Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray

Abstract

Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, coldtreated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using ‫ف‬ 1300 full-length Arabidopsis cDNAs to identify drought-and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought-and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought-or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought-and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis -acting DNA elements by combining the expression data with the genomic sequence data. INTRODUCTIONSequencing projects are producing large quantities of genomic and cDNA sequences for a number of organisms. In the model plant Arabidopsis, the complete genomic sequences of two chromosomes have been determined (Lin et al., 1999;Mayer et al., 1999), and the entire genomic sequence was completed by the end of 2000. Expressed sequence tag (EST) projects have also provided a major contribution with the discovery of expressed genes (Höfte et al., 1993;Newman et al., 1994;Cooke et al., 1996;Asamizu et al., 2000). A recent release of dbEST (the EST database of the National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov) contained partial cDNA sequences in which more than half of the total gene complement (i.e., ‫ف‬ 28,000 genes) is represented (as estimated from the gene content of the entirely sequenced chromosome 2 in Arabidopsis [Lin et al., 1999]). A major challenge for the coming decade is the functional analysis of this large set of genes.Recently, microarray technology has become a useful tool for the analysis of genome-scale gene expression (Schena et al., 1995;Eisen and Brown, 1999). This DNA chip-based technology arrays cDNA sequences on a glass slide at a density Ͼ 1000 genes/cm 2 . These arrayed sequences are hybridized simultaneously to a two-color fluorescently labeled cDNA probe pair prepared from RNA samples of different cell or tissue types, allowing direct and large-scale comparati...

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