Modelling metabolic fluxes of tomato stems reveals that nitrogen shapes central metabolism for defence against Botrytis cinerea.

Among plant pathogens, the necrotrophic fungus Botrytis cinerea is one of the most prevalent, leading to severe crop damage. Studies related to its colonization of different plant species have reported variable host metabolic responses to infection. In tomato, high N availability leads to decreased susceptibility. Metabolic flux analysis can be used as an integrated method to better understand which metabolic adaptations lead to effective host defence and resistance. Here, we investigated the metabolic response of tomato infected by B. cinerea in symptomless stem tissues proximal to the lesions, with a reconstructed metabolic model constrained with a large and consistent metabolic dataset acquired under four different N supplies, throughout 7 days post inoculation (dpi). An overall comparison of 48 flux solution vectors of Botrytis- and mock-inoculated plants showed that fluxes were higher in Botrytis-inoculated plants, and the difference increased with a reduction in available N, accompanying an unexpected increase in radial growth. Despite higher fluxes, such as those involved in cell wall synthesis and other pathways, fluxes related to glycolysis, the TCA cycle, amino acids and protein synthesis were limited under very low N, which might explain the enhanced susceptibility. Limiting starch synthesis and enhancing fluxes towards redox and specialized metabolism also contributed to defence independent of N supply.

Multi-regulated GDP-l-galactose phosphorylase calls the tune in ascorbate biosynthesis.

Ascorbate is involved in numerous vital processes, in particular in response to abiotic but also biotic stresses whose frequency and amplitude increase with climate change. Ascorbate levels vary greatly depending on species, tissues, or stages of development, but also in response to stress. Since its discovery, the ascorbate biosynthetic pathway has been intensely studied and it appears that GDP-l-galactose phosphorylase (GGP) is the enzyme with the greatest role in the control of ascorbate biosynthesis. Like other enzymes of this pathway, its expression is induced by various environmental and also developmental factors. Although mRNAs encoding it are among the most abundant in the transcriptome, the protein is only present in very small quantities. In fact, GGP translation is repressed by a negative feedback mechanism involving a small open reading frame located upstream of the coding sequence (uORF). Moreover, its activity is inhibited by a PAS/LOV type photoreceptor, the action of which is counteracted by blue light. Consequently, this multi-level regulation of GGP would allow fine control of ascorbate synthesis. Indeed, experiments varying the expression of GGP have shown that it plays a central role in response to stress. This new understanding will be useful for developing varieties adapted to future environmental conditions.

Ecological and metabolic implications of the nurse effect of Maihueniopsis camachoi in the Atacama Desert.

Plant-plant positive interactions are key drivers of community structure. Yet, the underlying molecular mechanisms of facilitation processes remain unexplored. We investigated the 'nursing' effect of Maihueniopsis camachoi, a cactus that thrives in the Atacama Desert between c. 2800 and 3800 m above sea level. We hypothesised that an important protective factor is thermal amelioration of less cold-tolerant species with a corresponding impact on molecular phenotypes. To test this hypothesis, we compared plant cover and temperatures within the cactus foliage with open areas and modelled the effect of temperatures on plant distribution. We combined eco-metabolomics and machine learning to test the molecular consequences of this association. Multiple species benefited from the interaction with M. camachoi. A conspicuous example was the extended distribution of Atriplex imbricata to colder elevations in association with M. camachoi (400 m higher as compared to plants in open areas). Metabolomics identified 93 biochemical markers predicting the interaction status of A. imbricata with 79% accuracy, independently of year. These findings place M. camachoi as a key species in Atacama plant communities, driving local biodiversity with an impact on molecular phenotypes of nursed species. Our results support the stress-gradient hypothesis and provide pioneer insights into the metabolic consequences of facilitation.

Enzyme-based kinetic modelling of ASC-GSH cycle during tomato fruit development reveals the importance of reducing power and ROS availability.

