Arabidopsis WRKY6 And PHO1 Modulate ABA-Mediated Regulation Of Seed Germination And Early Seedling Development

Seed germination is a crucial process for plant growth and development, and it also significantly influences crop production. It is well-known that abscisic acid (ABA) is a key phytohormone involved in regulation of seed germination. The focus of this dissertation work is to investigate the molecular mechnism of WRKY6 and PHO1 participating in ABA-regulated seed germination and early seedling growth in Arabidopsis.The expression of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA, indicating that WRKY6 was involved in ABA-mediated regulation of seed germination and early seedling development. The wrky6 mutants were ABA insensitive, whereas the WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAVI was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) experiments showed that WRKY6 could bind to the RAVI promoter in vitro and in vivo. Further genetic analysis showed that overexpression of RAVI in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes. Similar to ravl mutant, the ravl wrky6-2 double mutant showed an ABA-hypersensitive phenotype. These results demonstrate that the Arabidopsis WRKY6 transcription factor plays important roles in ABA-mediated regulation of seed germination by direct down-regulation of RAVI expression.It was also found that the expression of PHO1 was enhanced during seed germination and significantly repressed by exogenous ABA. The phol mutants showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The transcription of PHO1 was repressed in the ABI5-overexpressing line and elevated in the abi5 mutant.The EMSA and ChIP assays showed that ABI5 can bind to PHO1 promoter in vitro and in vivo, indicating that ABI5 negatively regulated PHO1 expression. Further genetic analysis showed that disruption of PHO1 in abi5 mutant abolished the ABA-insensitive phenotypes of abi5 mutant, demonstrating that PHO1 is epistatic to ABI5. The phol mutants accumulated more reactive oxygen species (ROS), and the ABA-mediated inhibition of seed germination in phol mutants was released by addition of reduced GSH, suggesting that PHO1 was involved in ABA-regulated seed germination at least partially via ROS-dependent pathway. These results demonstrate that the Arabidopsis PHO1 is the main target of ABI5 for ABA-mediated regulation of seed germination and early seedling development.In summary, the experimental results presented in this dissertation demonstrate that there exsits a WRKY6-RAV1-ABI5-PHO1 regulatory pathway in ABA-mediated regulation of seed germination and early seedling development in Arabidopsis. These novel findings increased our understanding in ABA-regulation of seed germination and early seedling development for higher plants.