Drought and salt tolerance characteristics of rice plants transformed with the SNAC1 transcription factor

Abstract

Abiotic stresses are one of the major problems for restricting food production around the world. Plant survival and crop production has been hampered due to environmental stresses such as salinity and drought. Some plants may however show tolerance to abiotic stress through activating different genes including those for transcription factors. In the current study, stress tolerance was shown to be increased in rice which was transformed with the transcription factor SNAC1 (stress responsive NAC1). My purpose was to increase the stress tolerance in existing commercial and farmer popular high yielding rice varieties, which directly influences the lives and livelihood of farmers. The SNAC1 transcription factor was isolated and cloned from the salt tolerant rice landrace, Pokkali, under the constitutive promoter, CaMV35S. It was at first overexpressed and characterized in the tissue culture responsive indica rice cultivar Binnatoa, at the two developmental stages at seedling and maturity for both the stresses salinity and drought. Transgenic lines were confirmed by PCR and Southern blot hybridization indicating stable transgene integration into the genome. Assays for leaf senescence and chlorophyll content at 100mM and 200mM salt and survival rates at 200 mM salt and drought condition showed that transgenic lines were significantly increased in their stress tolerance compared to wildtype at seedling stage. At reproductive stages, the transgenic lines showed significantly enhanced spikelet fertility, yield and 1000 grain weight compared to wildtype under both salinity and drought stresses. Thus, the transgenic rice overexpressing SNAC1 showed significantly improved tolerance to salinity and drought stresses at the vegetative as well as reproductive stages. After characterization in indica rice, the SNAC1 gene was transformed in three high yielding rice varieties (BRRI Dhan-55, BRRI Dhan-56 and BRRI Dhan-49) for stress inducible expression under rd29A promoter. All high yielding varieties were transformed through tissue culture independent Agrobacterium-mediated in planta transformation method which bypasses the problems associated with tissue culture-based indica rice transformation methods. Transgenic lines showed correct sized bands in PCR and integration of the SNAC1 gene into the genome were confirmed by Southern blot hybridization. Significantly higher transgene expression (SNAC1) was found by real time (qRT) PCR. Transgenic lines showed 3:1 segregation ratio at T2 generation following the Mendelian law of inheritance. Transgenic lines also showed significantly higher level of stress tolerance in salinity and drought condition compared to their respective wildtype plants. A comparative assay was done in BRRI Dhan-45 (salt sensitive) and BRRI Dhan-56 (moderately drought tolerant) transgenic lines showing that the level of tolerance differ in different genotypes. Transgenic lines of BRRI Dhan-56 showed more stress tolerance than BRRI Dhan-49 in seedling and reproductive stages and both varieties showed significantly higher stress tolerance compared to their respective wildtype plants. To understand the promoter effects on SNAC1 gene expression comparative assay was done in BRRI Dhan-55 transgenic lines between SNAC1 expressed under stress inducible promoter and constitutively SNAC1 overexpression lines. It was found that at both seedling and reproductive stages under both salinity and drought stresses, induced expression of SNAC1 provided significantly more tolerance in plants. In this study, six up-regulated genes were selected from reported microarray data and tested for expression in BRRI Dhan-55 SNAC1 transgenic lines under inducible promoter. It was found that the expression of all 6 genes was induced under salt stress condition. The increased expression of these SNAC1 downstream genes likely helped in providing a higher level of stress tolerance in abiotic stresses. The underlying pathway and possible interactions of the selected genes were also predicted by literature and network analysis. This work provided a promising approach for the genetic improvement in the salinity and drought tolerance of commercial, high yielding indica rice cultivars through transformation with the SNAC1 transcription factor under stress inducible promoter.