Leaf drought adaptive response in winter oilseed rape is altered at the onset of senescence: a study combining NMR relaxometry, multi-omics and microscopy.

Climate change is bringing more frequent and intense droughts, reducing overall water availability and adversely affecting crops. There is a need to improve our understanding of the tissular and cellular adaptation mechanisms that are critical for plant water conservation strategies. Here, we have used NMR relaxometry in combination with microscopy and multi-omic analysis to study the effects of progressive soil drought on winter oilseed rape (WOSR, Brassica napus L., cv. Aviso) leaves. This study reveals the structural and metabolic adjustments these leaves operate to maintain cell homeostasis. Our results are original in showing that the adaptive responses are altered in leaves at the onset of senescence, associated with changes in metabolic plasticity and mesophyll structures. Thus, long-term responses in young leaves involving osmotic adjustment were combined with the maintenance of tissue hydration and cell growth, contributing to high survival and recovery capacity. For the first time, short-term responses observed in early senescent-old leaves were associated with early drought-induced dehydration of the spongy layer. However, this dehydration was not followed by osmotic adjustment and did not allow maintenance of leaf tissue turgor. These findings open further studies on the genetic variability of drought responses related to identified short- and long-term structural and metabolic plasticity traits in Brassica species.

Leaf status and environmental signals jointly regulate proline metabolism in winter oilseed rape.

Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

The contrasting N management of two oilseed rape genotypes reveals the mechanisms of proteolysis associated with leaf N remobilization and the respective contributions of leaves and stems to N storage and remobilization during seed filling.

BACKGROUND: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds. This weak N use efficiency is associated with a low foliar N remobilization, leading to a significant return of N to the soil and a risk of pollution. Contrary to what is observed during senescence in the vegetative stages, N remobilization from stems and leaves is considered efficient during monocarpic senescence. However, the contribution of stems towards N management and the cellular mechanisms involved in foliar remobilization remain largely unknown. To reach this goal, the N fluxes at the whole plant level from bolting to mature seeds and the processes involved in leaf N remobilization and proteolysis were investigated in two contrasting genotypes (Aviso and Oase) cultivated under ample or restricted nitrate supply. RESULTS: During seed filling in both N conditions, Oase efficiently allocated the N from uptake to seeds while Aviso favoured a better N remobilization from stems and leaves towards seeds. Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling. Under N limitation, Aviso had a better N remobilization from leaves to stems before the onset of seed filling. Afterwards, the higher N remobilization from stems and leaves of Aviso led to a higher final N amount in seeds. This high leaf N remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. By using an original method based on the determination of Rubisco degradation in the presence of inhibitors of proteases, efficient proteolysis associated with cysteine proteases and proteasome activities was identified as the mechanism of N remobilization. CONCLUSION: The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms. In addition, the stem may act as transient storage organs in the case of an asynchronism between leaf N remobilization and N demand for seed filling.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

MAIN CONCLUSION: Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

A profiling approach of the natural variability of foliar N remobilization at the rosette stage gives clues to understand the limiting processes involved in the low N use efficiency of winter oilseed rape.

Oilseed rape, a crop requiring a high level of nitogen (N) fertilizers, is characterized by low N use efficiency. To identify the limiting factors involved in the N use efficiency of winter oilseed rape, the response to low N supply was investigated at the vegetative stage in 10 genotypes by using long-term pulse-chase (15)N labelling and studying the physiological processes of leaf N remobilization. Analysis of growth and components of N use efficiency allowed four profiles to be defined. Group 1 was characterized by an efficient N remobilization under low and high N conditions but by a decrease of leaf growth under N limitation. Group 2 showed a decrease in leaf growth under low N supply that was associated with a low N remobilization efficiency under both N supplies despite a high remobilization of soluble proteins. In response to N limitation, Group 3 is characterized by an increase in N use efficiency and leaf N remobilization compared with high N that is not sufficient to sustain the leaf biomass production at a similar level to non-limited plants. Genotypes of Group 4 subjected to low nitrate were able to maintain leaf growth to the same level as under high N. The profiling approach indicated that enhancement of amino acid export and soluble protein degradation was crucial for N remobilization improvement. At the whole-plant level, N fluxes revealed that Group 4 showed a high N remobilization in source leaves combined with a better N utilization in young leaves. Consequently, an enhanced N remobilization limits N loss in fallen leaves, but this remobilized N needs to be efficiently utilized in young leaves to improve N use efficiency.

Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions.

Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.

Metabolome and water homeostasis analysis of Thellungiella salsuginea suggests that dehydration tolerance is a key response to osmotic stress in this halophyte.

