Characterizing the grape transcriptome. Analysis of expressed sequence tags from multiple Vitis species and development of a compendium of gene expression during berry development

Abstract

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of > or =98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species.

Figure 1.

A controlled vocabulary for description of the Vitis species cDNA libraries. Libraries are organized into three main categories, (A) genotype, (B) development, and (C) stress, which can be further subdivided as shown. Terms for the Cabernet Sauvignon Leaf (CA12LI) are shown by way of example, with detailed information for all libraries available in Supplemental Tables I and II.

Figure 2.

Distribution of V. vinifera unigenes with putative functions assigned through annotation using gene ontology. Molecular function (A), biological process (B), and cellular component (C). Assignments are based on the data available at the TIGR Vitis vinifera Gene Index (http://www.tigr.org/tdb/tgi/vvgi/) version 3.1 (November, 2003).

Figure 3.

Hierarchical cluster of grape cDNA libraries and TCs based on EST distribution. A, One-dimensional clustering of 29 grape cDNA libraries. B, Two-dimensional hierarchical clustering of 665 differentially expressed transcripts versus the 29 cDNA libraries shown in A. All 665 differentially expressed transcripts in B have a true positive rate of >98.4% (Stekel et al., 2000). Two-dimensional clustering is based on a Pearson's correlation coefficient matrix. Band intensity designates relative transcript abundance in a given library, as inferred from EST frequency within each TC. Black and white designate least and most abundant transcripts, respectively. ND, Not determined; NA, not available.

Figure 4.

Expression of GRIP genes based on normalized EST frequency. A, Putative cell-wall-associated GRIP genes. B, Putative stress-response-associated GRIP genes. Gene expression is represented as percentage of distribution of ESTs in each of the corresponding libraries. ND, Developmental stage not determined. NCBI accession and contig numbers are given in Table V.

Figure 5.

Overview of berry developmental and stages sampled by EST sequencing in the UC Davis flower-berry developmental series. SI, stage I; SII, stage II; SIII, stage III. 1, Flower prebloom; 2, flower bloom; 3, berry stage I; 4, berry stage II green hard; 5, berry stage II green soft; 6, berry stage III, 19 brix. Brix is a measure of soluble solids and a standard measure of ripening.

Figure 6.

Hierarchical cluster of differentially expressed berry transcripts. EST frequencies used in this analysis corresponds to those derived from sequencing of the UC Davis flower-berry developmental series (see Fig. 5). Contig (CTG) numbers on the vertical axis correspond to the 87 differentially expressed transcripts described in the compendium of berry gene expression. Color designates relative transcript abundance in a given library, with red and blue being the most and least abundant, respectively. Individual transcripts span the vertical axis. cDNA libraries are organized in a temporal series, horizontally from left to right, as follows: FpB, flower prebloom; FB, flower bloom; SI, stage 1 berries; SIIgh, stage II green hard berries; SIIgs, stage II green soft berries; SIII, stage III berries (postveraison).

Figure 7.

PCA of 10 independent flower and berry EST data sets. F, Flower; FpB, flower prebloom; FB, flower bloom; SI, stage I berries; Pv, preveraison berries (approximately equivalent to stage I); SIIgh, stage II green hard berries; SIIgs, stage II green soft berries; SIII, stage III berries (postveraison); V, veraison; R, ripe (postveraison). Note that libraries that sampling similar developmental stages have highly similar principal components. Symbols are as follows: white diamond, chitinases; white square, nsLTPs; gray circle, allergens; white triangle, metallothioneins; *, putative transcription factors; white circle, Hsp; -, hidroxyproline-rich cell wall protein; gray diamond, photosynthesis-related; black inverted triangle, ripening induced proteins; gray square, plasma-membrane-associated proteins; black circle, ACC oxidase; black circle, caffeic acid OMT; black square, PPO; black triangle, chalcone isomerase; gray triangle, myo-inositol 1-P synthase; gray inverted triangle, aquaporin; and black diamond, no hit.