall, the results constitute interesting pathways for screening genotypes with high NRE and NUE.

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.

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using ¹⁵N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.

In coming decades, increasing temperatures are expected to impact crop yield and seed quality. To develop low input systems, the effects of temperature and sulfur (S) nutrition in oilseed rape, a high S demanding crop, need to be jointly considered. In this study, we investigated the effects of temperatures (High Temperature (HT), 33 °C/day, 19 °C/night vs. Control Temperature (Ctrl T), 20°C/day, 15°C/day) and S supply (High S (HS), 500 µm SO42- vs. Low S (LS), 8.7 µM SO42-) during seed filling on (i) yield components (seed number, seed dry weight (SDW) and seed yield), (ii) grain composition (nitrogen (N) and S contents) and quality (fatty acid (FA) composition and seed storage protein (SSP) accumulation) and (iii) germination characteristics (pre-harvest sprouting, germination rates and abnormal seedlings). Abscisic acid (ABA), soluble sugar contents and seed conductivity were also measured. HT and LS decreased the number of seeds per plant. SDW was less affected due to compensatory effects since the number of seeds decreased under stress conditions. While LS had negative effects on seed composition by reducing the FA contents and increasing the ratio S-poor SSPs (12S globulins)/S-rich SSPs (2S albumins) ratio, HT had positive effects by increasing S and FA contents and decreasing the C18:2/C18:3 ratio and the 12S/2S protein ratio. Seeds produced under HT showed high pre-harvest sprouting rates along with decreased ABA contents and high rates of abnormal seedlings. HT and LS restriction significantly accelerated germination times. High conductivity, which indicates poor seed storage capacity, was higher in HT seeds. Consistently, the lower ratio of (raffinose +stachyose)/sucrose in HT seeds indicated low seed storage capacity. We demonstrated the effects of HT and LS on grain and on germination characteristics. These results suggest that hormonal changes might control several seed characteristics simultaneously.