The ascorbate-glutathione (ASC-GSH) cycle is at the heart of redox metabolism, linking the major redox buffers with central metabolism through the processing of reactive oxygen species (ROS) and pyridine nucleotide metabolism. Tomato fruit development is underpinned by changes in redox buffer contents and their associated enzyme capacities, but interactions between them remain unclear. Based on quantitative data obtained for the core redox metabolism, we built an enzyme-based kinetic model to calculate redox metabolite concentrations with their corresponding fluxes and control coefficients. Dynamic and associated regulations of the ASC-GSH cycle throughout the whole fruit development were analysed and pointed to a sequential metabolic control of redox fluxes by ASC synthesis, NAD(P)H and ROS availability depending on the developmental phase. Furthermore, we highlighted that monodehydroascorbate reductase and the availability of reducing power were found to be the main regulators of the redox state of ASC and GSH during fruit growth under optimal conditions. Our kinetic modelling approach indicated that tomato fruit development displayed growth phase-dependent redox metabolism linked with central metabolism via pyridine nucleotides and H2 O2 availability, while providing a new tool to the scientific community to investigate redox metabolism in fruits.

Comparative constraint-based modelling of fruit development across species highlights nitrogen metabolism in the growth-defence trade-off.

Although primary metabolism is well conserved across species, it is useful to explore the specificity of its network to assess the extent to which some pathways may contribute to particular outcomes. Constraint-based metabolic modelling is an established framework for predicting metabolic fluxes and phenotypes and helps to explore how the plant metabolic network delivers specific outcomes from temporal series. After describing the main physiological traits during fruit development, we confirmed the correlations between fruit relative growth rate (RGR), protein content and time to maturity. Then a constraint-based method is applied to a panel of eight fruit species with a knowledge-based metabolic model of heterotrophic cells describing a generic metabolic network of primary metabolism. The metabolic fluxes are estimated by constraining the model using a large set of metabolites and compounds quantified throughout fruit development. Multivariate analyses showed a clear common pattern of flux distribution during fruit development with differences between fast- and slow-growing fruits. Only the latter fruits mobilise the tricarboxylic acid cycle in addition to glycolysis, leading to a higher rate of respiration. More surprisingly, to balance nitrogen, the model suggests, on the one hand, nitrogen uptake by nitrate reductase to support a high RGR at early stages of cucumber and, on the other hand, the accumulation of alkaloids during ripening of pepper and eggplant. Finally, building virtual fruits by combining 12 biomass compounds shows that the growth-defence trade-off is supported mainly by cell wall synthesis for fast-growing fruits and by total polyphenols accumulation for slow-growing fruits.

Nitrogen-mediated metabolic patterns of susceptibility to Botrytis cinerea infection in tomato (Solanum lycopersicum) stems.

MAIN CONCLUSION: Severe N stress allows an accumulation of C-based compounds but impedes that of N-based compounds required to lower the susceptibility of tomato stem to Botrytis cinerea. Botrytis cinerea, a necrotrophic filamentous fungus, forms potentially lethal lesions on the stems of infected plants. Contrasted levels of susceptibility to B. cinerea were obtained in a tomato cultivar grown on a range of nitrate concentration: low N supply resulted in high susceptibility while high N supply conferred a strong resistance. Metabolic deviations and physiological traits resulting from both infection and nitrogen limitation were investigated in the symptomless stem tissue surrounding the necrotic lesion. Prior to infection, nitrogen-deficient plants showed reduced levels of nitrogen-based compounds such as amino acids, proteins, and glutathione and elevated levels of carbon-based and defence compounds such as α-tomatine and chlorogenic acid. After B. cinerea inoculation, all plants displayed a few common responses, mainly alanine accumulation and galactinol depletion. The metabolome of resistant plants grown under high N supply showed no significant change after inoculation. On the contrary, the metabolome of susceptible plants grown under low N supply showed massive metabolic adjustments, including changes in central metabolism around glutamate and respiratory pathways, suggesting active resource mobilization and production of energy and reducing power. Redox and defence metabolisms were also stimulated by the infection in plants grown under low N supply; glutathione and chlorogenic acid accumulated, as well as metabolites with more controversial defensive roles, such as polyamines, GABA, branched-chain amino acids and phytosterols. Taken together, the results showed that nitrogen deficiency, although leading to an increase in secondary metabolites even before the pathogen attack, must have compromised the constitutive levels of defence proteins and delayed or attenuated the induced responses. The involvement of galactinol, alanine, cycloartenol and citramalate in the tomato stem response to B. cinerea is reported here for the first time.

From fruit growth to ripening in plantain: a careful balance between carbohydrate synthesis and breakdown.

