Functional Characterization of Novel E3 Ubiquitin Ligases RING DOMAIN OF UNKNOWN FUNCTION 1117 1 (RDUF1) and RDUF2 in Light-Mediated Regulation of Seedling Photomorphogenesis in Arabidopsis

Modak, Annayasa (2025) Functional Characterization of Novel E3 Ubiquitin Ligases RING DOMAIN OF UNKNOWN FUNCTION 1117 1 (RDUF1) and RDUF2 in Light-Mediated Regulation of Seedling Photomorphogenesis in Arabidopsis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Annayasa Modak (19RS082)) 19RS082.pdf - Submitted Version Restricted to Repository staff only Download (14MB)

Abstract

Light has a significant impact on plant growth and development. Plants have evolved exquisite sensory arrangements for monitoring the qualities and intensities of Light. The CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)-ELONGATED HYPOCOTYL5 (HY5) module controls plant seedling photomorphogenesis. Maintaining HY5 protein levels is most important for photomorphogenic growth. This study reported the novel role of two E3 ubiquitin ligases, RING DOMAIN OF UNKNOWN FUNCTION 1117 1 (RDUF1) and RDUF2, as components of the COP1-HY5 circuit in Arabidopsis. The loss-of-function mutants of RDUF1 and RDUF2 showed longer hypocotyls with reduced photopigment accumulation compared to wild-type Columbia. Contrastingly, the overexpressing transgenic lines exhibited shorter hypocotyls with more photopigment accumulation in a wavelength-independent manner. HY5 directly binds to the RDUF1 and RDUF2 promoters through the G-box, activating their expression in response to light. Epistatic analysis and biochemical data showed that RDUF1 and RDUF2 genetically interact with and stabilize the HY5 protein. Conversely, COP1 ubiquitinated RDUF1 and RDUF2 in the dark, subjecting them to degradation to keep HY5 levels low and promote skotomorphogenic growth. However, in response to light, photoreceptor-mediated inhibition of COP1 activity increased the accumulation of RDUF1 and RDUF2, enhancing HY5 protein stability and photomorphogenesis. Conversely, in agreement with previous reports, our yeast-one-hybrid assay also reveals that PIF3 and PIF4 directly bind to RDUF1/RDUF2 promoters through the G-box, negatively regulating their expression in dark. Besides, our thorough genetic analysis suggests that RDUF1/RDUF2 functions upstream of both PIF3 and PIF4 and probably downregulates PIF3 and PIF4 protein to promote photomorphogenesis. Together, this study highlights the novel role of RDUF1 and RDUF2 as positive regulators of photomorphogenesis via regulating two alternative pathways, on the one hand by stabilizing HY5 and on the other by negatively regulating PIF3/PIF4 in promoting seedling photomorphogenic growth in Arabidopsis.

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