Multi-omics quantitative data of tomato fruit unveils regulation modes of least variable metabolites.

BACKGROUND: The composition of ripe fruits depends on various metabolites which content evolves greatly throughout fruit development and may be influenced by the environment. The corresponding metabolism regulations have been widely described in tomato during fruit growth and ripening. However, the regulation of other metabolites that do not show large changes in content have scarcely been studied. RESULTS: We analysed the metabolites of tomato fruits collected on different trusses during fruit development, using complementary analytical strategies. We identified the 22 least variable metabolites, based on their coefficients of variation. We first verified that they had a limited functional link with the least variable proteins and transcripts. We then posited that metabolite contents could be stabilized through complex regulations and combined their data with the quantitative proteome or transcriptome data, using sparse partial-least-square analyses. This showed shared regulations between several metabolites, which interestingly remained linked to early fruit development. We also examined regulations in specific metabolites using correlations with individual proteins and transcripts, which revealed that a stable metabolite does not always correlate with proteins and transcripts of its known related pathways. CONCLUSIONS: The regulation of the least variable metabolites was then interpreted regarding their roles as hubs in metabolic pathways or as signalling molecules.

Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco.

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.

Maize metabolome and proteome responses to controlled cold stress partly mimic early-sowing effects in the field and differ from those of Arabidopsis.

In Northern Europe, sowing maize one-month earlier than current agricultural practices may lead to moderate chilling damage. However, studies of the metabolic responses to low, non-freezing, temperatures remain scarce. Here, genetically-diverse maize hybrids (Zea mays, dent inbred lines crossed with a flint inbred line) were cultivated in a growth chamber at optimal temperature and then three decreasing temperatures for 2 days each, as well as in the field. Leaf metabolomic and proteomic profiles were determined. In the growth chamber, 50% of metabolites and 18% of proteins changed between 20 and 16°C. These maize responses, partly differing from those of Arabidopsis to short-term chilling, were mapped on genome-wide metabolic maps. Several metabolites and proteins showed similar variation for all temperature decreases: seven MS-based metabolite signatures and two proteins involved in photosynthesis decreased continuously. Several increasing metabolites or proteins in the growth-chamber chilling conditions showed similar trends in the early-sowing field experiment, including trans-aconitate, three hydroxycinnamate derivatives, a benzoxazinoid, a sucrose synthase, lethal leaf-spot 1 protein, an allene oxide synthase, several glutathione transferases and peroxidases. Hybrid groups based on field biomass were used to search for the metabolite or protein responses differentiating them in growth-chamber conditions, which could be of interest for breeding.

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.

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

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.

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.

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.

Untargeted Analysis of Semipolar Compounds by LC-MS and Targeted Analysis of Fatty Acids by GC-MS/GC-FID: From Plant Cultivation to Extract Preparation.

The way plants are grown and samples are harvested, prepared, and extracted has a profound impact on the output of a metabolomics experiment. In this chapter, we detail the experimental procedures from plant cultivation to extract preparation, in order to avoid difficulties that could result in contamination or undesired changes of the analytes. Two plant organs are mentioned as examples: tomato fruits (Solanum lycopersicum) and flax seeds (Linum usitatissimum). Extractions designed for the untargeted analysis of semipolar compounds by liquid chromatography-mass spectrometry (LC-MS) and targeted analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS) or gas chromatography with flame ionization detector (GC-FID) are described.

Bryophyte gas-exchange dynamics along varying hydration status reveal a significant carbonyl sulphide (COS) sink in the dark and COS source in the light.

Carbonyl sulphide (COS) is a potential tracer of gross primary productivity (GPP), assuming a unidirectional COS flux into the vegetation that scales with GPP. However, carbonic anhydrase (CA), the enzyme that hydrolyses COS, is expected to be light independent, and thus plants without stomata should continue to take up COS in the dark. We measured net CO2 (AC ) and COS (AS ) uptake rates from two astomatous bryophytes at different relative water contents (RWCs), COS concentrations, temperatures and light intensities. We found large AS in the dark, indicating that CA activity continues without photosynthesis. More surprisingly, we found a nonzero COS compensation point in light and dark conditions, indicating a temperature-driven COS source with a Q10 (fractional change for a 10°C temperature increase) of 3.7. This resulted in greater AS in the dark than in the light at similar RWC. The processes underlying such COS emissions remain unknown. Our results suggest that ecosystems dominated by bryophytes might be strong atmospheric sinks of COS at night and weaker sinks or even sources of COS during daytime. Biotic COS production in bryophytes could result from symbiotic fungal and bacterial partners that could also be found on vascular plants.

