Coexpression analysis of tomato genes and experimental verification of coordinated expression of genes found in a functionally enriched coexpression module

Abstract

Gene-to-gene coexpression analysis is a powerful approach to infer the function of uncharacterized genes. Here, we report comprehensive identification of coexpression gene modules of tomato (Solanum lycopersicum) and experimental verification of coordinated expression of module member genes. On the basis of the gene-to-gene correlation coefficient calculated from 67 microarray hybridization data points, we performed a network-based analysis. This facilitated the identification of 199 coexpression modules. A gene ontology annotation search revealed that 75 out of the 199 modules are enriched with genes associated with common functional categories. To verify the coexpression relationships between module member genes, we focused on one module enriched with genes associated with the flavonoid biosynthetic pathway. A non-enzyme, non-transcription factor gene encoding a zinc finger protein in this module was overexpressed in S. lycopersicum cultivar Micro-Tom, and expression levels of flavonoid pathway genes were investigated. Flavonoid pathway genes included in the module were up-regulated in the plant overexpressing the zinc finger gene. This result demonstrates that coexpression modules, at least the ones identified in this study, represent actual transcriptional coordination between genes, and can facilitate the inference of tomato gene function.

Figure 1

Global topology of the tomato coexpression network. (A) Network density, (B) number of nodes, and (C) number of edges, at varied PCC cutoff values. Inserts are magnified curves within a cutoff range from 0.85 to 1.0. Network density showed the minimal value at PCC cutoff 0.91. Arrows in the inserts indicate this cutoff value.

Figure 2

Distribution of characteristic parameters of the identified coexpression modules. Distribution of (A) number of member probes per module and (B) NB value. NB value is defined as a ratio of a number of edges within the module and a total number of edges between module members and all possible nodes irrespective of membership in the module. Median value of the number of member probes per module is 7. More than 40% of the modules have NB values >0.8, indicating that intra-modular connectivity is high in the coexpression modules.

Figure 3

Distribution of GO category significantly enriched within a coexpression module. Note that GO categories of ‘chloroplast’ and ‘plastid’ are frequently associated with the same genes. GO categories of ‘cytosol’, ‘ribosome’, ‘structural molecule activity’, and ‘protein metabolism’ are frequently associated with the same genes.

Figure 4

Experimental verification of the coexpression relationship between members of module 64. (A) Expression profiles of 15 member genes (corresponding to 17 probes, see Supplementary data 2). 5W, five week; 3W, three week; MG, mature green; Y, yellow; O, orange; R, red. (B) Sequence of a full-length cDNA corresponding to the probe Les.2294.2.A1_at (LEFL2003DB10, GenBank accession number AK326277). Gray-shaded letters indicate a unigene sequence used to design Les.2294.2.A1_at. Boxed ATG indicates the start codon. Underlined TGA indicates the stop codon. Dotted line indicates cDNA sequence corresponding to zinc finger domain. (C) Flavonoid biosynthesis pathway (left) and coexpression network of module 64 (right, correlation coefficient cutoff at 0.6). ZnF, zinc finger; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate–CoA ligase; CCR, cinnamoyl-CoA reductase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; GT, glycosyltransferase; F3′H, flavonoid 3′-hydroxylase; EP, expressed protein; Cyt B561, cytochrome b-561; AADH, allyl alcohol dehydrogenase; dKae, dihydrokaempferol; dQue, dihydroquercetin; Kae, kaempferol; Que, quercetin. In the network graph, black edges indicate PCC ≥0.8, and grey edges indicate PCC from 0.6 to 0.8. (D) Changes in expression levels of flavonoid biosynthesis genes in module 64. Expression level of each gene is indicated as a relative value to the level in control line. Black and grey bars indicate control lines and ZnF-overexpression lines, respectively. Each of the four grey bars indicates independent ZnF-overexpression plant.

Figure 5

Intracellular localization of (A) GFP protein and (B) GFP-ZnF fusion protein. N, nucleus. Scale bar, 50 µm. Localization pattern of GFP-ZnF is the same as free GFP, suggesting that ZnF protein is localized to cytosol.