Functional Analysis Of A C3H2C3-type RING Protein NtRCP1in Nicotiana Tobacum

Ubiquitin (Ub)/26S pathway-mediated protein degradation is an essential proteolytic system in eukaryotes. The ubiquitin protein is conjugated to the Lys residues of specific substrate via the sequential action of three enzymes:ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) and subsequently recognized and degradated by26S proteasome. E3ligase is the most important protein of them, which recruits the proper substrate and determines the specificity of ubiquitination. Really interesting new gene (RING) proteins belong to a subgroup of the E3ligases, which comprises a diverse of proteins and play important role in various cellular processes, including abiotic or biotic stress responses, circadian rhythms, photomorphogenesis, cell cycles, and differentiation. Whereas a number of RING proteins have been identified and functional characterized in Arabidopsis, only one C3HC4-type RING gene has been cloned in tobacco. In this study, we have characterized the function of an unknown C3H2C3-type RING protein NtRCPl (Nicotiana tobacum RING-domain-containing protein1) from tobacco BY-2cells by molecular biology, biochemistry, cellular biology and reversed genetics approaches. The provided information is of important for understanding the biological functions of the RING family proteins.NtRCPl was identified in a cytokinesis-related cDNAs from tobacco BY-2cells using fission yeast as a functional system. Enzymatic analysis demonstrated that NtRCPl is a functional E3ubiquitin ligase, which catalyzed the formation of a high molecular-weight ubiquitin ladder in the presence of E1and E2. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control, while the RNAi plants showed no significant difference to the wild-type plant although a2-to4-d delay in flowering time could be observed. Histological analysis revealed that the shoot apical meristem (SAM) of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division, indicating that NtRCPl may stimulates cell division at the SAM site. In tobacco plant, the expression of NtRCP1appeared ubiquitously in all the tissues tested. In the shoot apices, the expression level of NtRCPl was higher in the reproductive shoot apices than in the vegetative ones. Overexpression of NtRCPl in BY-2suspension cells promoted cell division, which was a consequence of the shortened G2phase in the cell cycle. Together, our data suggest that NtRCPl may act as a regulator of the phase transition, possibly through its role in cell cycle regulation during vegetative/reproductive development in tobacco plant.Accumulating evidence indicates that RING finger proteins participate both in growth development and in response to biotic and/or abiotic stresses. As we observed that the expression of NtRCP1was induced by abitic stresses, we further analyzed the function of NtRCP1in response to environmental cues. Our data showed that overexpression of NtRCPl conferred enhanced oxidative stress tolerance in transgenic tobacco plants by increasing the activity of Ascorbate peroxidase, indicating that NtRCP1may also function as a regulator of the stress-responsive protein(s) involved in reactive oxygen species (ROS) homeostasis. Collectively, our results show that NtRCP1may act as a dual-function RING protein in tobacco, which plays roles in both the vegetative/reproductive phase transition and in response to abiotic stresses. Our data provide not only information for understanding the roles of RING family proteins, but also a candidate gene in molecular breeding for stress tolerance and growth phase regulation in tobacco.