Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

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.

PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

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.

The multistress-induced Translocator protein (TSPO) differentially modulates storage lipids metabolism in seeds and seedlings.

Translocator proteins (TSPO) are conserved membrane proteins extensively studied in mammals, but their function is still unclear. Angiosperm TSPO are transiently induced by abiotic stresses in vegetative tissues. We showed previously that constitutive expression of the Arabidopsis TSPO (AtTSPO) could be detrimental to the cell. Degradation of AtTSPO requires an active autophagy pathway. We show here that genetic modifications of TSPO expression in plant and yeast cells reduce the levels of cytoplasmic lipid droplets (LD). Transgenic Arabidopsis seedlings overexpressing AtTSPO contain less LD as compared with wild type (WT). LD levels were increased in Arabidopsis AtTSPO knockout (KO) seedlings. Deletion of the Schizosaccharomyces pombe TSPO resulted in an increase in LD level in the cell. As compared with the WT, the mutant strain was more sensitive to cerulenin, an inhibitor of fatty acids and sterol biosynthesis. We found that in contrast with seedlings, overexpression of AtTSPO (OE) resulted in an up to 50% increase in seeds fatty acids as compared with WT. A time course experiment revealed that after 4 days of seed imbibition, the levels of triacylglycerol (TAG) was still higher in the OE seeds as compared with WT or KO seeds. However, the de novo synthesis of phospholipids and TAG after 24 h of imbibition was substantially reduced in OE seeds as compared with WT or KO seeds. Our findings support a plant TSPO role in energy homeostasis in a tissue-specific manner, enhancing fatty acids and LD accumulation in mature seeds and limiting LD levels in seedlings.

Metabolomic characterization of sunflower leaf allows discriminating genotype groups or stress levels with a minimal set of metabolic markers.

INTRODUCTION: Plant and crop metabolomic analyses may be used to study metabolism across genetic and environmental diversity. Complementary analytical strategies are useful for investigating metabolic changes and searching for biomarkers of response or performance. METHODS AND OBJECTIVES: The experimental material consisted in eight sunflower lines with two line status, four restorers (R, used as males) and four maintainers (B, corresponding to females) routinely used for sunflower hybrid varietal production, respectively to complement or maintain the cytoplasmic male sterility PET1. These lines were either irrigated at full soil capacity (WW) or submitted to drought stress (DS). Our aim was to combine targeted and non-targeted metabolomics to characterize sunflower leaf composition in order to investigate the effect of line status genotypes and environmental conditions and to find the best and smallest set of biomarkers for line status and stress response using a custom-made process of variables selection. RESULTS: Five hundred and eighty-eight metabolic variables were measured by using complementary analytical methods such as 1H-NMR, MS-based profiles and targeted analyses of major metabolites. Based on statistical analyses, a limited number of markers were able to separate WW and DS samples in a more discriminant manner than previously published physiological data. Another metabolic marker set was able to discriminate line status. CONCLUSION: This study underlines the potential of metabolic markers for discriminating genotype groups and environmental conditions. Their potential use for prediction is discussed.

Mycotoxin Biosynthesis and Central Metabolism Are Two Interlinked Pathways in Fusarium graminearum, as Demonstrated by the Extensive Metabolic Changes Induced by Caffeic Acid Exposure.

Fusarium graminearum is a major plant pathogen that causes devastating diseases of cereals and produces type B trichothecene (TCTB) mycotoxins in infected grains. A comprehensive understanding of the molecular and biochemical mechanisms underlying the regulation of TCTB biosynthesis is required for improving strategies to control the TCTB contamination of crops and ensuring that these strategies do not favor the production of other toxic metabolites by F. graminearum Elucidation of the association of TCTB biosynthesis with other central and specialized processes was the focus of this study. Combined 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analyses were used to compare the exo- and endometabolomes of F. graminearum grown under toxin-inducing and -repressing caffeic acid conditions. Ninety-five metabolites were putatively or unambiguously identified, including 26 primary and 69 specialized metabolites. Our data demonstrated that the inhibition of TCTB production induced by caffeic acid exposure was associated with significant changes in the secondary and primary metabolism of F. graminearum, although the fungal growth was not affected. The main metabolic changes were an increase in the accumulation of several polyketides, including toxic ones, alterations in the tricarboxylic organic acid cycle, and modifications in the metabolism of several amino acids and sugars. While these findings provide insights into the mechanisms that govern the inhibition of TCTB production by caffeic acid, they also demonstrate the interdependence between the biosynthetic pathway of TCTB and several primary and specialized metabolic pathways. These results provide further evidence of the multifaceted role of TCTB in the life cycle of F. graminearumIMPORTANCEFusarium graminearum is a major plant pathogen that causes devastating diseases of cereal crops and produces type B trichothecene (TCTB) mycotoxins in infected grains. The best way to restrict consumer exposure to TCTB is to limit their production before harvest, which requires increasing the knowledge on the mechanisms that regulate their biosynthesis. Using a metabolomics approach, we investigated the interconnection between the TCTB production pathway and several fungal metabolic pathways. We demonstrated that alteration in the TCTB biosynthetic pathway can have a significant impact on other metabolic pathways, including the biosynthesis of toxic polyketides, and vice versa. These findings open new avenues for identifying fungal targets for the design of molecules with antimycotoxin properties and therefore improving sustainable strategies to fight against diseases caused by F. graminearum Our data further demonstrate that analyses should consider all fungal toxic metabolites rather than the targeted family of mycotoxins when assessing the efficacy of control strategies.

