The auxin responsive AP2/ERF transcription factor CROWN ROOTLESS5 is involved in crown root initiation in rice through the induction of OsRR1, a type-A response regulator of cytokinin signaling

Cytokinin is known to have negative effects on de novo auxin-induced root formation. However, the regulatory mechanisms of root initiation by both cytokinin and auxin are poorly understood. In this study, we characterized a rice mutant, termed crown rootless5 (crl5), which produced fewer crown roots and displayed impaired initiation of crown root primordia. The expression of CRL5, which encodes a member of the large AP2/ERF transcription factor family protein, was observed in the stem region where crown root initiation occurs. Exogenous auxin treatment induced CRL5 expression without de novo protein biosynthesis, which also required the degradation of AUX/IAA proteins. A putative auxin response element in the CRL5 promoter region specifically interacted with a rice ARF, demonstrating that CRL5 may be a direct target of an ARF, similar to CRL1/ADVENTITIOUS ROOTLESS1 (ARL1) that also regulates crown root initiation. A crl1 crl5 double mutant displayed an additive phenotype, indicating that these two genes function in different genetic pathways for crown root initiation. In addition, ProACT: CRL5/WT showed a cytokinin-resistant phenotype for crown root initiation, and also up-regulated the expression of two negative regulators of cytokinin signaling, OsRR1 and OsRR2, which were downregulated in crl5. Transgenic plants that over-expressed OsRR1 under the control of the CRL5 promoter in a crl5 mutant background produced a higher number of crown roots than the crl5 plant. Taken together, these results indicate that auxin-induced CRL5 promotes crown root initiation through repression of cytokinin signaling by positively regulating type-A RR, OsRR1.