Identification of differentially expressed genes in wheat undergoing gradual water deficit stress using a subtractive hybridisation approach

Drought stress leads to changes in expression of a large number of genes in plants. In order to identify and characterise genes involved in wheat adaptation to water deficit, differentially expressed genes in wheat plants subjected to a progressive drought stress were isolated through suppressive subtractive hybridisation. Approximately 2500 clones were isolated from each of the forward (drought versus well-watered) and reverse (well-watered versus drought) subtracted libraries. A total of 600 expressed sequence tags (ESTs) (500 from the forward and 100 from the reverse library) were obtained. None of the ESTs were common to both libraries and several previously uncharacterised ESTs were identified. The success of the subtractive hybridisation was confirmed using microarray and Northern analysis. A set of 300 unique ESTs was subjected to microarray analysis to study differential gene expression between leaves of well-watered wheat plants and plants subjected to water deficit stress. Overall, 30% of the genes were significantly up-regulated and 18% were significantly down-regulated under water deficit stress. Northern analysis was conducted using a subset of genes from the microarray experiment showing differential expression under drought and confirmed their differential expression with progressive reduction in leaf relative water content. We demonstrate that subtractive hybridisation is an effective means of enriching for genes up- and down-regulated in wheat during a slowly developed drought stress akin to that experienced in field situations.