MAP-BASED CLONING AND MOLECULAR CHARACTERIZATION OF ARABIDOPSIS shiny MUTANTS shi2, shi6 and shi3/sta1

In effort to identify novel genetic components that play important roles in abiotic stress response and tolerance, a forward genetic mutant screen was established by using a highly sensitive imaging system monitoring luciferase reporter gene expression driven by the stress-inducible promoter from the Arabidopsis SOT12 gene encoding a sulfotransferase. A number of mutants showing increased luciferase expression in response to abiotic stresses were isolated and named as shiny (shi) mutants. Among all shiny mutants, shi2, shi6 and shi3/sta1 were selected for map-based cloning and further detailed characterization in this study. All three mutants harbor single recessive mutations resulting in elevated expression of the luciferase reporter gene with or without the NaCl treatment when compared to wild type. Map-based cloning of shi2 identified a mutation in a gene encoding a DEAD (Asp-Glu-Ala-Asp) box RNA helicase. SHI2 contains the conserved DEAD box and other motifs characteristic of RNA helicases and belongs to the largest family of helicase proteins known to be involved in gene expression from transcription to translation. Our results revealed that shi2 mutant is more sensitive to ABA in seed germination, exhibits inhibited growth at low temperature, and is hypersensitive to LiCl when compared to the wild type. GFP fusion experiments establish that SHI2 is nuclear localized and plays a role in pre-mRNA splicing in Arabidopsis. Map-based cloning of shi6 revealed a mutation in a gene known to be involved in embryo development. SHI6 is a large gene encoding a protein containing 2171 amino acids with two tandem repeats of DEAD box-like RNA helicase motifs. shi6 mutant displays a dwarf phenotype at normal growth condition, is more sensitive to ABA in seed germination, and shows inhibited growth at low temperature. Genetic complementation confirmed that the mutation in shi6 is indeed responsible for the mutant phenotypes. Four alleles of shi3/sta1 were isolated from the shiny mutants. SHI3/STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein which functions in pre-mRNA splicing. SHI3/STA1 is previously reported to play an important role in pre-mRNA splicing in Arabidopsis, which is further supported by our experimental results. shi3/sta1 displays a dwarf phenotype under normal growth condition, serrated leaves with pointed leaf tips, hypersensitive to ABA in seed germination, growth inhibition at low temperature, and is also sensitive to LiCl treatment. All these three SHINY genes, SHI2, SHI6, and SHI3/STA1, are essential genes for plant growth and development in Arabidopsis. Null T-DNA knockout mutants of SHI6 and SHI3/STA1 are embryo lethal, and null T-DNA knockout mutant of SHI2 seems to have deleterious effects on pollen development. Through detailed molecular and biochemical characterizations, the roles of these SHINY genes in gene transcription and co-transcriptional processes were studied. The possible function of these genes in abiotic stress response and tolerance is discussed.