Regulation of anthocyanin and sugar accumulation in grape berry through carbon limitation and exogenous ABA application.

To optimize vineyard management practices to adapt viticulture to climate change, knowledge of the regulation mechanism of metabolite accumulation under carbon source limitation and abscisic acid (ABA) application in grapes should be deepened. Here, carbon source limitations were imposed by reducing leaf area from 12 to 2 leaves per vine (at pea sized stage, - 2L-P; or one week prior to veraison - 2L-V) and phloem girdling between the second and third leaf from bottom to top (one week prior to veraison - 12L-girdling) were compared for their effects on berry composition. All three modalities significantly reduced sugar, anthocyanin and ABA content in comparison with berries under sufficient carbon supply (12 leaves per vine - 12L), with 2L-V being the greatest. Allowing leaf area to partially recover (2L-R) or berry ABA application (400 mg. L-1) one week before veraison increased the ratio of anthocyanin to sugar under source limitation. Combined with the analysis of berry metabolites and transcript abundances, our results indicate that source limitation and exogenous ABA co-regulated anthocyanins content through differential gene expression.

Identifying early metabolite markers of successful graft union formation in grapevine.

Grafting is an important horticultural technique used for many crop species. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. In general, visible phenotypes of grafted plants (size, root number, etc.) are poorly correlated with grafting success, but some studies have suggested that some polyphenols could be used as markers of graft incompatibility several months or years after grafting. However, much of the previous studies into metabolite markers of grafting success have not included all the controls necessary to unequivocally validate the markers proposed. In this study, we quantified 73 primary and secondary metabolites in nine hetero-grafts and six homo-grafted controls 33 days after grafting at the graft interface and in both the scion and rootstock woody tissues. Certain biomarker metabolites typical of a high stress status (such as proline, GABA and pallidol) were particularly accumulated at the graft interface of the incompatible scion/rootstock combination. We then used correlation analysis and generalized linear models to identify potential metabolite markers of grafting success measured one year after grafting. Here we present the first attempt to quantitatively predict graft compatibility and identify marker metabolites (especially asparagine, trans-resveratrol, trans-piceatannol and α-viniferin) 33 days after grafting, which was found to be particularly informative for homo-graft combinations.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Metabolite profiling during graft union formation reveals the reprogramming of primary metabolism and the induction of stilbene synthesis at the graft interface in grapevine.

BACKGROUND: Grafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruits and vegetables. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine 1 month after grafting. Table-top grafting was done on over-wintering stems (canes) of grapevine and the graft interface tissues (containing some woody stem tissues and callus) were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses. RESULTS: A total of 54 compounds from primary and secondary metabolism (19 amino acids, five primary and 30 secondary compounds metabolites) and the activity of two enzymes were measured. The graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Some amino acids and stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues. CONCLUSIONS: This study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes.

Identification and functional study of a mild allele of SlDELLA gene conferring the potential for improved yield in tomato.

Parthenocarpy, or pollination-independent fruit set, is an attractive trait for fruit production and can be induced by increased responses to the phytohormone gibberellin (GA), which regulates diverse aspects of plant development. GA signaling in plants is negatively regulated by DELLA proteins. A loss-of-function mutant of tomato DELLA (SlDELLA), procera (pro) thus exhibits enhanced GA-response phenotypes including parthenocarpy, although the pro mutation also confers some disadvantages for practical breeding. This study identified a new milder hypomorphic allele of SlDELLA, procera-2 (pro-2), which showed weaker GA-response phenotypes than pro. The pro-2 mutant contains a single nucleotide substitution, corresponding to a single amino acid substitution in the SAW subdomain of the SlDELLA. Accumulation of the mutated SlDELLA transcripts in wild-type (WT) resulted in parthenocarpy, while introduction of intact SlDELLA into pro-2 rescued mutant phenotypes. Yeast two-hybrid assays revealed that SlDELLA interacted with three tomato homologues of GID1 GA receptors with increasing affinity upon GA treatment, while their interactions were reduced by the pro and pro-2 mutations. Both pro and pro-2 mutants produced higher fruit yields under high temperature conditions, which were resulted from higher fruit set efficiency, demonstrating the potential for genetic parthenocarpy to improve yield under adverse environmental conditions.

Integrating proteomics and enzymatic profiling to decipher seed metabolism affected by temperature in seed dormancy and germination.

Temperature is an important environmental factor affecting seed dormancy and germination. The mechanism by which temperature induces germination in dormant seeds is however still unclear. Proteomic study has been performed in dormant sunflower seeds during imbibition at permissive and non-permissive temperatures for germination, 20 and 10 °C, respectively. Proteome analysis showed an increase of proteins belonging to metabolism and energy from the first hours of imbibition followed by a decrease of proteins involved in protein metabolism and seed storage in germinating compared to non-germinating seeds. Proteomic study was completed by polysome and proteasome activity assessment and enzymatic profiling on several altered proteins involved in metabolism and energy. Results showed that 20 °C treatment induced the activation of both protein synthesis and degradation processes, the latter being related to proteasome activity during the germination sensu stricto, and to other degradation processes such as proteases during the post-germination. Interestingly, enzymatic profiles showed that TCA cycle and glycolysis were more active in non-germinating seeds in the phase I of the germination sensu stricto. This result suggests the regulation of central metabolism activity in germinating seeds. The control of energy production during imbibition seems to be involved in molecular networks controlling seed dormancy and germination.

Wine yeast phenomics: A standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions.

