In silico analysis and heterologous expression of OsNAC121 shed light on its structure and function in flowering and osmotic stress

Abstract

Plant-specific NAC transcription factors (TFs) are key master regulators in multiple vital physiological processes like development, organogenesis, stress tolerance and senescence. Identifying suitable TF is crucial for crop improvement program via biotechnological intervention. In rice (Oryza sativa L.) plant, only 38 out of 151 NAC TFs have been characterized till date. In this study, we have deciphered the in silico structure and in vivo function of OsNAC121 through heterologous expression in Escherichia coli and tobacco systems, and documented its potential role in flowering and osmotic stress. Like a typical NAC TF, OsNAC121 has a highly conserved NAC domain at the N-terminal half, featuring the subdomains A-E with the signature NAC fold comprising the twisted β-barrel between the two α-helices, and a highly variable C-terminal random coil. Analyses revealed that OsNAC121 binds to the consensus NAC binding DNA sequence (NACBS) in silico. In this study we have observed that the bacterially expressed truncated OsNAC121 protein forms tetramers in vitro, but structural modeling and DNA docking strongly support the dimeric form as the biologically relevant DNA-binding unit. Further bioinformatics analysis unravelled that R79 residue and the ⁸⁶WKAT⁸⁹ motif are pivotal for binding to the NACBS. Transgenic tobacco plants constitutively expressing OsNAC121 had elongated stem with reduced stem girth, grew faster, and flowered early, suggesting a role of OsNAC121 in determining the fate of meristematic cells. Transgenic tobacco plants also exhibited susceptibility to both drought and salinity stresses characterized by loss of chlorophyll, stunted height and smaller leaves. Therefore, we conclude that the OsNAC121 plays a crucial role in plant development, flowering time, and stress biology. Research in autologous host rice will elucidate the exact signalling pathway of OsNAC121 involving phytohormones and identify its interacting partners.