Thellungiella salsuginea, a Brassicaceae species closely related to Arabidopsis thaliana, is tolerant to high salinity. The two species were compared under conditions of osmotic stress to assess the relationships between stress tolerance, the metabolome, water homeostasis and growth performance. A broad range of metabolites were analysed by metabolic fingerprinting and profiling, and the results showed that, despite a few notable differences in raffinose and secondary metabolites, the same metabolic pathways were regulated by salt stress in both species. The main difference was quantitative: Thellungiella had much higher levels of most metabolites than Arabidopsis whatever the treatment. Comprehensive quantification of organic and mineral solutes showed a relative stability of the total solute content regardless of the species or treatment, meaning that little or no osmotic adjustment occurred under stress. The reduction in osmotic potential observed in plants under stress was found to result from a passive loss of water. Thellungiella shoots contain less water than Arabidopsis shoots, and have the ability to lose more water, which could contribute to maintain a water potential gradient between soil and plant. Significant differences between Thellungiella and Arabidopsis were also observed in terms of the physicochemical properties of their metabolomes, such as water solubility and polarity. On the whole, the Thellungiella metabolome appears to be more compatible with dehydration. Osmotic stress was also found to impact the metabolome properties in both species, increasing the overall polarity. Together, the results suggest that Thellungiella copes with osmotic stress by tolerating dehydration, with its metabolic configuration lending itself to osmoprotective strategies rather than osmo-adjustment.

Metabolome and water status phenotyping of Arabidopsis under abiotic stress cues reveals new insight into ESK1 function.

Metabolomic investigation of the freezing-tolerant Arabidopsis mutant esk1 revealed large alterations in polar metabolite content in roots and shoots. Stress metabolic markers were found to be among the most significant metabolic markers associated with the mutation, but also compounds related to growth regulation or nutrition. The metabolic phenotype of esk1 was also compared to that of wild type (WT) under various environmental constraints, namely cold, salinity and dehydration. The mutant was shown to express constitutively a subset of metabolic responses which fits with the core of stress metabolic responses in the WT. But remarkably, the most specific metabolic responses to cold acclimation were not phenocopied by esk1 mutation and remained fully inducible in the mutant at low temperature. Under salt stress, esk1 accumulated lower amounts of Na(+) in leaves than the WT, and under dehydration stress its metabolic profile and osmotic potential were only slightly impacted. These phenotypes are consistent with the hypothesis of an altered water status in esk1, which actually exhibited basic lower water content (WC) and transpiration rate (TR) than the WT. Taken together, the results suggest that ESK1 does not function as a specific cold acclimation gene, but could rather be involved in water homeostasis.

A Comparative Study of Proteolytic Mechanisms during Leaf Senescence of Four Genotypes of Winter Oilseed Rape Highlighted Relevant Physiological and Molecular Traits for NRE Improvement.

Winter oilseed rape is characterized by a low N use efficiency related to a weak leaf N remobilization efficiency (NRE) at vegetative stages. By investigating the natural genotypic variability of leaf NRE, our goal was to characterize the relevant physiological traits and the main protease classes associated with an efficient proteolysis and high leaf NRE in response to ample or restricted nitrate supply. The degradation rate of soluble proteins and D1 protein (a thylakoid-bound protein) were correlated to N remobilization, except for the genotype Samouraï which showed a low NRE despite high levels of proteolysis. Under restricted nitrate conditions, high levels of soluble protein degradation were associated with serine, cysteine and aspartic proteases at acidic pH. Low leaf NRE was related to a weak proteolysis of both soluble and thylakoid-bound proteins. The results obtained on the genotype Samouraï suggest that the timing between the onset of proteolysis and abscission could be a determinant. The specific involvement of acidic proteases suggests that autophagy and/or senescence-associated vacuoles are implicated in N remobilization under low N conditions. The data revealed that the rate of D1 degradation could be a relevant indicator of leaf NRE and might be used as a tool for plant breeding.

Effects of the inhibitor of the γ-aminobutyrate-transaminase, vinyl-γ-aminobutyrate, on development and nitrogen metabolism in Brassica napus seedlings.

γ-aminobutyrate-transaminase (EC 2.6.1.19) catalyzes the first step of the catabolism of γ-aminobutyric acid (GABA), a non-protein amino acid well-known to accumulate in plant in response to environmental stimuli. Recent studies reinforce more and more the role of its metabolism in carbon and/or nitrogen metabolisms and as a signalling molecule in developmental processes. Here we investigated the effects of inhibition of γ-aminobutyrate-transaminase (GABA-T) in seedlings of Brassica napus, using vinyl-GABA (VGB) as a specific inhibitor of GABA-T to prevent enzyme activity. Root growth was reduced by 44% in VGB-treated seedlings but was less inhibited when VGB was associated with exogenous GABA and was not reduced with exogenous GABA alone. Measurements of GABA content in seedlings grown on VGB, GABA or VGB + GABA demonstrated that GABA level in root was not linked with the root length reduction, suggesting that GABA was not the sole component acting in root growth inhibition. Besides, metabolic profiling revealed that in root, VGB-treatment caused a twofold increase in content of almost all amino acids, except for alanine whose content was 19-fold higher than in control. In order to test the involvement of alanine accumulation on growth we studied the effects of exogenous alanine. High alanine content slightly reduced root growth suggesting that VGB-induced alanine accumulation was not responsible for root length reduction. We conclude that root growth inhibition in plants whose GABA catabolism was impaired could result at least partly from the disruption of the primary metabolism as a whole rather than direct effect of GABA on cellular growth process.