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.

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.

Validation of carbon isotopologue distribution measurements by GC-MS and application to 13C-metabolic flux analysis of the tricarboxylic acid cycle in Brassica napus leaves.

The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of 13C-Metabolic Flux Analysis at isotopically non-stationary steady-state. This approach requires a high level of accuracy for the measurement of Carbon Isotopologue Distribution in plant metabolites. But this accuracy has still not been evaluated at the isotopologue level for GC-MS, leading to uncertainties for the metabolic fluxes calculated based on these fragments. Here, we developed a workflow to validate the measurements of CIDs from plant metabolites with GC-MS by producing tailor-made E. coli standard extracts harboring a predictable binomial CID for some organic and amino acids. Overall, most of our TMS-derivatives mass fragments were validated with these standards and at natural isotope abundance in plant matrices. Then, we applied this validated MS method to investigate the light/dark regulation of plant TCA cycle by incorporating U-13C-pyruvate to Brassica napus leaf discs. We took advantage of pathway-specific isotopologues/isotopomers observed between two and six hours of labeling to show that the TCA cycle can operate in a cyclic manner under both light and dark conditions. Interestingly, this forward cyclic flux mode has a nearly four-fold higher contribution for pyruvate-to-citrate and pyruvate-to-malate fluxes than the phosphoenolpyruvate carboxylase (PEPc) flux reassimilating carbon derived from some mitochondrial enzymes. The contribution of stored citrate to the mitochondrial TCA cycle activity was also questioned based on dynamics of 13C-enrichment in citrate, glutamate and succinate and variations of citrate total amounts under light and dark conditions. Interestingly, there was a light-dependent 13C-incorporation into glycine and serine showing that decarboxylations from pyruvate dehydrogenase complex and TCA cycle enzymes were actively reassimilated and could represent up to 5% to net photosynthesis.

Dataset for the metabolic and physiological characterization of seeds from oilseed rape (Brassica napus L.) plants grown under single or combined effects of drought and clubroot pathogen Plasmodiophora brassicae.

Faced with the challenges of adapting agriculture to climate change, seed production should have increased resilience to abiotic stress factors and the expected proliferation of pathogens. This concerns both the nutritional quality and seed vigor, two crucial factors in seedling establishment and yield. Both qualities are acquired during seed development, but how environment influences the genetic and physiological determinisms of these qualities remains to be elucidated. With a world production of 71 Mt of seeds per year, oilseed rape (Brassica napus) is the third largest oleaginous crop. But its productivity must cope with several abiotic stresses, among which drought is one of the main constraints in current and future climate scenarios. In addition, clubroot disease, caused by the pathogen Plasmodiophora brassicae, leads to severe yield losses for the Brassica crops worldwide. Clubroot provokes the formation of galls on the infected roots that can restrict the flow of water and nutrients within the plant throughout the growth cycle. In order to get new insights into the impact of single or combined constraints on seed qualities, metabolic profiling assays were run for a collection of 330 seed samples (including developing, mature and imbibed seeds) harvested from plants of two B. napus cultivars ("Express" and "Montego") that were grown under either drought conditions, the presence of P. brassicae, or a combination of both stresses. Metabolites were identified and quantified by UPLC or GC. In addition, monitoring germination traits was conducted for 60 mature seed lots under in vitro conditions using an automated phenotyping platform. The present dataset contains the raw contents for 42 metabolites (nmol.mg-1 of seed dry weight) filtered and analyzed with statistical tests as well as germination speed and percentages. This dataset is available under accession at Data INRAE. These data will contribute to a better understanding of the crosstalk between the plant responses to water deprivation and/or pathogen attack and how it compromises seed quality. A better understanding of the molecular and physiological responses of the seed to (a)biotic stress on a molecular and physiological will be a first step to meet scientific and technological challenges of adapting seeds to their environment.

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.

Characterization of the salt stress vulnerability of three invasive freshwater plant species using a metabolic profiling approach.

The effects of salt stress on freshwater plants has been little studied up to now, despite the fact that they are expected to present different levels of salt sensitivity or salt resistance depending on the species. The aim of this work was to assess the effect of NaCl at two concentrations on three invasive freshwater species, Elodea canadensis, Myriophyllum aquaticum and Ludwigia grandiflora, by examining morphological and physiological parameters and using metabolic profiling. The growth rate (biomass and stem length) was reduced for all species, whatever the salt treatment, but the response to salt differed between the three species, depending on the NaCl concentration. For E. canadensis, the physiological traits and metabolic profiles were only slightly modified in response to salt, whereas M. aquaticum and L. grandiflora showed great changes. In both of these species, root number, photosynthetic pigment content, amino acids and carbohydrate metabolism were affected by the salt treatments. Moreover, we are the first to report the salt-induced accumulation of compatible solutes in both species. Indeed, in response to NaCl, L. grandiflora mainly accumulated sucrose. The response of M. aquaticum was more complex, because it accumulated not only sucrose and myo-inositol whatever the level of salt stress, but also amino acids such as proline and GABA, but only at high NaCl concentrations. These responses are the metabolic responses typically found in terrestrial plants.

Manipulating feeding stimulation to protect crops against insect pests?

Enhancing natural mechanisms of plant defense against herbivores is one of the possible strategies to protect cultivated species against insect pests. Host plant feeding stimulation, which results from phagostimulant and phagodeterrent effects of both primary and secondary metabolites, could play a key role in levels of damage caused to crop plants. We tested this hypothesis by comparing the feeding intensity of the pollen beetle Meligethes aeneus on six oilseed rape (Brassica napus) genotypes in a feeding experiment, and by assessing the content of possible phagostimulant and phagodeterrent compounds in tissues targeted by the insect (flower buds). For this purpose, several dozens of primary and secondary metabolites were quantified by a set of chromatographic techniques. Intergenotypic variability was found both in the feeding experiment and in the metabolic profile of plant tissues. Biochemical composition of the perianth was in particular highly correlated with insect damage. Only a few compounds explained this correlation, among which was sucrose, known to be highly phagostimulating. Further testing is needed to validate the suggested impact of the specific compounds we have identified. Nevertheless, our results open the way for a crop protection strategy based on artificial selection of key determinants of insect feeding stimulation.