Martine Lemaire-Chamley

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.

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.

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

This study analyzes the mechanism by which cutin is deposited. GDSL1, which belongs to the GDSL esterase/acylhydrolase family of plant proteins, is found to play a key role in cutin deposition during fruit cuticle development. 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.

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.

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.

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 β-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 8th 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.

Identification of differentially expressed genes during early development of tomato fruit. Characterisation of a novel cDNA coding for a RAD23 protein.

Before the onset of ripening, tomato fruit development comprises three distinct phases: fruit set, a cell division phase and a cell expansion phase. In this study, we used the method of mRNA differential display in order to isolate tomato genes specifically expressed during these early phases of fruit development. Among 40 differen-tial bands, nine cDNAs were selected for further investigations based on their identification after nucleotide sequencing. We isolated the full-length cDNA corresponding to one of these fragments, coding for RAD23, a protein involved in the excision repair system, thus providing new sequence information on a poorly characterised protein in plants. All the isolated cDNAs were mapped on the tomato genome and their expression studied by northern blot and semi-quantitative RT–PCR during early fruit development and in vegetative organs of tomato plants. The sequence data are deposited in the GenBank under the accession numbers: AJ270956 (mo5-3C11/1), AJ270957 (mo5-3G12/4), AJ270958 (mo5-3G17), AJ270959 (mo5-3T12), AJ270960 (mo1-6A1), AJ270961 (mo1-6T1), AJ270962 (mo5-10G1), AJ270963 (mo6-20G1), AJ270964 (mo6-MGT2) and AJ243875 (LeRAD23-8 full-length cDNA).