Control of OsARF3a by OsKANADI1 contributes to lemma development in rice

In rice (Oryza sativa), the lemma and palea protect the internal organs of the floret，provide nutrients for seed development, and determine grain size. We previously revealed that a trans-acting small interfering RNA targeting AUXIN RESPONSE FACTORS (tasiR-ARF) regulates lemma polarity establishment via post-transcriptional repression of AUXIN RESPONSE FACTORS (ARFs) in rice. TasiR-ARF formation requires RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). However, the underlying molecular mechanism of the tasiR-ARF-ARF regulon in lemma development remains unclear. Here, by genetic screening for suppressors of the thermosensitive mutant osrdr6-1, we identified three suppressors, huifu 1 (hf1), hf9, and hf17. Mapping-by-sequencing revealed that HF1 encodes a MYB transcription factor belonging to the KANADI1 family. The hf1 mutation partially rescued the osrdr6-1 lemma defect but not the defect in tasiR-ARF levels. DNA affinity purification sequencing analysis identified 17 725 OsKANADI1-associated sites, most of which contain the SPBP-box binding motif (RGAATAWW) and are located in the promoter, protein-coding, intron, and intergenic regions. Moreover, we found that OsKANADI1 could directly bind to the intron of OsARF3a in vitro and in vivo and promote OsARF3a expression at the transcriptional level. In addition, hf9 and hf17 are intragenic suppressors containing mutations in OsRDR6 that partially rescue tasiR-ARF levels by restoring OsRDR6 protein levels. Collectively, our results demonstrate that OsKANADI1 and tasiR-ARFs synergistically maintain the proper expression of OsARF3a and thus contribute to rice lemma development.