In this study, we aimed to investigate for the first time different fruit development stages in plantain banana in order gain insights into the order of appearance and dominance of specific enzymes and fluxes. We examined fruit development in two plantain banana cultivars during the period between 2-12 weeks after bunch emergence using high-throughput proteomics, quantification of major metabolites, and analyses of metabolic fluxes. Starch synthesis and breakdown are processes that take place simultaneously. During the first 10 weeks fruits accumulated up to 48% of their dry weight as starch, and glucose 6-phosphate and fructose were important precursors. We found a unique amyloplast transporter and hypothesize that it facilitates the import of fructose. We identified an invertase originating from the Musa balbisiana genome that would enable carbon flow back to growth and starch synthesis and maintain a high starch content even during ripening. Enzymes associated with the initiation of ripening were involved in ethylene and auxin metabolism, starch breakdown, pulp softening, and ascorbate biosynthesis. The initiation of ripening was cultivar specific, with faster initiation being particularly linked to the 1-aminocyclopropane-1-carboxylate oxidase and 4-alpha glucanotransferase disproportionating enzymes. Information of this kind is fundamental to determining the optimal time for picking the fruit in order to reduce post-harvest losses, and has potential applications for breeding to improve fruit quality.

The Evolution of Leaf Function during Development Is Reflected in Profound Changes in the Metabolic Composition of the Vacuole.

During its development, the leaf undergoes profound metabolic changes to ensure, among other things, its growth. The subcellular metabolome of tomato leaves was studied at four stages of leaf development, with a particular emphasis on the composition of the vacuole, a major actor of cell growth. For this, leaves were collected at different positions of the plant, corresponding to different developmental stages. Coupling cytology approaches to non-aqueous cell fractionation allowed to estimate the subcellular concentrations of major compounds in the leaves. The results showed major changes in the composition of the vacuole across leaf development. Thus, sucrose underwent a strong allocation, being mostly located in the vacuole at the beginning of development and in the cytosol at maturity. Furthermore, these analyses revealed that the vacuole, rather rich in secondary metabolites and sugars in the growth phases, accumulated organic acids thereafter. This result suggests that the maintenance of the osmolarity of the vacuole of mature leaves would largely involve inorganic molecules.

Modelling predicts tomatoes can be bigger and sweeter if biophysical factors and transmembrane transports are fine-tuned during fruit development.

The trade-off between yield and quality, a major problem for the production of fleshy fruits, involves fruit expansive growth and sugar metabolism. Here we developed an integrative model by coupling a biophysical model of fleshy fruit growth processes, including water and carbon fluxes and organ expansion, with an enzyme-based kinetic model of sugar metabolism to better understand the interactions between these two processes. The integrative model was initially tested on tomato fruit, a model system for fleshy fruit. The integrative model closely simulated the biomass and major carbon metabolites of tomato fruit developing under optimal or stress conditions. The model also performed robustly when simulating the fruit size and sugar concentrations of different tomato genotypes including wild species. The validated model was used to explore ways of uncoupling the size-sweetness trade-off in fruit. Model-based virtual experiments suggested that larger sweeter tomatoes could be obtained by simultaneously manipulating certain biophysical factors and transmembrane transports. The integrative fleshy fruit model provides a promising tool to facilitate the targeted bioengineering and breeding of tomatoes and other fruits.

Regulation of sugar metabolism genes in the nitrogen-dependent susceptibility of tomato stems to Botrytis cinerea.

BACKGROUND AND AIMS: The main soluble sugars are important components of plant defence against pathogens, but the underlying mechanisms are unclear. Upon infection by Botrytis cinerea, the activation of several sugar transporters, from both plant and fungus, illustrates the struggle for carbon resources. In sink tissues, the metabolic use of the sugars mobilized in the synthesis of defence compounds or antifungal barriers is not fully understood. METHODS: In this study, the nitrogen-dependent variation of tomato stem susceptibility to B. cinerea was used to examine, before and throughout the course of infection, the transcriptional activity of enzymes involved in sugar metabolism. Under different nitrate nutrition regimes, the expression of genes that encode the enzymes of sugar metabolism (invertases, sucrose synthases, hexokinases, fructokinases and phosphofructokinases) was determined and sugar contents were measured before inoculation and in asymptomatic tissues surrounding the lesions after inoculation. KEY RESULTS: At high nitrogen availability, decreased susceptibility was associated with the overexpression of several genes 2 d after inoculation: sucrose synthases Sl-SUS1 and Sl-SUS3, cell wall invertases Sl-LIN5 to Sl-LIN9 and some fructokinase and phosphofructokinase genes. By contrast, increased susceptibility corresponded to the early repression of several genes that encode cell wall invertase and sucrose synthase. The course of sugar contents was coherent with gene expression. CONCLUSIONS: The activation of specific genes that encode sucrose synthase is required for enhanced defence. Since the overexpression of fructokinase is also associated with reduced susceptibility, it can be hypothesized that supplementary sucrose cleavage by sucrose synthases is dedicated to the production of cell wall components from UDP-glucose, or to the additional implication of fructose in the synthesis of antimicrobial compounds, or both.

