The serine-glycine-one-carbon metabolic network orchestrates changes in nitrogen and sulfur metabolism and shapes plant development.

L-serine (Ser) and L-glycine (Gly) are critically important for the overall functioning of primary metabolism. We investigated the interaction of the phosphorylated pathway of Ser biosynthesis (PPSB) with the photorespiration-associated glycolate pathway of Ser biosynthesis (GPSB) using Arabidopsis thaliana PPSB-deficient lines, GPSB-deficient mutants, and crosses of PPSB with GPSB mutants. PPSB-deficient lines mainly showed retarded primary root growth. Mutation of the photorespiratory enzyme Ser-hydroxymethyltransferase 1 (SHMT1) in a PPSB-deficient background resumed primary root growth and induced a change in the plant metabolic pattern between roots and shoots. Grafting experiments demonstrated that metabolic changes in shoots were responsible for the changes in double mutant development. PPSB disruption led to a reduction in nitrogen (N) and sulfur (S) contents in shoots and a general transcriptional response to nutrient deficiency. Disruption of SHMT1 boosted the Gly flux out of the photorespiratory cycle, which increased the levels of the one-carbon (1C) metabolite 5,10-methylene-tetrahydrofolate and S-adenosylmethionine. Furthermore, disrupting SHMT1 reverted the transcriptional response to N and S deprivation and increased N and S contents in shoots of PPSB-deficient lines. Our work provides genetic evidence of the biological relevance of the Ser-Gly-1C metabolic network in N and S metabolism and in interorgan metabolic homeostasis.

Different metabolic adaptation strategies after overwintering in Eutrema sp. and Arabidopsis accessions under field conditions in Germany.

Successful overwintering is a prerequisite for high fitness in temperate perennials and winter annuals and is highly dependent on increased freezing tolerance and timely balancing of deacclimation with growth resumption in spring. To assess fitness costs associated with overwintering and elucidate metabolic mechanisms underlying winter survival and the switch from acclimated freezing tolerance to growth resumption, we performed a comparative field study using 14 Eutrema salsugineum accessions, E. halophilum, E. botschantzevii and 11 Arabidopsis thaliana accessions differing in freezing tolerance. Winter survival and reproductive fitness parameters were recorded and correlated with phenological stage and metabolite status during growth resumption in spring. The results revealed considerable intraspecific variation in winter survival, but survival rates of the extremophyte Eutrema were not inherently better. In both Eutrema and A. thaliana, improved winter survival was associated with reduced reproductive fitness. Metabolic analysis by GC-MS revealed intrinsic differences in the primary metabolism of the two genera during deacclimation. Eutrema contained higher levels of several amino and chlorogenic acids, while Arabidopsis had higher levels of several sugars and sugar conjugates. In both genera, increased levels of several soluble sugars were associated with increased winter survival, whereas myo-inositol has different roles in overwintering of Eutrema and A. thaliana. In addition, differences in amino acid metabolism and polyhydroxy acids levels after winter survival were found. The results provide strong evidence for a trade-off between increased winter survival and reproductive fitness in both Eutrema and Arabidopsis and document inherent differences in their metabolic strategies to survive winter.

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids.

The high-value carotenoid astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type-like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

Establishment of a GC-MS-based 13 C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism.

13 C-Metabolic flux analysis (13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 C-positionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)-derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPc-mediated CO2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

Improvement of prediction ability by integrating multi-omic datasets in barley.

