We are integrating a tomato fruit transcript expression profiling initiative with the cell wall proteome analysis, in both wild type ripening fruit and those of the ripening impaired mutants ripening inhibitor (rin), non-ripening (Nor) and never ripe (Nr). The transcriptome data is being generated with the 8,700 unigene TOM1 cDNA array, the long oligonucleotide 12,000 unigene (TOM2) microarrays and two RNASeq platforms (454 and Illumina).

Following the identification of genes and their cognate proteins through comparison of MS-derived protein sequence analysis with the complement of the tomato microarray, microarray and proteomics data of this gene set are directly compared to identify: (a) genes showing significant expression changes by proteomic analysis but not by microarray analysis, or vice versa, through fruit development, or in comparison with mutant fruits; (b) genes showing significant differences between changes in transcript and cognate protein levels.

Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes (transcripts, proteins or metabolites) are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection.

Light microscope image of a cross section through a tomato fruit pericarp, which comprises several tissue types: outer epidermis, collenchyma, parenchyma, vascular tissues, and inner epidermis.

We are using laser capture microdissection (LMD), coupled with transcript profiling using RNAseq to identify tissue type specific transcripts and molecular pathways, in to gain new insights into aspects of tissue-specific gene expression, and consequently tissue and organ physiology. In this regard, we are particularly interested in defining tissue-specific secretomes. In addition, this deeper mining of the transcriptome is extremely valuable for tomato gene annotation; for example, revealing substantial alternative splicing, which in turn is critical for enhancing the proteome analyses.

Tomato fruit pericarp section after removal of the vascular tissue using LMD.

To date, a total of 1,456,024 high quality sequences have been generated, distributed among the tissue libraries. Following sequence assembly, 20,976 tomato unigenes (assembled from at least five reads) were associated with one or more of the tissues.