Biomass composition explains fruit relative growth rate and discriminates climacteric from non-climacteric species.

Fleshy fruits are very varied, whether in terms of their composition, physiology, or rate and duration of growth. To understand the mechanisms that link metabolism to phenotypes, which would help the targeting of breeding strategies, we compared eight fleshy fruit species during development and ripening. Three herbaceous (eggplant, pepper, and cucumber), three tree (apple, peach, and clementine) and two vine (kiwifruit and grape) species were selected for their diversity. Fruit fresh weight and biomass composition, including the major soluble and insoluble components, were determined throughout fruit development and ripening. Best-fitting models of fruit weight were used to estimate relative growth rate (RGR), which was significantly correlated with several biomass components, especially protein content (R=84), stearate (R=0.72), palmitate (R=0.72), and lignocerate (R=0.68). The strong link between biomass composition and RGR was further evidenced by generalized linear models that predicted RGR with R-values exceeding 0.9. Comparison of the fruit also showed that climacteric fruit (apple, peach, kiwifruit) contained more non-cellulosic cell-wall glucose and fucose, and more starch, than non-climacteric fruit. The rate of starch net accumulation was also higher in climacteric fruit. These results suggest that the way biomass is constructed has a major influence on performance, especially growth rate.

Model-assisted comparison of sugar accumulation patterns in ten fleshy fruits highlights differences between herbaceous and woody species.

BACKGROUND AND AIMS: Sugar concentration is a key determinant of fruit quality. Soluble sugars and starch concentrations in fruits vary greatly from one species to another. The aim of this study was to investigate similarities and differences in sugar accumulation strategies across ten contrasting fruit species using a modelling approach. METHODS: We developed a coarse-grained model of primary metabolism based on the description of the main metabolic and hydraulic processes (synthesis of compounds other than sugar and starch, synthesis and hydrolysis of starch, and water dilution) involved in the accumulation of soluble sugars during fruit development. KEY RESULTS: Statistical analyses based on metabolic rates separated the species into six groups according to the rate of synthesis of compounds other than sugar and starch. Herbaceous species (cucumber, tomato, eggplant, pepper and strawberry) were characterized by a higher synthesis rate than woody species (apple, nectarine, clementine, grape and kiwifruit). Inspection of the dynamics of the processes involved in sugar accumulation revealed that net sugar importation, metabolism and dilution processes were remarkably synchronous in most herbaceous plants, whereas in kiwifruit, apple and nectarine, processes related to starch metabolism were temporally separated from other processes. Strawberry, clementine and grape showed a distinct dynamic compared with all other species. CONCLUSIONS: Overall, these results provide fresh insights into species-specific regulatory strategies and into the role of starch metabolism in the accumulation of soluble sugars in fleshy fruits. In particular, inter-specific differences in development period shape the co-ordination of metabolic processes and affect priorities for carbon allocation across species. The six metabolic groups identified by our analysis do not show a clear separation into climacteric and non-climacteric species, possibly suggesting that the metabolic processes related to sugar concentration are not greatly affected by ethylene-associated events.

Transcriptomic and proteomic data in developing tomato fruit.

Transcriptomic and proteomic analyses were performed on three replicates of tomato fruit pericarp samples collected at nine developmental stages, each replicate resulting from the pooling of at least 15 fruits. For transcriptome analysis, Illumina-sequenced libraries were mapped on the tomato genome with the aim to obtain absolute quantification of mRNA abundance. To achieve this, spikes were added at the beginning of the RNA extraction procedure. From 34,725 possible transcripts identified in the tomato, 22,877 were quantified in at least one of the nine developmental stages. For the proteome analysis, label-free liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used. Peptide ions, and subsequently the proteins from which they were derived, were quantified by integrating the signal intensities obtained from extracted ion currents (XIC) with the MassChroQ software. Absolute concentrations of individual proteins were estimated for 2375 proteins by using a mixed effects model from log10-transformed intensities and normalized to the total protein content. Transcriptomics data are available via GEO repository with accession number GSE128739. The raw MS output files and identification data were deposited on-line using the PROTICdb database (http://moulon.inra.fr/protic/tomato_fruit_development) and MS proteomics data have also been deposited to the ProteomeXchange with the dataset identifier PXD012877. The main added value of these quantitative datasets is their use in a mathematical model to estimate protein turnover in developing tomato fruit.

