Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

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.

Involvement of SUT1 and SUT2 Sugar Transporters in the Impairment of Sugar Transport and Changes in Phloem Exudate Contents in Phytoplasma-Infected Plants.

Phytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. However, how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomatoes infected by "Candidatus Phytoplasma solani" at the beginning of the symptomatic stage, the first symptoms appearing in the newly emerged leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. In symptomatic sink leaves, leaf curling was associated with higher starch accumulation and the expression of defense genes. The analysis of leaf midribs of symptomatic leaves indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism differed from these in noninfected plants. The phytoplasma also multiplied in the three lower source leaves, even if it was not associated with the symptoms. In these leaves, the rate of phloem sucrose exudation was lower for infected plants. Metabolite profiling of phloem sap-enriched exudates revealed that glycolate and aspartate levels were affected by the infection. Their levels were also affected in the noninfected SUT1- and SUT2-antisense lines. The findings suggest the role of sugar transporters in the responses to infection and describe the consequences of impaired sugar transport on the primary metabolism.

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).

Resolution of quantitative resistance to clubroot into QTL-specific metabolic modules.

Plant disease resistance is often under quantitative genetic control. Thus, in a given interaction, plant cellular responses to infection are influenced by resistance or susceptibility alleles at different loci. In this study, a genetic linkage analysis was used to address the complexity of the metabolic responses of Brassica napus roots to infection by Plasmodiophora brassicae. Metabolome profiling and pathogen quantification in a segregating progeny allowed a comparative mapping of quantitative trait loci (QTLs) involved in resistance and in metabolic adjustments. Distinct metabolic modules were associated with each resistance QTL, suggesting the involvement of different underlying cellular mechanisms. This approach highlighted the possible role of gluconasturtiin and two unknown metabolites in the resistance conferred by two QTLs on chromosomes C03 and C09, respectively. Only two susceptibility biomarkers (glycine and glutathione) were simultaneously linked to the three main resistance QTLs, suggesting the central role of these compounds in the interaction. By contrast, several genotype-specific metabolic responses to infection were genetically unconnected to resistance or susceptibility. Likewise, variations of root sugar profiles, which might have influenced pathogen nutrition, were not found to be related to resistance QTLs. This work illustrates how genetic metabolomics can help to understand plant stress responses and their possible links with disease.

Evolution of DMSP (dimethylsulfoniopropionate) biosynthesis pathway: Origin and phylogenetic distribution in polyploid Spartina (Poaceae, Chloridoideae).

DMSP (dimethylsulfoniopropionate) is an ecologically important sulfur metabolite commonly produced by marine algae and by some higher plant lineages, including the polyploid salt marsh genus Spartina (Poaceae). The molecular mechanisms and genes involved in the DMSP biosynthesis pathways are still unknown. In this study, we performed comparative analyses of DMSP amounts and molecular phylogenetic analyses to decipher the origin of DMSP in Spartina that represents one of the major source of terrestrial DMSP in coastal marshes. DMSP content was explored in 14 Spartina species using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Putative genes encoding the four enzymatic steps of the DMSP biosynthesis pathway in Spartina were examined and their evolutionary dynamics were studied. We found that the hexaploid lineage containing S. alterniflora, S. foliosa and S. maritima and their derived hybrids and allopolyploids are all able to produce DMSP, in contrast to species in the tetraploid clade. Thus, examination of DMSP synthesis in a phylogenetic context implicated a single origin of this physiological innovation, which occurred in the ancestor of the hexaploid Spartina lineage, 3-6MYA. Candidate genes specific to the Spartina DMSP biosynthesis pathway were also retrieved from Spartina transcriptomes, and provide a framework for future investigations to decipher the molecular mechanisms involved in this plant phenotypic novelty that has major ecological impacts in saltmarsh ecosystems.

Metabolic and physiological adjustment of Suaeda maritima to combined salinity and hypoxia.