BACKGROUND: Genomic prediction (GP) based on single nucleotide polymorphisms (SNP) has become a broadly used tool to increase the gain of selection in plant breeding. However, using predictors that are biologically closer to the phenotypes such as transcriptome and metabolome may increase the prediction ability in GP. The objectives of this study were to (i) assess the prediction ability for three yield-related phenotypic traits using different omic datasets as single predictors compared to a SNP array, where these omic datasets included different types of sequence variants (full-SV, deleterious-dSV, and tolerant-tSV), different types of transcriptome (expression presence/absence variation-ePAV, gene expression-GE, and transcript expression-TE) sampled from two tissues, leaf and seedling, and metabolites (M); (ii) investigate the improvement in prediction ability when combining multiple omic datasets information to predict phenotypic variation in barley breeding programs; (iii) explore the predictive performance when using SV, GE, and ePAV from simulated 3'end mRNA sequencing of different lengths as predictors. RESULTS: The prediction ability from genomic best linear unbiased prediction (GBLUP) for the three traits using dSV information was higher than when using tSV, all SV information, or the SNP array. Any predictors from the transcriptome (GE, TE, as well as ePAV) and metabolome provided higher prediction abilities compared to the SNP array and SV on average across the three traits. In addition, some (di)-similarity existed between different omic datasets, and therefore provided complementary biological perspectives to phenotypic variation. Optimal combining the information of dSV, TE, ePAV, as well as metabolites into GP models could improve the prediction ability over that of the single predictors alone. CONCLUSIONS: The use of integrated omic datasets in GP model is highly recommended. Furthermore, we evaluated a cost-effective approach generating 3'end mRNA sequencing with transcriptome data extracted from seedling without losing prediction ability in comparison to the full-length mRNA sequencing, paving the path for the use of such prediction methods in commercial breeding programs.

Specific CBF transcription factors and cold-responsive genes fine-tune the early triggering response after acquisition of cold priming and memory.

Plants need to adapt to fluctuating temperatures throughout their lifetime. Previous research showed that Arabidopsis memorizes a first cold stress (priming) and improves its primed freezing tolerance further when subjected to a second similar stress after a lag phase. This study investigates primary metabolomic and transcriptomic changes during early cold priming or triggering after 3 days at 4°C interrupted by a memory phase. DREB1 family transcription factors DREB1C/CBF2, DREB1D/CBF4, DREB1E/DDF2, and DREB1F/DDF1 were strongly significantly induced throughout the entire triggering. During triggering, genes encoding Late Embryogenesis Abundant (LEA), antifreeze proteins or detoxifiers of reactive oxygen species (ROS) were higher expressed compared with priming. Examples of early triggering responders were xyloglucan endotransglucosylase/hydrolase genes encoding proteins involved in cell wall remodeling, while late responders were identified to act in fine-tuning the stress response and developmental regulation. Induction of non-typical members of the DREB subfamily of ERF/AP2 transcription factors, the relatively small number of induced CBF regulon genes and a slower accumulation of selected cold stress associated metabolites indicate that a cold triggering stimulus might be sensed as milder stress in plants compared with priming. Further, strong induction of CBF4 throughout triggering suggests a unique function of this gene for the response to alternating temperatures.

Acclimatisation of guard cell metabolism to long-term salinity.

Stomatal movements are enabled by changes in guard cell turgor facilitated via transient accumulation of inorganic and organic ions imported from the apoplast or biosynthesized within guard cells. Under salinity, excess salt ions accumulate within plant tissues resulting in osmotic and ionic stress. To elucidate whether (a) Na+ and Cl- concentrations increase in guard cells in response to long-term NaCl exposure and how (b) guard cell metabolism acclimates to the anticipated stress, we profiled the ions and primary metabolites of leaves, the apoplast and isolated guard cells at darkness and during light, that is, closed and fully opened stomata. In contrast to leaves, the primary metabolism of guard cell preparations remained predominantly unaffected by increased salt ion concentrations. Orchestrated reductions of stomatal aperture and guard cell osmolyte synthesis were found, but unlike in leaves, no increases of stress responsive metabolites or compatible solutes occurred. Diverging regulation of guard cell metabolism might be a prerequisite to facilitate the constant adjustment of turgor that affects aperture. Moreover, the photoperiod-dependent sucrose accumulation in the apoplast and guard cells changed to a permanently replete condition under NaCl, indicating that stress-related photosynthate accumulation in leaves contributes to the permanent closing response of stomata under stress.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Transcriptional, hormonal, and metabolic changes in susceptible grape berries under powdery mildew infection.

