Functional analysis of the structural domain of ARF proteins in rice (Oryza sativa L.)

Auxin response factors (ARFs) are key regulators of plant growth and development. Through interaction with auxin/indole acetic acid (Aux/IAA) proteins, they influence the expression of auxin response genes. An ARF gene family has been predicted in rice, but the functions of the individual structural domains of the OsARFs remain obscure. Bioinformatics was used to analyse the position of the DNA-binding domain (DBD), middle region (MR), and C-terminal dimerization domain (CTD) of OsARFs, and experimentally confirmed the presence of a classical monopartite nuclear localization signal (NLS) in the DBD. The DBD was shown to contribute to nuclear localization of OsARF proteins in addition to its known DNA-binding function. Interactions between 14 integrated OsARFs and 15 OsIAA proteins were tested using yeast two-hybrid assays. It was found that eight OsARF activators interacted with the 15 OsIAA proteins, while six OsARF repressors did not. The interactions between the MR+CTD or CTD of 10 OsARFs and 15 OsIAA proteins were also tested and the results were consistent with those of each intact OsARF, although some slight differences in interaction intensity were observed by α-galactosidase quantitative assays. The truncated CTD of OsARF11 did not interact with any OsIAA, implying that the CTD is required for ARF-IAA dimerization, and that the MR influences the interaction intensity in yeast. A subset of the interactions in yeast were also observed in tobacco plants using firefly luciferase complementation imaging assays, indicating that these interactions are specific in plants, and might have a special role in the auxin signalling response. This study provides new insight into the structure of OsARF proteins and ARF-Aux/IAA interactions.