Auxin signaling plays a key role in the regulation of various growth and developmental processes in higher plants. Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin-response genes. The osarf 24-1 mutant contains a truncation of domain IV in the C-terminal dimerization domain of a rice ARF protein, OsARF24. This mutant showed auxin-deficient phenotypes and reduced sensitivity to auxin. However, OsARF24 protein contains an SPL-rich repression domain in its middle region and acts as a transcriptional repressor. These results imply that the C-terminal dimerization domain, especially the C-terminal half of domain IV, is essential for the proper regulation of OsARF24 function as a transcriptional repressor in rice.
Auxins are endogenous phytohormones that play important roles in regulating a wide variety of cellular and developmental processes. Analyses of auxin-insensitive mutants have provided solid evidence to support the models of auxin function proposed by conventional physiological experiments and have also provided new insights and ideas about auxin. Recent molecular genetic studies, mainly on Arabidopsis (Arabidopsis thaliana L.), have made significant progress in elucidating the auxin signaling pathway. The binding of a bioactive auxin such as indole-3-acetic acid (IAA) to members of the TIR1/ AFB family of F-box proteins triggers the degradation of Aux/IAA transcriptional repressors, thereby allowing auxin response factor (ARF) transcription factors, which show either activator or repressor activity, to regulate the expression of auxin-response genes [1-5].
A typical ARF protein contains a conserved N-terminal DNA-binding domain, a non-conserved middle region, and a conserved C-terminal dimerization domain [3,6,7]. The DNA-binding domain of ARF protein binds with specificity to TGTCTC auxin response elements (AuxREs) in promoters of auxin-response genes to regulate their expression [
The ARF proteins are encoded by a multigene family in plants, and in rice (Oryza sativa L.), 25 OsARF genes have been identified [
Seeds of wild-type rice (Oryza sativa L. “Nipponbare”)the osarf24-1 mutant, and transformants (described below) were sterilized in 1% NaClO for 30 min and sown on Murashige and Skoog agar medium. Seedlings were grown in a growth chamber at 28˚C under continuous light for 2 weeks. For morphological characterization, seedlings were transplanted and grown in the paddy field at the Experimental Farm of Ishikawa Prefectural University. For gene expression analyses, seedlings were selected for uniformity of growth and adapted to hydroponic culture for 2 days before treatment. IAA treatment (20 mM) was carried out by adding IAA to the culture medium.
Total RNA was extracted from whole seedlings of wildtype and mutant rice and from mature leaves of transgenic rice by using an RNeasy Plant Mini Kit (Qiagen, Venlo, Netherlands). Single-strand cDNAs were synthesized by using the Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA, USA). Quantitative RT-PCR was performed with an iCycler iQ real-time PCR system (BioRad Laboratories, Hercules, CA, USA). The primer sequences were 5’-CAGGAAGCTGGTGT GTTGTC-3’ and 5’-CTTGATCAGGCGTGGCTGTG-3’ for OsARF23, 5’-AATGACGCCTGACATCACAC-3’ and 5’-GCTTG ATAAGACTCGATGAGG-3’ for OsARF24, 5’-ACCA AGAGCCGCTCAATGAG-3’ and 5’-ATCACACGTG GGCGAACATC-3’ for OsIAA1, 5’-GATGAACAGGC GGTCGCTGC-3’ and 5’-GGCTC CGGTAGTAGCTTG TG-3’ for OsGH3-1, and 5’-CGCC AGTTTGGTCGCT CTCGATTTCG-3’ and 5’-TCAGGA GCTCCGTGCTC TTCTGGTAC-3’ for Histone H3. These primers specifically amplified the target gene sequences. Expression levels were normalized against the values obtained for Histone H3, which was used as an internal reference gene. For the gene expression experiments, we performed 3 biological repeats.
Full-length OsARF24 cDNA was inserted in the sense orientation into the pET-32a expression vector (Novagen, Madison, WI, USA) to generate a thioredoxin fusion protein when expressed in BL21(DE3) E. coli cells (Stratagene, La Jolla, CA, USA). The recombinant protein was purified by using Talon Metal Affinity Resin (Clontech Co., Palo Alto, CA). OsIAA1 promoter fragments containing WT or MT AuxRE were amplified by PCR with rice genomic DNA. The primer sequences were 5’-GGTTGAAATTGGAACGATGTG-3’ and 5’-G GAACTTTCATCTACTACTAC-3’ for OsIAA1 AuxRE (WT), and 5’-TTTGGATTCTCCATTATGAGAAAATC AAAACATGGTTTTTT-3’ and 5’-TTAATAAAAAAC CATGTTTTGATTTTCTCATAATGGAGAATCC-3’ for generating the AuxRE mutation (MT). The amplified fragments were cloned into pBluescript II SK (Stratagene) and their identities were confirmed by sequence analysis. The PCR-amplified fragments were excised with restriction endonucleases, purified by 10% PAGE, and labeled with biotin using a Biotin 3’ End DNA Labeling Kit (Pierce, Rockford, IL, USA). The electrophoresis mobility shift assay was performed by using a LightShift Chemiluminescent EMSA Kit (Pierce).