Grapevine (Vitis vinifera) berries are extremely sensitive to infection by the biotrophic pathogen Erysiphe necator, causing powdery mildew disease with deleterious effects on grape and wine quality. The combined analysis of the transcriptome and metabolome associated with this common fungal infection has not been previously carried out in any fruit. In order to identify the molecular, hormonal, and metabolic mechanisms associated with infection, healthy and naturally infected V. vinifera cv. Carignan berries were collected at two developmental stages: late green (EL33) and early véraison (EL35). RNA sequencing combined with GC-electron impact ionization time-of-flight MS, GC-electron impact ionization/quadrupole MS, and LC-tandem MS analyses revealed that powdery mildew-susceptible grape berries were able to activate defensive mechanisms with the involvement of salicylic acid and jasmonates and to accumulate defense-associated metabolites (e.g. phenylpropanoids, fatty acids). The defensive strategies also indicated organ-specific responses, namely the activation of fatty acid biosynthesis. However, defense responses were not enough to restrict fungal growth. The fungal metabolic program during infection involves secretion of effectors related to effector-triggered susceptibility, carbohydrate-active enzymes and activation of sugar, fatty acid, and nitrogen uptake, and could be under epigenetic regulation. This study also identified potential metabolic biomarkers such as gallic, eicosanoic, and docosanoic acids and resveratrol, which can be used to monitor early stages of infection.

Differentiation of the High Night Temperature Response in Leaf Segments of Rice Cultivars with Contrasting Tolerance.

High night temperatures (HNT) affect rice yield in the field and induce chlorosis symptoms in leaves in controlled chamber experiments. However, little is known about molecular changes in leaf segments under these conditions. Transcript and metabolite profiling were performed for leaf segments of six rice cultivars with different HNT sensitivity. The metabolite profile of the sheath revealed a lower metabolite abundance compared to segments of the leaf blade. Furthermore, pre-adaptation to stress under control conditions was detected in the sheath, whereas this segment was only slightly affected by HNT. No unique significant transcriptomic changes were observed in the leaf base, including the basal growth zone at HNT conditions. Instead, selected metabolites showed correlations with HNT sensitivity in the base. The middle part and the tip were most highly affected by HNT in sensitive cultivars on the transcriptomic level with higher expression of jasmonic acid signaling related genes, genes encoding enzymes involved in flavonoid metabolism and a gene encoding galactinol synthase. In addition, gene expression of expansins known to improve stress tolerance increased in tolerant and sensitive cultivars. The investigation of the different leaf segments indicated highly segment specific responses to HNT. Molecular key players for HNT sensitivity were identified.

Characterization of the Heat-Stable Proteome during Seed Germination in Arabidopsis with Special Focus on LEA Proteins.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

Untargeted metabolomics as a hypothesis-generation tool in plant protection product discovery: Highlighting the potential of trehalose and glycerol metabolism of fungal conidiospores as novel targets.

INTRODUCTION: The production of high quality and safe food represents a main priority for the agri-food sector in the effort to sustain the exponentially growing human population. Nonetheless, there are major challenges that require the discovery of new, alternative, and improved plant protection products (PPPs). Focusing on fungal plant pathogens, the dissection of mechanisms that are essential for their survival provides insights that could be exploited towards the achievement of the aforementioned aim. In this context, the germination of fungal spores, which are essential structures for their dispersal, survival, and pathogenesis, represents a target of high potential for PPPs. To the best of our knowledge, no PPPs that target the germination of fungal spores currently exist. OBJECTIVES: Within this context, we have mined for changes in the metabolite profiles of the model fungus Aspergillus nidulans FGSC A4 conidiospores during germination, in an effort to discover key metabolites and reactions that could potentially become targets of PPPs. METHODS: Untargeted GC/EI-TOF/MS metabolomics and multivariate analyses were employed to monitor time-resolved changes in the metabolomes of germinating A. nidulans conidiospores. RESULTS: Analyses revealed that trehalose hydrolysis plays a pivotal role in conidiospore germination and highlighted the osmoregulating role of the sugar alcohols, glycerol, and mannitol. CONCLUSION: The ineffectiveness to introduce active ingredients that exhibit new mode(s)-of-action as fungicides, dictates the urge for the discovery of PPPs, which could be exploited to combat major plant protection issues. Based on the crucial role of trehalose hydrolysis in conidiospore dormancy breakage, and the subsequent involvement of glycerol in their germination, it is plausible to suggest their biosynthesis pathways as potential novel targets for the next-generation antifungal PPPs. Our study confirmed the applicability of untargeted metabolomics as a hypothesis-generation tool in PPPs' research and discovery.

