Marie-Françoise Niogret

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.

The fate of osmo-accumulated proline in leaf discs of Rape (Brassica napus L.) incubated in a medium of low osmolarity

Abstract When rape leaf discs were submitted in vitro to an upshock osmotic stress they accumulated proline. We used leaf discs treated for 16–20 h in the light with either sucrose 800 mM (−2.3 MPa) or PEG 6000 (400 g/kg H 2 O) (−1.69 MPa) to study their capacity to mobilize proline once transferred to media of higher osmotic potential. It was found that proline metabolism took place with no lag provided the external pressure was increased stepwise by 0.3 MPa. The mean rate of proline metabolization, which was lower than the rate of accumulation during the upshock, was dependent on the level of proline available at the beginning of the transfer and related to the external osmotic potential. In the recovery medium, following a hypersomotic stress with sucrose, a significant quantity of proline leakage occurred, indicating organic solute efflux during osmotic adjustment of leaf discs experiencing hypo-osmotic stress. These released solutes were taken up by the leaf disc and mobilized during the later stages of recovery. Fluxes of carbohydrates and proline are much less prominent in PEG-treated discs. The kinetics performed with sucrose and PEG-treated leaf discs showed that a significant fraction of the osmo-accumulated proline was not available for mobilization during recovery. It is suggested that, at the cellular level, this proline could be stored in the vacuole. This contrasts with the main fraction, which presumably accumulated in the cytosol/plastids. Such compartmentation seems to be related to the upshock osmotic stress response, because turgid leaf discs loaded with exogenous l -proline exhibited a high rate of proline mobilization when transferred to the reference medium not supplemented with proline. As demonstrated by the changes, firstly, in the level of individual free amino acids during the recovery of the leaf discs, and secondly induced by added citrate and glutamate on the rate of proline withdrawal, proline metabolization is partly reliant on conversion to, and the subsequent metabolism of glutamate. However changes in the activities at the level of transcription and protein synthesis are also involved, as shown by the addition of various inhibitors of protein synthesis or proline analogs.

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 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.

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.