Characterization of GMO or glyphosate effects on the composition of maize grain and maize-based diet for rat feeding.

INTRODUCTION: In addition to classical targeted biochemical analyses, metabolomic analyses seem pertinent to reveal expected as well as unexpected compositional differences between plant genetically modified organisms (GMO) and non-GMO samples. Data previously published in the existing literature led to divergent conclusions on the effect of maize transgenes on grain compositional changes and feeding effects. Therefore, a new study examining field-grown harvested products and feeds derived from them remains useful. OBJECTIVES: Our aim was to use a metabolomics approach to characterize grain and grain-based diet compositional changes for two GMO events, one involving Bacillus thuringiensis toxin to provide insect resistance and the other one conferring herbicide tolerance by detoxification of glyphosate. We also investigated the potential compositional modifications induced by the use of a glyphosate-based herbicide on the transgenic line conferring glyphosate tolerance. RESULTS: The majority of statistically significant differences in grain composition, evidenced by the use of 1H-NMR profiling of polar extracts and LC-ESI-QTOF-MS profiling of semi-polar extracts, could be attributed to the combined effect of genotype and environment. In comparison, transgene and glyphosate effects remained limited in grain for the compound families studied. Some but not all compositional changes observed in grain were also detected in grain-based diets formulated for rats. CONCLUSION: Only part of the data previously published in the existing literature on maize grains of plants with the same GMO events could be reproduced in our experiment. All spectra have been deposited in a repository freely accessible to the public. Our grain and diet characterization opened the way for an in depth study of the effects of these diets on rat health.

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.

A DEMETER-like DNA demethylase governs tomato fruit ripening.

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

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.

The peach HECATE3-like gene FLESHY plays a double role during fruit development.

Tight control of cell/tissue identity is essential for a correct and functional organ patterning, an important component of overall fruit development and eventual maturation and ripening. Despite many investigations regarding the molecular determinants of cell identity in fruits of different species, a useful model able to depict the regulatory networks governing this relevant part of fruit development is still missing. Here we described the peach fruit as a system to link the phenotype of a slow ripening (SR) selection to an altered transcriptional regulation of genes involved in determination of mesocarp cell identity providing insight toward molecular regulation of fruit tissue formation. Morpho-anatomical observations and metabolomics analyses performed during fruit development on the reference cultivar Fantasia, compared to SR, revealed that the mesocarp of SR maintained typical immaturity traits (e.g. small cell size, high amino acid contents and reduced sucrose) throughout development, along with a strong alteration of phenylpropanoid contents, resulting in accumulation of phenylalanine and lignin. These findings suggest that the SR mesocarp is phenotypically similar to a lignifying endocarp. To test this hypothesis, the expression of genes putatively involved in determination of drupe tissues identity was assessed. Among these, the peach HEC3-like gene FLESHY showed a strongly altered expression profile consistent with pit hardening and fruit ripening, generated at a post-transcriptional level. A double function for FLESHY in channelling the phenylpropanoid pathway to either lignin or flavour/aroma is suggested, along with its possible role in triggering auxin-ethylene cross talk at the start of ripening.

Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics.

At natural (13)C abundance, metabolomics based on heteronuclear NMR is limited by sensitivity. We have recently demonstrated how hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) assisted by cross-polarization (CP) provides a reliable way of enhancing the sensitivity of heteronuclear NMR in dilute mixtures of metabolites. In this Technical Note, we evaluate the precision of this experimental approach, a critical point for applications to metabolomics. The higher the repeatability, the greater the likelihood that one can detect small biologically relevant differences between samples. The average repeatability of our state-of-the-art D-DNP NMR equipment for samples of metabolomic relevance (20 mg dry weight tomato extracts) is 3.6% for signals above the limit of quantification (LOQ) and 6.4% when all the signals above the limit of detection (LOD) are taken into account. This first report on the repeatability of D-DNP highlights the compatibility of the technique with the requirements of metabolomics and confirms its potential as an analytical tool for such applications.

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.

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance.

Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.

Aluminium stress disrupts metabolic performance of Plantago almogravensis plantlets transiently.