Regulation of Pyridine Nucleotide Metabolism During Tomato Fruit Development Through Transcript and Protein Profiling.

Central metabolism is the engine of plant biomass, supplying fruit growth with building blocks, energy, and biochemical cofactors. Among metabolic cornerstones, nicotinamide adenine dinucleotide (NAD) is particularly pivotal for electron transfer through reduction-oxidation (redox) reactions, thus participating in a myriad of biochemical processes. Besides redox functions, NAD is now assumed to act as an integral regulator of signaling cascades involved in growth and environmental responses. However, the regulation of NAD metabolism and signaling during fruit development remains poorly studied and understood. Here, we benefit from RNAseq and proteomic data obtained from nine growth stages of tomato fruit (var. Moneymaker) to dissect mRNA and protein profiles that link to NAD metabolism, including de novo biosynthesis, recycling, utilization, and putative transport. As expected for a cofactor synthesis pathway, protein profiles failed to detect enzymes involved in NAD synthesis or utilization, except for nicotinic acid phosphoribosyltransferase (NaPT) and nicotinamidase (NIC), which suggested that most NAD metabolic enzymes were poorly represented quantitatively. Further investigations on transcript data unveiled differential expression patterns during fruit development. Interestingly, among specific NAD metabolism-related genes, early de novo biosynthetic genes were transcriptionally induced in very young fruits, in association with NAD kinase, while later stages of fruit growth rather showed an accumulation of transcripts involved in later stages of de novo synthesis and in NAD recycling, which agreed with augmented NAD(P) levels. In addition, a more global overview of 119 mRNA and 78 protein significant markers for NAD(P)-dependent enzymes revealed differential patterns during tomato growth that evidenced clear regulations of primary metabolism, notably with respect to mitochondrial functions. Overall, we propose that NAD metabolism and signaling are very dynamic in the developing tomato fruit and that its differential regulation is certainly critical to fuel central metabolism linking to growth mechanisms.

Get the Balance Right: ROS Homeostasis and Redox Signalling in Fruit.

Plant central metabolism generates reactive oxygen species (ROS), which are key regulators that mediate signalling pathways involved in developmental processes and plant responses to environmental fluctuations. These highly reactive metabolites can lead to cellular damage when the reduction-oxidation (redox) homeostasis becomes unbalanced. Whilst decades of research have studied redox homeostasis in leaves, fundamental knowledge in fruit biology is still fragmentary. This is even more surprising when considering the natural profusion of fruit antioxidants that can process ROS and benefit human health. In this review, we explore redox biology in fruit and provide an overview of fruit antioxidants with recent examples. We further examine the central role of the redox hub in signalling during development and stress, with particular emphasis on ascorbate, also referred to as vitamin C. Progress in understanding the molecular mechanisms involved in the redox regulations that are linked to central metabolism and stress pathways will help to define novel strategies for optimising fruit nutritional quality, fruit production and storage.

Modeling Protein Destiny in Developing Fruit.

Protein synthesis and degradation are essential processes that regulate cell status. Because labeling in bulky organs, such as fruits, is difficult, we developed a modeling approach to study protein turnover at the global scale in developing tomato (Solanum lycopersicum) fruit. Quantitative data were collected for transcripts and proteins during fruit development. Clustering analysis showed smaller changes in protein abundance compared to mRNA abundance. Furthermore, protein and transcript abundance were poorly correlated, and the coefficient of correlation decreased during fruit development and ripening, with transcript levels decreasing more than protein levels. A mathematical model with one ordinary differential equation was used to estimate translation (kt ) and degradation (kd ) rate constants for almost 2,400 detected transcript-protein pairs and was satisfactorily fitted for >1,000 pairs. The model predicted median values of ∼2 min for the translation of a protein, and a protein lifetime of ∼11 d. The constants were validated and inspected for biological relevance. Proteins involved in protein synthesis had higher kt and kd values, indicating that the protein machinery is particularly flexible. Our model also predicts that protein concentration is more strongly affected by the rate of translation than that of degradation.