Respiration climacteric in tomato fruits elucidated by constraint-based modelling.

Tomato is a model organism to study the development of fleshy fruit including ripening initiation. Unfortunately, few studies deal with the brief phase of accelerated ripening associated with the respiration climacteric because of practical problems involved in measuring fruit respiration. Because constraint-based modelling allows predicting accurate metabolic fluxes, we investigated the respiration and energy dissipation of fruit pericarp at the breaker stage using a detailed stoichiometric model of the respiratory pathway, including alternative oxidase and uncoupling proteins. Assuming steady-state, a metabolic dataset was transformed into constraints to solve the model on a daily basis throughout tomato fruit development. We detected a peak of CO2 released and an excess of energy dissipated at 40 d post anthesis (DPA) just before the onset of ripening coinciding with the respiration climacteric. We demonstrated the unbalanced carbon allocation with the sharp slowdown of accumulation (for syntheses and storage) and the beginning of the degradation of starch and cell wall polysaccharides. Experiments with fruits harvested from plants cultivated under stress conditions confirmed the concept. We conclude that modelling with an accurate metabolic dataset is an efficient tool to bypass the difficulty of measuring fruit respiration and to elucidate the underlying mechanisms of ripening.

Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays.

The measurement of enzyme activities represents an important step towards the understanding of biological networks. Continuous or discontinuous assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, two protocols for phosphoenolpyruvate carboxylase activity (EC 4.1.1.31) in plant extracts are given. For this, an extraction protocol is also described. Then, an optimization protocol for enzyme assays using enzymatic, chemical, or biological standards is proposed. This protocol evaluates in one run the optimal extract dilution, the recovery of a standard, and the technical error in a given matrix. The interest of using biological standard in routine measurements is highlighted. © 2016 by John Wiley & Sons, Inc.

Metabolomic profiling in tomato reveals diel compositional changes in fruit affected by source-sink relationships.

A detailed study of the diurnal compositional changes was performed in tomato (Solanum lycopersicum cv. Moneymaker) leaves and fruits. Plants were cultivated in a commercial greenhouse under two growth conditions: control and shaded. Expanding fruits and the closest mature leaves were harvested during two different day/night cycles (cloudy or sunny day). High-throughput robotized biochemical phenotyping of major compounds, as well as proton nuclear magnetic resonance and mass spectrometry metabolomic profiling, were used to measure the contents of about 70 metabolites in the leaves and 60 metabolites in the fruits, in parallel with ecophysiological measurements. Metabolite data were processed using multivariate, univariate, or clustering analyses and correlation networks. The shaded carbon-limited plants adjusted their leaf area, decreased their sink carbon demand and showed subtle compositional modifications. For source leaves, several metabolites varied along a diel cycle, including those directly linked to photosynthesis and photorespiration. These metabolites peaked at midday in both conditions and diel cycles as expected. However, transitory carbon storage was limited in tomato leaves. In fruits, fewer metabolites showed diel fluctuations, which were also of lower amplitude. Several organic acids were among the fluctuating metabolites. Diel patterns observed in leaves and especially in fruits differed between the cloudy and sunny days, and between the two conditions. Relationships between compositional changes in leaves and fruits are in agreement with the fact that several metabolic processes of the fruit appeared linked to its momentary supply of sucrose.

Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit.

Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co-factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits.

Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion.

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.

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.

In-silico analysis of water and carbon relations under stress conditions. A multi-scale perspective centered on fruit.

Fruit development, from its early stages, is the result of a complex network of interacting processes, on different scales. These include cell division, cell expansion but also nutrient transport from the plant, and exchanges with the environment. In the presence of nutrient limitation, in particular, the plant reacts as a whole, by modifying its architecture, metabolism, and reproductive strategy, determining the resources available for fruit development, which in turn affects the overall source-sink balance of the system. Here, we present an integrated model of tomato that explicitly accounts for early developmental changes (from cell division to harvest), and use it to investigate the impact of water deficit and carbon limitation on nutrient fluxes and fruit growth, in both dry and fresh mass. Variability in fruit response is analyzed on two different scales: among trusses at plant level, and within cell populations at fruit level. Results show that the effect of stress on individual cells strongly depends on their age, size, and uptake capabilities, and that the timing of stress application, together with the fruit position on the plant, is crucial in determining the final phenotypic outcome. Water deficit and carbon depletion impacted either source size, source activity, or sink strength with contrasted effects on fruit growth. An important prediction of the model is the major role of symplasmic transport of carbon in the early stage of fruit development, as a catalyst for cell and fruit growth.

