Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: An approach based on gene down regulation

Functional characterization of water deficit stress responsive genes is important to understand their role in stress tolerance. RNAi-based silencing of gene of interest and studying the stress response of knockdown plants under stress can be one of the potential options for assessing functional significance of these genes. Several genes showing higher transcript expression under water deficit stress were cloned earlier from a stress adapted crop species, groundnut. In this study, a few selected gene homologs have been characterized in Nicotiana tabacum and Arabidopsis. Using post transcriptional gene silencing (PTGS) based RNAi approach we developed N. tabacum knockdown lines for three of the genes namely alcohol dehydrogenase (ADH), trans caffeoyl coA-3-O-methyl transferase (CcoAOMT) and flavonol-3-O-glucosyl transferase (F3OGT). By quantitative RT-PCR we demonstrated that the RNAi lines showed significant reduction in target gene transcripts. We followed a stress imposition protocol that allows the plants to experience initial gradual acclimation stress and subsequently severe stress for a definite period. The RNAi knockdown lines generated against ADH and F3OGT, when subjected to water deficit stress showed susceptible symptoms signifying the relevance of these genes under stress. Knockdown of CcoAOMT showed higher chlorophyll degradation and less cell viability upon stress compared to control plants. Further, the Arabidopsis mutant lines clearly showed susceptibility to salinity and water deficit stresses validating relevance of these three genes under abiotic stresses.