DNA microarrays: methodology, data evaluation and application in the analysis of plant defense signaling.

Abstract

A major challenge for science in the 21st century will be to obtain an integrated understanding of living organisms. We are witnessing a fundamental change in how this goal is pursued. The reductionist approach, trying to assign function to individual genes and proteins, is now followed by systems biology with the use of high-throughput techniques to extract comprehensive datasets and integrating the data to model the organism as a functional network of interacting components. One of the essential techniques is DNA microarray technology, which allows the analysis of the entire transcriptome in a single hybridization experiment. Since the pioneering work of Schena et al. on Arabidopsis in 1995 (1), microarray hybridization technology has developed into a powerful tool for the identification of differentially-regulated genes in plants and other organisms. Inherent characteristics of the technology such as miniaturization, automation and parallelism allow the determination of transcript concentrations from thousands of genes in a single experiment with high accuracy and sensitivity. The quantitative determination of transcript concentrations with microarrays is also an auspicious way toward the elucidation of plant signaling pathways. By comparing the concentrations of individual mRNAs present in samples originating from different genotypes, developmental stages, or growth conditions, genes can be identified that are differentially expressed and, hence, may have specific metabolic or morphogenetic functions. The analysis of transcription patterns has proven to be valuable in attributing function to novel sequences. Any similarity in expression patterns observed between known genes and sequences of unknown function may indicate functional homology. The first large-scale expression profiles of light- and dark-grown Arabidopsis thaliana seedlings, for example, revealed numerous genes that were highly regulated by light, but did not have a match in the nucleotide sequence databases (2, 3). The co-regulation with well-characterized light-inducible genes may indicate as yet undefined photomorphogenetic functions for the respective proteins. Another example is provided by the work of Seki et al. (4), who used a cDNA microarray of about 1,300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB IA, a transcription factor that controls stress-inducible gene expression. More than 60 transcripts were found to be upregulated, 50 of which were derived from novel genes that had not previously been reported as being drought-or cold-inducible. Twelve stress-inducible genes were recognized as targets of DREB1A, and six of them were novel. The assumption that two genes with similar expression patterns have similar functions or act in the same pathway is now supported by many examples demonstrating tight coupling of gene function with a specific pattern of gene expression.