The entire OsARF24 coding region was inserted between the rice Actin promoter and the nopaline synthase polyadenylation signal of the hygromycin-resistant binary vector pAct-Hm2. This vector was modified from pBIH1 [
osarf24-1 is a mutant of OsARF24 caused by insertion of the Tos17 retrotransposon. osarf24-1 showed a reduction in plant height (the height of osarf24-1 was 91% that of the wild-type, n = 10, P < 0.001;
Sequence analysis revealed that osarf24-1 had an insertion of Tos17 in exon 13 (
aEach column represents mean ± s.d. of 10 independent plants.
Tos17 was inserted into the part of the gene encoding the C-terminal dimerization domain of the OsARF24 protein at the 773th leucine residue (
The treatment of rice seedlings with IAA induced an increase in the expression of auxin-response genes OsIAA1 and OsGH3-1 [20,21]. After 10 min, IAA treatment of wild-type seedlings increased the expression of both OsIAA1 and OsGH3-1 to 1.5 times the levels in untreated seedlings (
OsARF24 protein represses auxin-response genes constitutively.
Among the 25 ARF genes in rice, the deduced amino acid sequence of OsARF24 is most closely related (71.4% identity) to OsARF23 (previously designated as OsARF1 [
Quantitative reverse-transcription PCR analysis revealed that OsARF24 and OsARF23 were expressed at different levels in all the organs of wild-type rice that we tested, including the vegetative shoot apices, leaf sheaths, leaf blades, elongating internodes, roots, inflorescences (immature panicles), and panicles at flowering time (
Previous observations indicate that the expression levels of some ARF genes were not affected by IAA treatment, and those of the others were either increased or decreased [10,13]. Thus, we examined the effect of exogenously applied auxin on the expression of OsARF23 and OsARF24. IAA treatment increased the expression of both OsARF23 and OsARF24 to 1.5 times that in untreated plants within 10 min (
expression level of OsARF23 gradually increased until 60 min after IAA treatment to 2.5 times that in untreated plants, whereas the expression level of OsARF24 reached a maximum within 10 min and was maintained at that level until 60 min after IAA treatment. These results suggest that the expression of OsARF23 and OsARF24 is regulated by different mechanisms.
The AuxRE has been identified in the promoters of some early auxin-response genes, and ARFs bind the AuxRE to regulate the transcription of these genes [
To assess the activity of the OsARF24 gene product in vivo, we fused the full-length OsARF24 cDNA to the rice Actin promoter in the sense orientation (Act::OsARF24) and introduced the construct into wild-type rice by Agrobacterium-mediated gene transfer (
We also examined the expression of auxin-response genes in the Act::OsARF24 transformants. The steadystate levels of OsIAA1 and OsGH3-1 mRNA in the leaves of Act::OsARF24 transformants decreased to about 50% and 20%, respectively, of those in the leaves of wild-type plants (
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. In addition, the expression levels of auxin-response genes OsIAA1 and OsGH3-1 were decreased in these transformants. These results strongly support the hypothesis that OsARF24 acts as a repressor ARF. However, the Tos17 retrotransposon insertion mutant of OsARF24, osarf24-1, also showed both auxin-deficient
phenotypes and decreased levels and auxin responses of OsIAA1 and OsGH3-1 expression. Although another rice ARF, OsARF23, shows high amino acid sequence similarity with OsARF24, the expression of OsARF23 and OsARF24 is regulated by different mechanisms in various organs of wild-type rice plants and in response to IAA treatment. Based on these results, we consider that OsARF23 and OsARF24 do not function redundantly in rice.
Most ARF proteins contain a C-terminal dimerization domain related to domains III and IV in Aux/IAA proteins [4,7,10]. The C-terminal dimerization domains in both ARF and Aux/IAA proteins are protein-protein interaction domains that allow homoand heterodimerization of ARF proteins and hetero-dimerization among ARF and Aux/IAA proteins [4,9,10]. Although ARF repressors can dimerize via their C-terminal dimerization domains, ARF repressor-Aux/IAA and ARF represssorARF activator interactions are much weaker than ARF activator-Aux/IAA and ARF activator-ARF activator interacttions, and it remains unclear whether Aux/IAAs interact with ARF repressors, or whether ARF repressors interact with ARF activators, to regulate target gene expression in plants [3,10,12,14].
In the osarf24-1 mutant, a Tos17 insertion in OsARF24 altered the amino acid sequence in the C-terminal dimerization domain of the OsARF24 protein, and the C-terminal half of domain IV was truncated. This mutation may reduce the formation of repressor ARF dimers; however, this does not explain why the osarf24-1 mutant showed reduced sensitivity to auxin because both monomers and dimers of ARF repressors can target and repress the expression of auxin-response genes [
The osarf24-1 mutant contains a truncation of domain IV in the C-terminal dimerization domain of OsARF24 protein. This mutant showed auxin-deficient phenotypes and reduced sensitivity to auxin. However, wild-type OsARF24 protein contains an SPL-rich repression domain and acts as a repressor ARF. These results imply that the C-terminal dimerization domain, especially the C-terminal half of domain IV, is essential for the proper regulation of OsARF24 function as a transcriptional repressor in rice.
We thank Asako Tokida-Segawa for technical assistance, and the GenBank project of the National Institute of Agrobiological Science in Japan for providing osarf24-1 (NE4013) mutant seeds and OsARF24 cDNA (AK- 067061). T. S. was supported by Grants-in-Aid for Young Scientists (Nos. 19688001 and 24780005) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.