Background: GABA (γ-aminobutyric acid) is a non protein amino acid that has been reported to accumulate in a number of plant species when subjected to high salinity and many other environmental constraints. However, no experimental data are to date available on the molecular function of GABA and the involvement of its metabolism in salt stress tolerance in higher plants. Here, we investigated the regulation of GABA metabolism in Arabidopsis thaliana at the metabolite, enzymatic activity and gene transcription levels upon NaCl stress. Results: We identified the GABA transaminase (GABA-T), the first step of GABA catabolism, as the most responsive to NaCl. We further performed a functional analysis of the corresponding gene POP2 and demonstrated that the previously isolated loss-of-function pop2-1 mutant was oversensitive to ionic stress but not to osmotic stress suggesting a specific role in salt tolerance. NaCl oversensitivity was not associated with overaccumulation of Na⁺ and Cl^- but mutant showed a slight decrease in K⁺. To bring insights into POP2 function, a promoter-reporter gene strategy was used and showed that POP2 was mainly expressed in roots under control conditions and was induced in primary root apex and aerial parts of plants in response to NaCl. Additionally, GC-MS- and UPLC-based metabolite profiling revealed major changes in roots of pop2-1 mutant upon NaCl stress including accumulation of amino acids and decrease in carbohydrates content. Conclusions: GABA metabolism was overall up-regulated in response to NaCl in Arabidopsis. Particularly, GABA-T was found to play a pivotal function and impairment of this step was responsible for a decrease in salt tolerance indicating that GABA catabolism was a determinant of Arabidopsis salt tolerance. GABA-T would act in salt responses in linking N and C metabolisms in roots.

The compatible solute hypothesis posits that maintaining osmotic equilibrium under conditions of high salinity requires synthesis of organic compounds, uptake of potassium ions, and partial exclusion of NaCl. To assess whether osmotic adaptation in Limonium latifolium proceeds according to this hypothesis, a comprehensive analysis of solute accumulation during NaCl treatments was conducted. Determination of prevailing inorganic ions and establishment of the metabolic profiles for low M r organic substances revealed that contrary to the mentioned hypothesis the major contributors to osmolarity were constituted by inorganic solutes. Independent of salinity, only 25% of this osmolarity resulted from organic solutes such as Suc and hexoses. Proline (Pro), β-alanine betaine, and choline-O-sulfate were minor contributors to osmolarity. Compatible inositols also occurred, especially chiro-inositol, characterized for the first time in this species, to our knowledge. Principal component analysis showed that only a limited number of metabolic reconfigurations occurred in response to dynamic changes in salinity. Under such conditions only sugars, chiro-inositol, and Pro behave as active osmobalancers. Analysis of metabolic profiles during acclimatization to either mild salinity or nonsaline conditions showed that organic solute accumulation is predominantly controlled by constitutive developmental programs, some of which might be slightly modulated by salinity. Osmolarity provided under such conditions can be sufficient to maintain turgor in salinized seedlings. Compartmental analysis of Pro and β-alanine betaine in leaf tissues demonstrated that these solutes, mainly located in vacuoles under nonsaline conditions, could be partly directed to the cytosol in response to salinization. Thus they did not conform with the predictions of the compatible solute hypothesis.

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. 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. 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.

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.

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.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N⁵,N¹⁰-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.