Peptide filtering differently affects the performances of XIC-based quantification methods.

In bottom-up proteomics, data are acquired on peptides resulting from proteolysis. In XIC-based quantification, the quality of the estimation of protein abundance depends on how peptide data are filtered and on which quantification method is used to express peptide intensity as protein abundance. So far, these two questions have been addressed independently. Here, we studied to what extent the relative performances of the quantification methods depend on the filters applied to peptide intensity data. To this end, we performed a spike-in experiment using Universal Protein Standard to evaluate the performances of five quantification methods in five datasets obtained after application of four peptide filters. Estimated protein abundances were not equally affected by filters depending on the computation mode and the type of data for quantification. Furthermore, we found that filters could have contrasting effects depending on the quantification objective. Intensity modeling proved to be the most robust method, providing the best results in the absence of any filter. However, the different quantification methods can achieve similar performances when appropriate peptide filters are used. Altogether, our findings provide insights into how best to handle intensity data according to the quantification objective and the experimental design. SIGNIFICANCE: We believe that our results are of major importance because they address, as far as we know for the first time, the crossed-effects of peptide intensity data filtering and XIC-based quantification methods on protein quantification. While previous papers have dealt with peptide filtering independently of the quantification method, here we combined four peptide filters (based on peptide sharing between proteins, retention time variability, peptides occurrence and peptide intensity profiles) with five XIC-based quantification methods representing different modes of calculating protein abundances from peptide intensities. For these different combinations, we analyzed the quality of protein quantification in terms of precision, accuracy and linearity of response to increasing protein concentration using a spike-in experiment. We showed that not only filters effect on the estimation of protein abundances depend on the quantification methods but also that quantification methods can reach similar performances when appropriate peptide filters are used. Also, depending on the quantification objective, i.e. absolute or relative, filters can have contrasting effects and we demonstrated that protein quantification by the peptide intensity modeling was the most robust method.

Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation.

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an "energy escape valve," helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3ß-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.

Putting primary metabolism into perspective to obtain better fruits.

Background: One of the key goals of fruit biology is to understand the factors that influence fruit growth and quality, ultimately with a view to manipulating them for improvement of fruit traits. Scope: Primary metabolism, which is not only essential for growth but is also a major component of fruit quality, is an obvious target for improvement. However, metabolism is a moving target that undergoes marked changes throughout fruit growth and ripening. Conclusions: Agricultural practice and breeding have successfully improved fruit metabolic traits, but both face the complexity of the interplay between development, metabolism and the environment. Thus, more fundamental knowledge is needed to identify further strategies for the manipulation of fruit metabolism. Nearly two decades of post-genomics approaches involving transcriptomics, proteomics and/or metabolomics have generated a lot of information about the behaviour of fruit metabolic networks. Today, the emergence of modelling tools is providing the opportunity to turn this information into a mechanistic understanding of fruits, and ultimately to design better fruits. Since high-quality data are a key requirement in modelling, a range of must-have parameters and variables is proposed.

Fluxomics links cellular functional analyses to whole-plant phenotyping.

Fluxes through metabolic pathways reflect the integration of genetic and metabolic regulations. While it is attractive to measure all the mRNAs (transcriptome), all the proteins (proteome), and a large number of the metabolites (metabolome) in a given cellular system, linking and integrating this information remains difficult. Measurement of metabolome-wide fluxes (termed the fluxome) provides an integrated functional output of the cell machinery and a better tool to link functional analyses to plant phenotyping. This review presents and discusses sets of methodologies that have been developed to measure the fluxome. First, the principles of metabolic flux analysis (MFA), its 'short time interval' version Inst-MFA, and of constraints-based methods, such as flux balance analysis and kinetic analysis, are briefly described. The use of these powerful methods for flux characterization at the cellular scale up to the organ (fruits, seeds) and whole-plant level is illustrated. The added value given by fluxomics methods for unravelling how the abiotic environment affects flux, the process, and key metabolic steps are also described. Challenges associated with the development of fluxomics and its integration with 'omics' for thorough plant and organ functional phenotyping are discussed. Taken together, these will ultimately provide crucial clues for identifying appropriate target plant phenotypes for breeding.