Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of tomato - regeneration of epicuticular fruit wax

ABSTRACT: Mature green fruits of the tomato (Solanum lycopersicum L.) cultivar MicroTom were investigated (fruit developmental category II). For removal of the epicuticular wax layer a thin film of 1 g ml-1 aqueous gum arabic (Roth, Karlsruhe, Germany) as a fixative was applied to the fruit surface. After 1 h the dried polymer including the outermost epicuticular waxes was mechanically removed. This procedure was repeated once. During this experiment the tomato fruits remained on the tomato plant. Untreated (0 days) and gum arabic stripped tomato fruits harvested 2 days after treatment were compared. Samples contained exclusively the fruit peel, which was removed with a scalpel to a depth of approximately 1 mm. After sampling point the plant material was immediately frozen in liquid nitrogen and stored at -80 C until use.

ORGANISM(S): Lycopersicon esculentum

SUBMITTER: Jana Leide

PROVIDER: E-MEXP-2163 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in Arabidopsis and especially other plant species engaged with global proteome assays. Here, we present large-scale protein profiling of tomato fruit across two key tissues and five developmental stages. This was accomplished by means of a shotgun proteomic assay comprising prior multi-step peptide fractionation followed by nano flow liquid chromatography coupled with high-resolution mass-spectrometry. A total of 7738 individual proteins were identified and reliably measured at least in one of the analysed tissues or stages. The detection of 115 putative transcription factors (TFs) further demonstrated the depth of our assay since a small number of TF proteins are typically detected in plant proteomics assays. Among this TFs set, 61 were differentially expressed in fruit development including renowned ripening regulators and elements of ethylene signalling. Curiously, we measured more than 80% of the predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were detected including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique, protein level view of skin and flesh tissues in developing fruit. This study adds a different perspective to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species which is presently focused on transcription.

Project description:Colletotrichum coccodes secretes ammonium during germination and colonization of host tissue as a mechanism for dual activation of fungal pathogenicity factors and plant cell death via activation of host NADPH oxidase. Ammonium accumulation during colonization of tomato fruits by C. coccodes induced host nucleus integrity changes and program cell death (PCD). Transcriptome analysis at the leading edge of colonized tissue and ammonium treated tomato fruits revealed 82 and 237 common up-regulated and common down-regulated genes, respectively. The common-up-regulated genes showed over representation of pathogen related (PR) proteins, salicylic acid (SA) dependent and systemic acquired resistance (SAR) related genes and genes related to biotic stress. The down regulated genes showed over representation of Jasmonic acid (JA) dependent genes. Consistent with this observation the direct application of SA enhanced C. coccodes colonization, whereas the application of JA delayed colonization. Candidate genes were selected for QRT-PCR analysis of transcripts in normal and transgenic RBOH antisense plants and showed dependence on RBOH regulation. In all, the results suggest that ammonia accumulation during C. coccodes colonization activates SA and suppress JA pathways through activation of RBOH. The effects of ammonium on the expression of the selected up-and down regulated genes were examined in fruit of different maturation stages. Red, susceptible and green resistant, tomato fruit displayed similar behavior, suggesting that resistance of green fruits to C. coccodes colonization is not modulated by the JA and SA pathways but depends on other factors Ten micrograms of total RNA was processed for the microarray hybridizations using the Affymetrix GeneChip one-cycle target-labeling kit (Affymetrix). The four treatments were: C. coccodes "mat inoculation" compared to a wound control (24 hours after the tomato fruit peel was removed). 5 mM Ammonium treatment in PBS compared to PBS treatments as a control for ammonium treatment, the treatments applied every four hours. Each treatment had a duplicate. The biotinylated complementary RNA was fragmented and hybridized to the GeneChip Tomato Genome Array (10,038 tomato probe sets for more than 9,200 tomato genes). The arrays were washed, stained, and scanned at the biological services (Weizmann institute of science, Rehovot, Israel). CEL files for the Affymetrix microarray data arising from the microarray experiments were analyzed utilizing the Partek genomics software (Downey, 2006).

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Transcription profiling of tomato - regeneration of epicuticular fruit wax

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