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.

The Tomato Guanylate-Binding Protein SlGBP1 Enables Fruit Tissue Differentiation by Maintaining Endopolyploid Cells in a Non-Proliferative State.

Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.

A Chimeric TGA Repressor Slows Down Fruit Maturation and Ripening in Tomato.

The bZIP transcription factor (TF) SlTGA2.2 was previously highlighted as a possible hub in a network regulating fruit growth and transition to ripening (maturation phase). It belongs to a clade of TFs well known for their involvement in the regulation of the salicylic acid-dependent systemic acquired resistance. To investigate if this TGA TF plays a role in tomato fruit growth and maturation, we took advantage of the fruit-specific SlPPC2 promoter (PPC2pro) to target the expression of a SlTGA2.2-SRDX chimeric repressor in a developmental window restricted to early fruit growth and maturation. Here, we show that this SlTGA2.2-SRDX repressor alters early fruit development and metabolism, including chloroplast number and structure, considerably extends the time necessary to reach the mature green stage and slows down fruit ripening. RNA sequencing and plant hormone analyses reveal that PPC2pro:SlTGA2.2-SRDX fruits are maintained in an immature stage as long as PPC2pro is active, through early modifications of plant hormonal signaling and down-regulation of MADS-RIN and NAC-NOR ripening regulators. Once PPC2pro becomes inactive and therefore SlTGA2.2-SRDX expression is reduced, ripening can proceed, albeit at a slower pace than normal. Altogether, this work emphasizes the developmental continuum between fruit growth, maturation and ripening and provides a useful tool to alter and study the molecular bases of tomato fruit transition to ripening.

The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato.

Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signaling network to improve productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a-overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and of AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a-overexpressing fruit further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BR target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroads with auxin and light signaling.

MRSI vs CEST MRI to understand tomato metabolism in ripening fruit: is there a better contrast?

Besides structural information, magnetic resonance imaging (MRI) is crucial to reveal the presence and gradients of metabolites in organs constituted of several tissues. In plant science, such knowledge is key to better understand fruit development and metabolism. Routine methods based on fixation for cytological studies or dissection for metabolite measurements induce biases and plant sample destruction. Magnetic resonance spectroscopy imaging (MSRI) leads to one NMR spectrum per pixel while chemical exchange saturation transfer (CEST) MRI allows mapping metabolites having exchangeable protons. As both methods present different advantages and drawbacks, we compared them to map metabolites in ripe tomato fruits. We demonstrated that MRSI was difficult to interpret due to large spatial chemical shift variations while CEST MRI produced promising image mapping of the main carbohydrates and amino acids. It showed that glucose/fructose was mostly located in the locular tissue, whereas glutamate/glutamine/GABA was found inside the columella.Graphical abstract.

Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.

Tomato GDSL1 is required for cutin deposition in the fruit cuticle.

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

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.

Flexible tools for gene expression and silencing in tomato.

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

NMR-Based Tissular and Developmental Metabolomics of Tomato Fruit.

Fruit is a complex organ containing seeds and several interconnected tissues with dedicated roles. However, most biochemical or molecular studies about fleshy fruit development concern the entire fruit, the fruit without seeds, or pericarp only. We studied tomato (Solanum lycopersicum) fruit at four stages of development (12, 20, 35, and 45 days post-anthesis). We separated the seeds and the other tissues, exocarp, mesocarp, columella with placenta and locular tissue, and analyzed them individually using proton NMR metabolomic profiling for the quantification of major polar metabolites, enzymatic analysis of starch, and LC-DAD analysis of isoprenoids. Pericarp tissue represented about half of the entire fruit mass only. The composition of each fruit tissue changed during fruit development. An ANOVA-PCA highlighted common, and specific metabolite trends between tissues e.g., higher contents of chlorogenate in locular tissue and of starch in columella. Euclidian distances based on compositional data showed proximities within and between tissues. Several metabolic regulations differed between tissues as revealed by the comparison of metabolite networks based on correlations between compounds. This work stressed the role of specific tissues less studied than pericarp but that impact fruit organoleptic quality including its shape and taste, and fruit processing quality.

Identification of Two New Mechanisms That Regulate Fruit Growth by Cell Expansion in Tomato.

Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection. Their thorough characterization at organ, tissue and cellular levels revealed two major clusters controlling fruit growth and tissue morphogenesis either through (i) the growth of all fruit tissues through isotropic cell expansion or (ii) only the growth of the pericarp through anisotropic cell expansion. These likely correspond to new cell expansion modules controlling fruit growth and tissue morphogenesis in tomato. Our study therefore opens the way for the identification of new gene regulatory networks controlling tomato fruit growth and morphology.

Culture of the Tomato Micro-Tom Cultivar in Greenhouse.

Micro-Tom tomato cultivar is particularly adapted to the development of genomic approaches in tomato. Here, we describe the culture of this plant in greenhouse, including climate regulation, seed sowing and watering, vegetative development, plant maintenance, including treatment of phytosanitary problems, and reproductive development.

Regulation of the fruit-specific PEP carboxylase SlPPC2 promoter at early stages of tomato fruit development.

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the ß-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5' SlPPC2 promoter deletions fused to LUC in fruits at the 8(th) day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5' UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

Gene and metabolite regulatory network analysis of early developing fruit tissues highlights new candidate genes for the control of tomato fruit composition and development.

Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12-35 d postanthesis). Transcriptome and metabolome analyses were carried out with tomato microarrays and analytical methods including proton nuclear magnetic resonance and liquid chromatography-mass spectrometry, respectively. Pairwise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes (e.g. the correlations between glutamine, bZIP, and MYB transcription factors). Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

An integrative genomics approach for deciphering the complex interactions between ascorbate metabolism and fruit growth and composition in tomato.

Very few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and other metabolic pathways in the fruit, we undertook a fruit systems biology approach. To this end, we have produced tomato transgenic lines altered in ascorbate content and redox ratio by RNAi-targeting several key enzymes involved in ascorbate biosynthesis (2 enzymes) and recycling (2 enzymes). In the VTC (ViTamin C) Fruit project, we then generated phenotypic and genomic (transcriptome, proteome, metabolome) data from wild type and mutant tomato fruit at two stages of fruit development, and developed or implemented statistical and bioinformatic tools as a web application (named VTC Tool box) necessary to store, analyse and integrate experimental data in tomato. By using Kohonen's self-organizing maps (SOMs) to cluster the biological data, pair-wise Pearson correlation analyses and simultaneous visualization of transcript/protein and metabolites (MapMan), this approach allowed us to uncover major relationships between ascorbate and other metabolic pathways.

Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato.

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.