This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following their weight loss over the time. This dispositive was coupled with robotized enzymatic assays for measuring metabolites of enological interest in natural grape juices. Despite the small volume used, kinetic parameters and fermentation end products measured are similar with those observed in larger scale vats. The vessel used also offers the possibility to assay 32 volatiles compounds using a headspace solid-phase micro-extraction coupled to gas chromatography and mass spectrometry. The vessel shaking applied strongly impacted most of the phenotypes investigated due to oxygen transfer occuring in the first hours of the alcoholic fermentation. The impact of grape must and micro-oxygenation was investigated illustrating some relevant genetic x environmental interactions. By phenotyping a wide panel of commercial wine starters in five grape juices, broad phenotypic correlations between kinetics and metabolic end products were evidentiated. Moreover, a multivariate analysis illustrates that some grape musts are more able than others to discriminate commercial strains since some are less robust to environmental changes.

Metabotyping of 30 maize hybrids under early-sowing conditions reveals potential marker-metabolites for breeding.

INTRODUCTION: In Northern Europe, maize early-sowing used to maximize yield may lead to moderate damages of seedlings due to chilling without visual phenotypes. Genetic studies and breeding for chilling tolerance remain necessary, and metabolic markers would be particularly useful in this context. OBJECTIVES: Using an untargeted metabolomic approach on a collection of maize hybrids, our aim was to identify metabolite signatures and/or metabolites associated with chilling responses at the vegetative stage, to search for metabolites differentiating groups of hybrids based on silage-earliness, and to search for marker-metabolites correlated with aerial biomass. METHODS: Thirty genetically-diverse maize dent inbred-lines (Zea mays) crossed to a flint inbred-line were sown in a field to assess metabolite profiles upon cold treatment induced by a modification of sowing date, and characterized with climatic measurements and phenotyping. RESULTS: NMR- and LC-MS-based metabolomic profiling revealed the biological variation of primary and specialized metabolites in young leaves of plants before flowering-stage. The effect of early-sowing on leaf composition was larger than that of genotype, and several metabolites were associated to sowing response. The metabolic distances between genotypes based on leaf compositional data were not related to the genotype admixture groups, and their variability was lower under early-sowing than normal-sowing. Several metabolites or metabolite-features were related to silage-earliness groups in the normal-sowing condition, some of which were confirmed the following year. Correlation networks involving metabolites and aerial biomass suggested marker-metabolites for breeding for chilling tolerance. CONCLUSION: After validation in other experiments and larger genotype panels, these marker-metabolites can contribute to breeding.

Is Change in Ovary Carbon Status a Cause or a Consequence of Maize Ovary Abortion in Water Deficit during Flowering?

Flower or grain abortion causes large yield losses under water deficit. In maize (Zea mays), it is often attributed to a carbon limitation via the disruption of sucrose cleavage by cell wall invertases in developing ovaries. We have tested this hypothesis versus another linked to the expansive growth of ovaries and silks. We have measured, in silks and ovaries of well-watered or moderately droughted plants, the transcript abundances of genes involved in either tissue expansion or sugar metabolism, together with the concentrations and amounts of sugars, and with the activities of major enzymes of carbon metabolism. Photosynthesis and indicators of sugar export, measured during water deprivation, suggested sugar export maintained by the leaf. The first molecular changes occurred in silks rather than in ovaries and involved genes affecting expansive growth rather than sugar metabolism. Changes in the concentrations and amounts of sugars and in the activities of enzymes of sugar metabolism occurred in apical ovaries that eventually aborted, but probably after the switch to abortion of these ovaries. Hence, we propose that, under moderate water deficits corresponding to most European drought scenarios, changes in carbon metabolism during flowering time are a consequence rather than a cause of the beginning of ovary abortion. A carbon-driven ovary abortion may occur later in the cycle in the case of carbon shortage or under very severe water deficits. These findings support the view that, until the end of silking, expansive growth of reproductive organs is the primary event leading to abortion, rather than a disruption of carbon metabolism.

Transcriptional and metabolic alternations rebalance wheat grain storage protein accumulation under variable nitrogen and sulfur supply.

Wheat (Triticum aestivum L.) grain storage proteins (GSPs) are major determinants of flour end-use value. Biological and molecular mechanisms underlying the developmental and nutritional determination of GSP accumulation in cereals are as yet poorly understood. Here we timed the accumulation of GSPs during wheat grain maturation relative to changes in metabolite and transcript pools in different conditions of nitrogen (N) and sulfur (S) availability. We found that the N/S supply ratio modulated the duration of accumulation of S-rich GSPs and the rate of accumulation of S-poor GSPs. These changes are likely to be the result of distinct relationships between N and S allocation, depending on the S content of the GSP. Most developmental and nutritional modifications in GSP synthesis correlated with the abundance of structural gene transcripts. Changes in the expression of transport and metabolism genes altered the concentrations of several free amino acids under variable conditions of N and S supply, and these amino acids seem to be essential in determining GSP expression. The comprehensive data set generated and analyzed here provides insights that will be useful in adapting fertilizer use to variable N and S supply, or for breeding new cultivars with balanced and robust GSP composition.

Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under contrasting environments provides a roadmap for studies of fruit metabolism.

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

Analysis of enzyme activities.

The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to >50 enzymes in relatively large numbers of samples and in various biological materials. Most of these enzymes are involved in central metabolism and several pathways are entirely covered. Direct or indirect assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, protocols for UDP-glucose pyrophosphorylase (E.C. 2.7.7.9) operating in real time and for pyrophosphate:fructose-6-phosphate 1-phosphotransferase (E.C. 2.7.1.90) are presented.

Control of flowering and cell fate by LIF2, an RNA binding partner of the polycomb complex component LHP1.

Polycomb Repressive Complexes (PRC) modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein like heterochromatin protein1 (LHP1) is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2). LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA processing and Polycomb regulation.