Identification and characterization of drought stress responsive genes in faba bean (Vicia faba L.) by suppression subtractive hybridization

Faba bean (Vicia faba L.) ranks fourth in food legume crop production in the world. However, drought is a potential major constraint to faba bean production and improved faba bean cultivars and development of drought-resistant varieties play a key role in enhancing faba bean crop production. In this study, suppression subtractive hybridization (SSH) technique was used to study differential expression in response to water stress and to identify genes involved in molecular mechanism of drought tolerance. A forward subtractive cDNA library induced by water deficit conditions was constructed used Hara faba bean cultivar grown in pots and treated with either well-watered (WW) or water-stressed (WS). A total of 28 clones were identified as drought stress induced. After sequencing, ten unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis which showed homology to known drought responsive genes including heat shock protein (HSP), late embryogenic abundant (LEA), zinc finger protein transcription factors (ZFP), lipid transfer protein (LTP), chlorophyll a/b-binding protein (ChlBP), thioredoxin h (Trx h), and ATP synthase as well as some functionally unknown transcripts. Their expression was characterized in Leaf, root, flower, cotyledon, and stem tissue. Quantitative RT-PCR analysis revealed that eight genes were consistently up-regulated in Hara compared to Giza3 cultivar, known as drought-tolerant and sensitive respectively under water deficit treatment. The expression of six genes was differentially expressed in different stages of water stress faba bean plant. Drought responsive genes showed changed expression patterns, indicating that they may play important roles in faba bean water stress response. Furthermore, these results indicate that drought-induced genes are related to metabolic pathways and genetic regulation of stress and development and can serve as a foundation for future studies to elucidate drought stress mechanisms of faba bean.