Season Affects Yield and Metabolic Profiles of Rice (Oryza sativa) under High Night Temperature Stress in the Field.

Rice (Oryza sativa) is the main food source for more than 3.5 billion people in the world. Global climate change is having a strong negative effect on rice production. One of the climatic factors impacting rice yield is asymmetric warming, i.e., the stronger increase in nighttime as compared to daytime temperatures. Little is known of the metabolic responses of rice to high night temperature (HNT) in the field. Eight rice cultivars with contrasting HNT sensitivity were grown in the field during the wet (WS) and dry season (DS) in the Philippines. Plant height, 1000-grain weight and harvest index were influenced by HNT in both seasons, while total grain yield was only consistently reduced in the WS. Metabolite composition was analysed by gas chromatography-mass spectrometry (GC-MS). HNT effects were more pronounced in panicles than in flag leaves. A decreased abundance of sugar phosphates and sucrose, and a higher abundance of monosaccharides in panicles indicated impaired glycolysis and higher respiration-driven carbon losses in response to HNT in the WS. Higher amounts of alanine and cyano-alanine in panicles grown in the DS compared to in those grown in the WS point to an improved N-assimilation and more effective detoxification of cyanide, contributing to the smaller impact of HNT on grain yield in the DS.

Opposite fates of the purine metabolite allantoin under water and nitrogen limitations in bread wheat.

KEY MESSAGE: Degradation of nitrogen-rich purines is tightly and oppositely regulated under drought and low nitrogen supply in bread wheat. Allantoin is a key target metabolite for improving nitrogen homeostasis under stress. The metabolite allantoin is an intermediate of the catabolism of purines (components of nucleotides) and is known for its housekeeping role in nitrogen (N) recycling and also for its function in N transport and storage in nodulated legumes. Allantoin was also shown to differentially accumulate upon abiotic stress in a range of plant species but little is known about its role in cereals. To address this, purine catabolic pathway genes were identified in hexaploid bread wheat and their chromosomal location was experimentally validated. A comparative study of two Australian bread wheat genotypes revealed a highly significant increase of allantoin (up to 29-fold) under drought. In contrast, allantoin significantly decreased (up to 22-fold) in response to N deficiency. The observed changes were accompanied by transcriptional adjustment of key purine catabolic genes, suggesting that the recycling of purine-derived N is tightly regulated under stress. We propose opposite fates of allantoin in plants under stress: the accumulation of allantoin under drought circumvents its degradation to ammonium (NH4+) thereby preventing N losses. On the other hand, under N deficiency, increasing the NH4+ liberated via allantoin catabolism contributes towards the maintenance of N homeostasis.

NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance.

Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.

Plant Temperature Acclimation and Growth Rely on Cytosolic Ribosome Biogenesis Factor Homologs.