Influence of repeated short-term nitrogen limitations on leaf phenolics metabolism in tomato.

High concentrations of phenolics have been shown to play a role in plant resistance to pathogens. One way to obtain increased phenolic concentrations in plant tissues is to limit mineral nitrogen (N) availability; however, over long periods, this treatment will have a negative effect on plant growth. The aim of our study was to determine the effect of repeated short-term N limitations on plant growth and phenolic metabolism in leaves. Tomato plants (Solanum lycopersicum, cv. Pixie) were subjected to two successive 10-day N-limitation periods (0.15 mM NO(3)(-), 0.01 mM NH(4)(+)), followed by periods of full nutrient supply (15 mM NO(3)(-), 1.2 mM NH(4)(+)). Additionally, other plants were subjected to either of these two limitation periods, and a set of control plants was given a full nutrient supply during the entire period. The phenolic metabolism was monitored by measuring the leaf concentrations of chlorogenic acid, three flavonol glycosides (quercetin and kaempferol derivatives) and two major anthocyanins, together with the expression of eight structural genes and three transcription factors of the phenylpropanoid pathway. The relative growth rate of the plants decreased during the N-limitation periods but was restored as soon as N was resupplied. Each N-limitation period resulted in an up-regulation of the phenolic biosynthetic pathway, as demonstrated by an increase in the leaf phenolic concentration and an up-regulation of the related genes. The genes in the phenolic pathway were down-regulated immediately when N was resupplied; however, the leaf concentrations of several phenolics, particularly flavonol glycosides, were maintained at significantly higher levels than in the control plants for up to 17 days after the end of the first limitation. The amplitude of the increase in leaf phenolic concentration did not depend on the number of N-limitation periods to which the plant was subjected, which indicates that the plants did not acclimate to nitrogen limitation. Successive N-limitation periods resulted in additive increases in flavonol glycoside concentrations.

Impact of temporary nitrogen deprivation on tomato leaf phenolics.

Reducing the use of pesticides represents a major challenge of modern agriculture. Plants synthesize secondary metabolites such as polyphenols that participate in the resistance to parasites. The aim of this study was to test: (1) the impact of nitrogen deficiency on tomato (Solanum lycopersicum) leaf composition and more particularly on two phenolic molecules (chlorogenic acid and rutin) as well as on the general plant biomass; and (2) whether this effect continued after a return to normal nitrogen nutrition. Our results showed that plants deprived of nitrogen for 10 or 19 days contained higher levels of chlorogenic acid and rutin than control plants. In addition, this difference persisted when the plants were once again cultivated on a nitrogen-rich medium. These findings offer interesting perspectives on the use of a short period of deprivation to modulate the levels of compounds of interest in a plant.

The 'trade-off' between synthesis of primary and secondary compounds in young tomato leaves is altered by nitrate nutrition: experimental evidence and model consistency.

Plants allocate internal resources to fulfil essential, yet possibly conflicting, demands such as defence or growth, as hypothesized by the 'growth-differentiation balance theory' (GDB). This trade-off was examined in young tomato plants grown for 25 d using the nutrient film technique with seven nitrate concentrations ([NO(3)]). The modification of primary (growth-related: organic acids, carbohydrates) and secondary (defence-related: phenolics) metabolite concentrations in leaves was assessed. Then a simple model was devised to simulate the trade-off between growth and secondary metabolism in response to N nutrition. N affected growth and metabolite concentrations in the leaves. Dry biomass, leaf area, and concentrations of nitrate and organic acid (malic, citric) increased with rising [NO(3)], up to a threshold, above which they remained constant. Starch, sucrose, and organic N concentrations were invariant with [NO(3)]. Glucose, fructose, and phenolic (chlorogenic acid, rutin, and kaempferol-rutinoside) concentrations were highest at lowest [NO(3)]. They declined progressively with rising [NO(3)] until a threshold, above which they remained constant. Model predictions are in phase with experimental phenolic concentration data although the simulated metabolic rates differ from the GDBH proposals depicted in the literature. From the model output it is shown that a careful definition of the C reserve compounds is required.