Background and Aims: Suaeda maritima is a halophyte commonly found on coastal wetlands in the intertidal zone. Due to its habitat S. maritima has evolved tolerance to high salt concentrations and hypoxic conditions in the soil caused by periodic flooding. In the present work, the adaptive mechanisms of S. maritima to salinity combined with hypoxia were investigated on a physiological and metabolic level. Methods: To compare the adaptive mechanisms to deficient, optimal and stressful salt concentrations, S. maritima plants were grown in a hydroponic culture under low, medium and high salt concentrations. Additionally, hypoxic conditions were applied to investigate the impact of hypoxia combined with different salt concentrations. A non-targeted metabolic approach was used to clarify the biochemical pathways underlying the metabolic and physiological adaptation mechanisms of S. maritima . Key Results: Roots exposed to hypoxic conditions showed an increased level of tricarboxylic acid (TCA)-cycle intermediates such as succinate, malate and citrate. During hypoxia, the concentration of free amino acids increased in shoots and roots. Osmoprotectants such as proline and glycine betaine increased in concentrations as the external salinity was increased under hypoxic conditions. Conclusions: The combination of high salinity and hypoxia caused an ionic imbalance and an increase of metabolites associated with osmotic stress and photorespiration, indicating a severe physiological and metabolic response under these conditions. Disturbed proline degradation in the roots induced an enhanced proline accumulation under hypoxia. The enhanced alanine fermentation combined with a partial flux of the TCA cycle might contribute to the tolerance of S. maritima to hypoxic conditions.

Overexpression of ALDH10A8 and ALDH10A9 Genes Provides Insight into Their Role in Glycine Betaine Synthesis and Affects Primary Metabolism in Arabidopsis thaliana.

Betaine aldehyde dehydrogenases oxidize betaine aldehyde to glycine betaine in species that accumulate glycine betaine as a compatible solute under stress conditions. In contrast, the physiological function of betaine aldehyde dehydrogenase genes is at present unclear in species that do not accumulate glycine betaine, such as Arabidopsis thaliana. To address this question, we overexpressed the Arabidopsis ALDH10A8 and ALDH10A9 genes, which were identified to code for betaine aldehyde dehydrogenases, in wild-type A. thaliana. We analysed changes in metabolite contents of transgenic plants in comparison with the wild type. Using exogenous or endogenous choline, our results indicated that ALDH10A8 and ALDH10A9 are involved in the synthesis of glycine betaine in Arabidopsis. Choline availability seems to be a factor limiting glycine betaine synthesis. Moreover, the contents of diverse metabolites including sugars (glucose and fructose) and amino acids were altered in fully developed transgenic plants compared with the wild type. The plant metabolic response to salt and the salt stress tolerance were impaired only in young transgenic plants, which exhibited a delayed growth of the seedlings early after germination. Our results suggest that a balanced expression of the betaine aldehyde dehydrogenase genes is important for early growth of A. thaliana seedlings and for salt stress mitigation in young seedlings.

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.

Assessment of nutrient remobilization through structural changes of palisade and spongy parenchyma in oilseed rape leaves during senescence.

MAIN CONCLUSION: Differential palisade and spongy parenchyma structural changes in oilseed rape leaf were demonstrated. These dismantling processes were linked to early senescence events and associated to remobilization processes. During leaf senescence, an ordered cell dismantling process allows efficient nutrient remobilization. However, in Brassica napus plants, an important amount of nitrogen (N) in fallen leaves is associated with low N remobilization efficiency (NRE). The leaf is a complex organ mainly constituted of palisade and spongy parenchyma characterized by different structures and functions concerning water relations and carbon fixation. The aim of the present study was to demonstrate a specific structural evolution of these parenchyma throughout natural senescence in B. napus, probably linked to differential nutrient remobilization processes. The study was performed on 340 leaves from 32 plants during an 8-week development period under controlled growing conditions. Water distribution and status at the cellular level were investigated by low-field proton nuclear magnetic resonance (NMR), while light and electron microscopy were used to observe cell and plast structure. Physiological parameters were determined on all leaves studied and used as indicators of leaf development and remobilization progress. The results revealed a process of hydration and cell enlargement of leaf tissues associated with senescence. Wide variations were observed in the palisade parenchyma while spongy cells changed only very slightly. The major new functional information revealed was the link between the early senescence events and specific tissue dismantling processes.

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.

Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress.

BACKGROUND AND AIMS: Osmolytes are low-molecular-weight organic solutes, a broad group that encompasses a variety of compounds such as amino acids, tertiary sulphonium and quaternary ammonium compounds, sugars and polyhydric alcohols. Osmolytes are accumulated in the cytoplasm of halophytic species in order to balance the osmotic potential of the Na(+) and Cl(-) accumulated in the vacuole. The advantages of the accumulation of osmolytes are that they keep the main physiological functions of the cell active, the induction of their biosynthesis is controlled by environmental cues, and they can be synthesized at all developmental stages. In addition to their role in osmoregulation, osmolytes have crucial functions in protecting subcellular structures and in scavenging reactive oxygen species. SCOPE: This review discusses the diversity of osmolytes among halophytes and their distribution within taxonomic groups, the intrinsic and extrinsic factors that influence their accumulation, and their role in osmoregulation and osmoprotection. Increasing the osmolyte content in plants is an interesting strategy to improve the growth and yield of crops upon exposure to salinity. Examples of transgenic plants as well as exogenous applications of some osmolytes are also discussed. Finally, the potential use of osmolytes in protein stabilization and solvation in biotechnology, including the pharmaceutical industry and medicine, are considered.

Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

Structural changes in senescing oilseed rape leaves at tissue and subcellular levels monitored by nuclear magnetic resonance relaxometry through water status.

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.

Early osmotic adjustment responses in drought-resistant and drought-sensitive oilseed rape.

The impact of osmotic stress on growth, physiology, and metabolism of winter oilseed rape (Brassica napus L.) was investigated by detailed analysis of biomass traits, hormone metabolites and osmolytes in two genetically unrelated drought-tolerant genotypes and two unrelated drought-sensitive genotypes. Seedlings were grown in vitro under controlled conditions and osmotic stress was simulated by applying a gradual treatment with polyethylene glycol (PEG 6000), followed by hypo-osmotic treatment of variants used for metabolite determination. The results provide a basis for the identification of reliable selection criteria for drought resistance in oilseed rape. The in vitro cultivation system established during this study enabled effective discrimination of early osmotic stress responses between drought-resistant and -susceptible oilseed rape genotypes that also show large differences in relative seed yield under drought conditions in the field. Clear physiological and metabolic differences were observed between the drought-resistant and drought-sensitive genotypes, suggesting that osmotic adjustment is a key component of drought response in oilseed rape. Unexpectedly, however, the drought-resistant genotypes did not show typical hormonal adjustment and osmolyte accumulation, suggesting that they possess alternative physiological mechanisms enabling avoidance of stress symptoms.

U-13C-glucose incorporation into source leaves of Brassica napus highlights light-dependent regulations of metabolic fluxes within central carbon metabolism.

Plant central carbon metabolism comprises several important metabolic pathways acting together to support plant growth and yield establishment. Despite the emergence of 13C-based dynamic approaches, the regulation of metabolic fluxes between light and dark conditions has not yet received sufficient attention for agronomically relevant plants. Here, we investigated the impact of light/dark conditions on carbon allocation processes within central carbon metabolism of Brassica napus after U-13C-glucose incorporation into leaf discs. Leaf gas-exchanges and metabolite contents were weakly impacted by the leaf disc method and the incorporation of glucose. 13C-analysis by GC-MS showed that U-13C-glucose was converted to fructose for de novo biosynthesis of sucrose at similar rates in both light and dark conditions. However, light conditions led to a reduced commitment of glycolytic carbons towards respiratory substrates (pyruvate, alanine, malate) and TCA cycle intermediates compared to dark conditions. Analysis of 13C-enrichment at the isotopologue level and metabolic pathway isotopic tracing reconstructions identified the contribution of multiple pathways to serine biosynthesis in light and dark conditions. However, the direct contribution of the glucose-6-phosphate shunt to serine biosynthesis was not observed. Our results also provided isotopic evidences for an active metabolic connection between the TCA cycle, glycolysis and photorespiration in light conditions through a rapid reallocation of TCA cycle decarboxylations back to the TCA cycle through photorespiration and glycolysis. Altogether, these results suggest the active coordination of core metabolic pathways across multiple compartments to reorganize C-flux modes.

Low Nitrogen Input Mitigates Quantitative but Not Qualitative Reconfiguration of Leaf Primary Metabolism in Brassica napus L. Subjected to Drought and Rehydration.

In the context of climate change and the reduction of mineral nitrogen (N) inputs applied to the field, winter oilseed rape (WOSR) will have to cope with low-N conditions combined with water limitation periods. Since these stresses can significantly reduce seed yield and seed quality, maintaining WOSR productivity under a wide range of growth conditions represents a major goal for crop improvement. N metabolism plays a pivotal role during the metabolic acclimation to drought in Brassica species by supporting the accumulation of osmoprotective compounds and the source-to-sink remobilization of nutrients. Thus, N deficiency could have detrimental effects on the acclimation of WOSR to drought. Here, we took advantage of a previously established experiment to evaluate the metabolic acclimation of WOSR during 14 days of drought, followed by 8 days of rehydration under high- or low-N fertilization regimes. For this purpose, we selected three leaf ranks exhibiting contrasted sink/source status to perform absolute quantification of plant central metabolites. Besides the well-described accumulation of proline, we observed contrasted accumulations of some "respiratory" amino acids (branched-chain amino acids, lysineand tyrosine) in response to drought under high- and low-N conditions. Drought also induced an increase in sucrose content in sink leaves combined with a decrease in source leaves. N deficiency strongly decreased the levels of major amino acids and subsequently the metabolic response to drought. The drought-rehydration sequence identified proline, phenylalanine, and tryptophan as valuable metabolic indicators of WOSR water status for sink leaves. The results were discussed with respect to the metabolic origin of sucrose and some amino acids in sink leaves and the impact of drought on source-to-sink remobilization processes depending on N nutrition status. Overall, this study identified major metabolic signatures reflecting a similar response of oilseed rape to drought under low- and high-N conditions.

