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

Abstract

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.

Characterization of a <i>Tos</i>17 Insertion Mutant of Rice Auxin Signal Transcription Factor Gene, <i>OsARF</i>24

“…As previously mentioned, OsARF24 protein, which contains the SPL-rich repression domain, is considered to be an ARF that functions as a transcriptional repressor [13,14]. In our experiments, transgenic rice overexpressing the OsARF24 cDNA showed auxin-deficient phenotypes including dwarf stature, narrow leaf, and aberrant phyllotaxis.…”

“…The structures of OsARF23 and OsARF24 are similar to that of AtARF2/ HSS throughout their lengths: three domains found in repressor ARFs-a DNA-binding domain, an SPL-rich repression domain, and a C-terminal dimerization domain that is related in amino acid sequence to domains III and IV in Aux/IAA proteins-are highly conserved. Because the SPL-rich repression domain is a characteristic of repressor ARF proteins [24], both OsARF23 and OsARF24 are considered to function as transcriptional repressors [13,14].…”

“…Among them, 9 OsARF genes encode transcriptional activators and the other 16 OsARF genes encode transcriptional repressors [14]. However, the biological function of most OsARFs is poorly understood.…”

Irrepressible, truncated Auxin Response Factors

“…To date, these have been identified in four species, and it seems that the numbers of C-terminally truncated ARFs increased during evolution, as flowering plants tend to encode more of them than nonflowering plants. [14][15][16] With one exception, all of the naturally occurring truncated ARFs appear to contain repression middle domains. 16 In at least one case during early angiosperm evolution, in Amborellales and Nymphaeales, mutations leading to apparent C-terminal truncations of ARF3/ ETTIN and ARF4 have occurred, but the functional consequences are unknown.…”

An enumeration-based putative dyad predicting algorithm for promoter analysis in plants

“…This is especially the case since it has been shown that many genes are colinear in grass species (Gale, 2001). Furthermore, differences in dyad/motif content between relative species in the promoters of orthologous genes may throw light on the molecular genetic background which is involved in speciation events (Wang, 2010). This is the case for example between the two dicot species Arabidopsis and Medicago truncatula, where only 62% of orthologs predicted through phylogenetic analysis had a similar expression profile (Benedito, 2008).…”