Arabidopsis (Arabidopsis thaliana) REI1-LIKE (REIL) proteins, REIL1 and REIL2, are homologs of a yeast ribosome biogenesis factor that participates in late cytoplasmic 60S ribosomal subunit maturation. Here, we report that the inhibited growth of the reil1-1 reil2-1 mutant at 10°C can be rescued by the expression of amino-terminal FLUORESCENT PROTEIN (FP)-REIL fusions driven by the UBIQUITIN10 promoter, allowing the analysis of REIL function in planta. Arabidopsis REIL1 appears to be functionally conserved, based on the cytosolic localization of FP-REIL1 and the interaction of native REIL1 with the 60S subunit in wild-type plants. In contrast to its yeast homologs, REIL1 also was present in translating ribosome fractions. Systems analysis revealed that wild-type Arabidopsis remodels the cytosolic translation machinery when grown at 10°C by accumulating cytosolic ribosome subunits and inducing the expression of cytosolic ribosomal RNA, ribosomal genes, ribosome biogenesis factors, and translation initiation or elongation factors. In the reil1-1 reil2-1 mutant, all processes associated with inhibited growth were delayed, although the plants maintained cellular integrity or acquired freezing tolerance. REIL proteins also were implicated in plant-specific processes: nonacclimated reil1-1 reil2-1 exhibited cold-acclimation responses, including activation of the DREB/CBF regulon. In addition, acclimated reil1-1 reil2-1 plants failed to activate FLOWERING LOCUS T expression in mature leaves. Therefore, in the wild type, REIL function may contribute to temperature perception by suppressing premature cold responses during growth at nonstressful temperatures. In conclusion, we suggest that Arabidopsis REIL proteins influence cold-induced plant ribosome remodeling and enhance the accumulation of cytosolic ribosome subunits after cold shift either by de novo synthesis or by recycling them from the translating ribosome fraction.

Ion-dependent metabolic responses of Vicia faba L. to salt stress.

Salt-affected farmlands are increasingly burdened by chlorides, carbonates, and sulfates of sodium, calcium, and magnesium. Intriguingly, the underlying physiological processes are studied almost always under NaCl stress. Two faba bean cultivars were subjected to low- and high-salt treatments of NaCl, Na2 SO4 , and KCl. Assimilation rate and leaf water vapor conductance were reduced to approximately 25-30% without biomass reduction after 7 days salt stress, but this did not cause severe carbon shortage. The equimolar treatments of Na+ , K+ , and Cl- showed comparable accumulation patterns in leaves and roots, except for SO42- which did not accumulate. To gain a detailed understanding of the effects caused by the tested ion combinations, we performed nontargeted gas chromatography-mass spectrometry-based metabolite profiling. Metabolic responses to various salts were in part highly linearly correlated, but only a few metabolite responses were common to all salts and in both cultivars. At high salt concentrations, only myo-inositol, allantoin, and glycerophosphoglycerol were highly significantly increased in roots under all tested conditions. We discovered several metabolic responses that were preferentially associated with the presence of Na+ , K+ , or Cl- . For example, increases of leaf proline and decreases of leaf fumaric acid and malic acid were apparently associated with Cl- accumulation.

Molecular signatures associated with increased freezing tolerance due to low temperature memory in Arabidopsis.

Alternating temperatures require fast and coordinated adaptation responses of plants. Cold acclimation has been extensively investigated and results in increased freezing tolerance in Arabidopsis thaliana. Here, we show that the two Arabidopsis accessions, Col-0 and N14, which differ in their freezing tolerance, showed memory of cold acclimation, that is, cold priming. Freezing tolerance was higher in plants exposed to cold priming at 4°C, a lag phase at 20°C, and a second triggering cold stress (4°C) than in plants that were only cold primed. To our knowledge, this is the first report on cold memory improving plant freezing tolerance. The triggering response was distinguishable from the priming response at the levels of gene expression (RNA-Seq), lipid (ultraperformance liquid chromatography-mass spectrometry), and metabolite composition (gas chromatography-mass spectrometry). Transcriptomic responses pointed to induced lipid, secondary metabolism, and stress in Col-0 and growth-related functions in N14. Specific accumulation of lipids included arabidopsides with possible functions as signalling molecules or precursors of jasmonic acid. Whereas cold-induced metabolites such as raffinose and its precursors were maintained in N14 during the lag phase, they were strongly accumulated in Col-0 after the cold trigger. This indicates genetic differences in the transcriptomic and metabolic patterns during cold memory.

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.