T-DNA-activation-tagging mutagenesis to identify determinants of abiotic stress response in Thellungiella halophila and Arabidopsis thaliana.

摘要

Environmental stresses such as salinity and extreme temperatures negatively affect germination, growth and productivity of crops worldwide. To identify genes involved in abiotic stress tolerance, we carried out a large scale screening of T-DNA activation tagged mutants with stress-tolerant extremophile Thellungiella halophila (SHANGDONG, a halophyte) and model plant Arabidopsis thaliana (Col-0, a glycophyte) carrying a stress responsive TCH4 promoter fused luciferase (TCH4::LUC) reporter gene. Biochemical, genetic and molecular characterization of several mutants revealed several known and novel genes involved in plant development and stress signaling, e.g. HGC1 and CET1. T. halophila hgc1-1 (high germination capacity line 1) mutant showed decreased sensitivity to NaCl, mannitol and ABA during germination. HGC1 encodes a putative pyruvate kinase. The hgc1-1 mutant accumulates higher levels of HGC transcripts compared to wild type plants. A knock-out mutant of A. thaliana AtHGC1 (homolog of ThHGC1) exhibited hypersensitivity to NaCl and ABA during germination. Arabidopsis transgenic plants overexpressing AtHGC1 phenocopied the T. halophila hgc1-1 mutant in seed germination on media containing NaCl and ABA. Conservation of these gene sequences that maintain similar function in both the halophyte and glycophyte may be indicative that HGC1 is critical to the fitness of T. halophila to its native saline environment. TCH4::LUC reporter genetic screening led to the identification of cet1-1 (constitutively expression of TCH4::LUC) mutant, which showed constitutive expression of LUC. Northern blot revealed that endogenous TCH4 gene expression was higher in cet1-1 as compared with wild type plants. cet1-1 mutation resulted in morphological changes and enhanced thermotolerance. One of the reasons for increased heat tolerance of cet1-1 appears to be higher expression levels of HSP101 which encodes a necessary heat shock protein for thermotolerance. CET1 is an unknown gene and encodes a protein with unknown properties, and expresses in a wide range of tissues and developmental stages.