Identification of drought related regulatory elements in tomato: A bioinformatics approach
With the threat of climate change and increasing demands for water resources, agricultural production may be more susceptible to drought-related crop losses. Understanding the mechanisms by which plants respond to drought stress is essential. A recent microarray study (Gong et. al 2010) identified 670 differentially expressed genes in tomatoes. Our goal is to identify regulatory elements associated with these differentially expressed genes. In addition, we hope to determine whether these elements work as enhancers or silencers of expression and whether combinatorial gene regulation is occurring in this system. Original sequence accession numbers for the 670 genes were obtained from the Gong et al. paper. Full genomic sequences were extracted from the tomato genome (solgenomics.com). To identify specific regulatory motifs, the promoter region (3kb upstream) of all 670 gene sequences were analyzed using HOMER (Hypergeometric Optimization of Motif Enrichment) for denovo motif prediction. Sequences were analyzed between the -3kb to the +1 kb transcriptional start site. Strong candidates were selected and analyzed using JASPAR. In house programs were used to identify the total number of binding sites for particular motifs in each of the 670 genes and to identify the average number of binding sites for regulatory elements in each gene.
From the analysis performed using HOMER, JASPAR, and the in house software, 31 common motifs were found between all databases. These results yielded P-values ranging from 1e-257 to 1e-67, and conservation of all target motifs ranging from 100% to 32.33%. From the 31 motifs identified, 8 were chosen and further analyzed computationally for number of binding sites within a gene. Using JASPAR, these motifs were compared with known genes to identify orthologous genes and known functions. Similarities were found with several sequences with homology to yeast (Saccharomyces cerevisiae) genes which play roles in transcriptional response to environmental stimuli, transcriptional activation, and transcriptional silencing. Other orthologous genes found in Zea mays, Nicotiana, and Petunia had roles related to transcription regulation, wound or growth responses and production of secondary compounds. In summary, eight de novo regulatory sequences have been identified within the tomato genome and compared with homologous sequences with known functions in other organisms using a bioinformatics approach. Identification of these potential transcription factor binding motifs may provide a clearer understanding of the mechanisms regulating in the differential expression of genes in response to drought.