Brassinosteroid-Regulated Carotenoid Biosynthesis and Stomatal Development in Arabidopsis thaliana

Abstract

Plants are constantly exposed to various abiotic and biotic stress. Adaptation of plants to environmental changes is critical for their survival. Brassinosteroids (BRs), plant-specific steroid hormones, play pivotal roles in regulating plant growth and development. However, the regulation of metabolic pathways by BR is not well established. In addition, BR regulates stomatal development, but the opposite regulation in cotyledons and hypocotyls is highly controversial, so further research must be needed. In this dissertation, I investigated the regulatory mechanisms of carotenoid biosynthesis and stomatal development through BR signalling. In Part II, I showed that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated a higher level of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid contents. Interestingly, I demonstrated that BR transcriptionally suppressed 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. Surprisingly, I found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. While expression patterns of other enzymes involved downstream of the HPPD-mediated metabolic pathways were enhanced by light irradiation. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. I found BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the proximal region of the HPPD promoter. Moreover, HPPD expression was significantly reduced by the bzr1-1D gain-of-function mutation in the dark but not in the light, and the HPPD suppression by BR was increased. However, dark-induced HPPD expression did not cause carotenoid accumulation due to the down-regulation of other carotenoid biosynthetic genes in the dark. Together, my result suggests that BR regulates different physiological responses in dark and light through inhibition of HPPD expression. In Part III, I showed that BR and bikinin differently regulate stomatal development in Arabidopsis hypocotyls. Treatment of BR greatly induced stomatal development in hypocotyls. Meanwhile, in BR-deficient or BR-insensitive mutants, the stomatal phenotype in hypocotyls was more strongly suppressed than in wild-type plants. Interestingly, the inhibitor of BIN2, bikinin, which has a similar effect to BL on Arabidopsis, strongly suppressed stomatal development in cotyledons and hypocotyls. Moreover, the bin2-1 gain-of-function mutant exhibited increased stomatal development compared to wild-type in hypocotyls. These results indicated that bikinin has opposite effect to BL in stomatal development of hypocotyls, and BIN2 positively regulates stomatal development in hypocotyls. Furthermore, stomatal development in hypocotyls of the bzr1-1D gain-of-function is strongly reduced compared to wild-type. However, bzr1-1D mutation did not suppress the induced stomatal phenotype of bin2-1. Meanwhile, bzr1 hextuple (bzr-hw) exhibited induced stomatal development compared to wild-type. Furthermore, bzr-hw was insensitive to the application of BL. This indicates that BR induced stomatal development in hypocotyls may be the effect of transcriptional regulation by BZR1. From transcriptome analysis, I found that stomatal development-related genes were up-regulated by BL and down-regulated by bikinin, respectively. In some genes that related to stomatal development, expression was remarkably down-regulated by bikinin rather than BL. These results indicated that the transcriptional regulation is mainly mediated by BIN2 activity rather than BZR1. Taken together, my study suggests that BR signalling has a dual regulatory role in stomatal development.