Functional Analyses Of Ubiquitin-proteasome Related Regulators CSN5B And OsEIP1

The homeostasis of protein biosynthesis and degradation in vivo is essential for cell cycle undernormal conditions, and ubiquitin-proteasome system (UPS) is an important pathway of proteindegradation, which plays a crucial role in plant growth and development. Except for ubiquitin, E1(Ub-activating enzyme), E2(Ub-conjugating enzyme), E3(Ub ligase) and26S proteasome, UPS alsoincludes many positive or negative regulators on protein degradation. For example, Arabidopsis COP9signalosome (CSN), as the key complex of light regulators, functions on cell cycle andphotomorphogenesis, through not only modulating E3activities but also binding to proteasome directly.In the present report, we further find that CSN has an important role in the regulation of ascorbic acid(AsA, Vitamin C) synthesis in Arabidopsis. It has been well known that light regulates AsA synthesis,which increases in the light, presumably reflecting a need for antioxidants to detoxify reactivemolecules produced during photosynthesis, and decreases in the dark to keep redox equilibrium in vivo.In order to address how light is involved in AsA synthesis, we firstly examined that the protein levels ofthe AsA biosynthetic enzyme GDP-mannose pyrophosphorylase (VTC1) in Arabidopsis are associatedwith the changes of AsA contents in light and darkness, implying that VTC1stability onpost-transcriptional level is controlled by the switch of light on and off. Interestingly, we identified thatArabidopsis CSN subunit CSN5B interacts with the N-terminus of VTC1through in vivo and in vitroassays. Although the expression level of CSN5A is much more than this of CSN5B, it is CSN5B but notCSN5A that interacts with VTC1in Arabisopsis based on CoIP assay, strongly indicating that thecomponents of CSN complex, CSN5A and CSN5B, play unequal roles in plant development. Furtheranalyses with gel filtration profiling showed that the interaction of VTC1-CSN5B is also associatedwith the CSN complex, and VTC1locates in both the nucleus and the cytoplasm, which provides thepossibility of interaction between VTC1and CSN5B. Biochemical data showed that CSN5B promotesubiquitination-dependent VTC1degradation in darkness through the26S proteasome pathway.Consistent with the observations, csn5b mutants showed very high AsA levels, a double mutant ofcsn5b with the partial loss-of-function mutant vtc1-1contained AsA levels between those of vtc1-1andcsn5b, AsA content in csn5b is nearly changeless under continuous light or darkness. These resultsshowed that CSN5B mediates light modulation on AsA synthesis by affecting VTC1stability. Inaddition, csn5b mutants displayed better growth than wild-type with more fresh weight and longer rootsunder salt stress; and the ROS and MDA levels in csn5b mutants are less than wild-type under oxidanttreatment, which indicates that csn5b mutant has higher tolerance to salt and oxidative stresses. Thusour data reveal that CSN also participates in proteasome proteolysis pathway through binding targetproteins, subsequently affecting plant stress responses in Arabidopsis.There is limited research on UbL/UBA proteins in plant, which is also a kind of regulators in UPS.In the investigation, we find that OsEIP1is a typical UbL/UBA protein in rice, with ubiquitin-like(UBQ) domain in N-terminus and ubiquitin-associated (UBA) domain in C-terminus. The homologous protein RAD23in Arabidopsis provides an essential connection between the26S proteasome andubiquited proteins. In this study, the binding of OsEIP1with poly Ub chain indicates the essentialfunction of UBA protein OsEIP1in ubiquitination. Moreover, OsEIP1interacts with EUI, a negativeregulator of gibberellin metabolism in rice, this interaction is independent on ubiquitination and thetargeted proteins of OsEIP1might be specially recognized. OsEIP1overexpressional transgenic lines inrice were more tolerant to drought stress than wild-type, while artificial microRNA interferencetransgenic lines were more sensitive to drought than wild-type. Meanwhile, eui mutants were moresensitive to drought and EUI-OX transgenic seedlings enhanced tolerance to drought. These datapreliminarily indicate that OsEIP1modulates ubiquitination-proteasome pathway and drought stressresponses in rice.