Little is known about how tolerant plants cope with internalized aluminium (Al). Tolerant plants are known to deploy efficient detoxification mechanisms, however it is not known to what extent the primary and secondary metabolism is affected by Al. The aim of this work was to study the metabolic repercussions of Al stress in the tolerant plant Plantago almogravensis. P. almogravensis is well adapted to acid soils where high concentrations of free Al are found and has been classified as a hyperaccumulator. In vitro reared plantlets were used for this purpose in order to control Al exposure rigorously. The metabolome of P. almogravensis plantlets as well as its metabolic response to the supply of sucrose was characterized. The supply of sucrose leads to an accumulation of amino acids and secondary metabolites and consumption of carbohydrates that result from increased metabolic activity. In Al-treated plantlets the synthesis of amino acids and secondary metabolites is transiently impaired, suggesting that P. almogravensis is able to recover from the Al treatment within the duration of the trials. In the presence of Al the consumption of carbohydrate resources is accelerated. The content of some metabolic stress markers also demonstrates that P. almogravensis is highly adapted to Al stress.

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes.

Determination of the composition in sugars and organic acids in peach using mid infrared spectroscopy: comparison of prediction results according to data sets and different reference methods.

The prediction of internal quality properties, such as sweetness and acidity, in peach fruit by mid infrared spectroscopy is of interest for rapid determination. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was tested here on two populations of peach fruits issued from contrasting genitors providing a large phenotypic variability. Over two successive years, 284 samples in 2006 and 483 samples in 2007 were characterized for soluble solids content (SSC), titratable acidity (TA), glucose, fructose, sucrose, malic acid, and citric acid contents. Sugar and organic acid composition were determined by three methods: colorimetric enzymatic measurements (ENZ), high-performance liquid chromatography (HPLC), or proton NMR spectroscopy ((1)H NMR), depending on the samples. For all samples, fruit homogenates were analyzed in ATR-FTIR using the same methodology and the same spectrometer. The objective here was to evaluate the effect of reference methods on the prediction performance. The best results were generally observed for SSC and TA, the percentage of the root-mean-square error of cross validation (RMSECV%) ranging respectively between 5.8% and 8.7% and between 5.9% and 8.0%, depending on the samples. For individual sugars and organic acids, the best correlations were obtained between ATR-FTIR data and ENZ reference data followed by HPLC and (1)H NMR ones.

Deciphering genetic diversity and inheritance of tomato fruit weight and composition through a systems biology approach.

Integrative systems biology proposes new approaches to decipher the variation of phenotypic traits. In an effort to link the genetic variation and the physiological and molecular bases of fruit composition, the proteome (424 protein spots), metabolome (26 compounds), enzymatic profile (26 enzymes), and phenotypes of eight tomato accessions, covering the genetic diversity of the species, and four of their F1 hybrids, were characterized at two fruit developmental stages (cell expansion and orange-red). The contents of metabolites varied among the genetic backgrounds, while enzyme profiles were less variable, particularly at the cell expansion stage. Frequent genotype by stage interactions suggested that the trends observed for one accession at a physiological level may change in another accession. In agreement with this, the inheritance modes varied between crosses and stages. Although additivity was predominant, 40% of the traits were non-additively inherited. Relationships among traits revealed associations between different levels of expression and provided information on several key proteins. Notably, the role of frucktokinase, invertase, and cysteine synthase in the variation of metabolites was highlighted. Several stress-related proteins also appeared related to fruit weight differences. These key proteins might be targets for improving metabolite contents of the fruit. This systems biology approach provides better understanding of networks controlling the genetic variation of tomato fruit composition. In addition, the wide data sets generated provide an ideal framework to develop innovative integrated hypothesis and will be highly valuable for the research community.

Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development.

The PIN-FORMED (PIN) auxin efflux transport protein family has been well characterized in the model plant Arabidopsis thaliana, where these proteins are crucial for auxin regulation of various aspects of plant development. Recent evidence indicates that PIN proteins may play a role in fruit set and early fruit development in tomato (Solanum lycopersicum), but functional analyses of PIN-silenced plants failed to corroborate this hypothesis. Here it is demonstrated that silencing specifically the tomato SlPIN4 gene, which is predominantly expressed in tomato flower bud and young developing fruit, leads to parthenocarpic fruits due to precocious fruit development before fertilization. This phenotype was associated with only slight modifications of auxin homeostasis at early stages of flower bud development and with minor alterations of ARF and Aux/IAA gene expression. However, microarray transcriptome analysis and large-scale quantitative RT-PCR profiling of transcription factors in developing flower bud and fruit highlighted differentially expressed regulatory genes, which are potential targets for auxin control of fruit set and development in tomato. In conclusion, this work provides clear evidence that the tomato PIN protein SlPIN4 plays a major role in auxin regulation of tomato fruit set, possibly by preventing precocious fruit development in the absence of pollination, and further gives new insights into the target genes involved in fruit set.