Towards deciphering dynamic changes and evolutionary mechanisms involved in the adaptation to low salinities in Ectocarpus (brown algae).

Colonizations of freshwater by marine species are rare events, and little information is known about the underlying mechanisms. Brown algae are an independent lineage of photosynthetic and multicellular organisms from which few species inhabit freshwater. As a marine alga that is also found in freshwater, Ectocarpus is of particular interest for studying the transition between these habitats. To gain insights into mechanisms of the transition, we examined salinity tolerance and adaptations to low salinities in a freshwater strain of Ectocarpus on physiological and molecular levels. We show that this isolate belongs to a widely distributed and highly stress-resistant clade, and differed from the genome-sequenced marine strain in its tolerance of low salinities. It also exhibited profound, but reversible, morphological, physiological, and transcriptomic changes when transferred to seawater. Although gene expression profiles were similar in both strains under identical conditions, metabolite and ion profiles differed strongly, the freshwater strain exhibiting e.g. higher cellular contents of amino acids and nitrate, higher contents of n-3 fatty acids, and lower intracellular mannitol and sodium concentrations. Moreover, several stress markers were noted in the freshwater isolate in seawater. This finding suggests that, while high stress tolerance and plasticity may be prerequisites for the colonization of freshwater, genomic alterations have occurred that produced permanent changes in the metabolite profiles to stabilize the transition.

γ-Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots.

Environmental constraints challenge cell homeostasis and thus require a tight regulation of metabolic activity. We have previously reported that the γ-aminobutyric acid (GABA) metabolism is crucial for Arabidopsis salt tolerance as revealed by the NaCl hypersensitivity of the GABA transaminase (GABA-T, At3g22200) gaba-t/pop2-1 mutant. In this study, we demonstrate that GABA-T deficiency during salt stress causes root and hypocotyl developmental defects and alterations of cell wall composition. A comparative genome-wide transcriptional analysis revealed that expression levels of genes involved in carbon metabolism, particularly sucrose and starch catabolism, were found to increase upon the loss of GABA-T function under salt stress conditions. Consistent with the altered mutant cell wall composition, a number of cell wall-related genes were also found differentially expressed. A targeted quantitative analysis of primary metabolites revealed that glutamate (GABA precursor) accumulated while succinate (the final product of GABA metabolism) significantly decreased in mutant roots after 1 d of NaCl treatment. Furthermore, sugar concentration was twofold reduced in gaba-t/pop2-1 mutant roots compared with wild type. Together, our results provide strong evidence that GABA metabolism is a major route for succinate production in roots and identify GABA as a major player of central carbon adjustment during salt stress.

Evaluation of GC/MS-Based 13C-Positional Approaches for TMS Derivatives of Organic and Amino Acids and Application to Plant 13C-Labeled Experiments.

Analysis of plant metabolite 13C-enrichments with gas-chromatography mass spectrometry (GC/MS) has gained interest recently. By combining multiple fragments of a trimethylsilyl (TMS) derivative, 13C-positional enrichments can be calculated. However, this new approach may suffer from analytical biases depending on the fragments selected for calculation leading to significant errors in the final results. The goal of this study was to provide a framework for the validation of 13C-positional approaches and their application to plants based on some key metabolites (glycine, serine, glutamate, proline, α-alanine and malate). For this purpose, we used tailor-made 13C-PT standards, harboring known carbon isotopologue distributions and 13C-positional enrichments, to evaluate the reliability of GC-MS measurements and positional calculations. Overall, we showed that some mass fragments of proline_2TMS, glutamate_3TMS, malate_3TMS and α-alanine_2TMS had important biases for 13C measurements resulting in significant errors in the computational estimation of 13C-positional enrichments. Nevertheless, we validated a GC/MS-based 13C-positional approach for the following atomic positions: (i) C1 and C2 of glycine_3TMS, (ii) C1, C2 and C3 of serine_3TMS, and (iii) C1 of malate_3TMS and glutamate_3TMS. We successfully applied this approach to plant 13C-labeled experiments for investigating key metabolic fluxes of plant primary metabolism (photorespiration, tricarboxylic acid cycle and phosphoenolpyruvate carboxylase activity).