WOX11 and CRL1 act synergistically to promote crown root development by maintaining cytokinin homeostasis in rice.

Crown root morphogenesis is critical for normal growth and nutrition absorption in cereals. In rice, WUSCHEL-RELATED HOMEOBOX11 (WOX11) and CROWN ROOTLESS1 (CRL1) play vital roles in controlling crown root development. Despite their importance, it remains unclear whether and how the two regulators coordinate crown root formation. Eletrophoretic mobility shift assays (EMSAs), transient expression and chromatin immunoprecipitation qPCR (ChIP-qPCR) suggested that WOX11 and CRL1 directly bind to OsCKX4 to regulate its expression during crown root development. CRL1 enhances OsCKX4 activation through direct interaction with WOX11 at root emergence and elongation stages. Genetic dissection showed that wox11/crl1 double mutant exhibits a more severe root phenotype. OsCKX4 knock-out plants generated by CRISPR/Cas9 exhibited fewer crown roots number and higher cytokinin levels in root meristem. Increased expression of OsCKX4 could partially complement the crown root phenotypes of both crl1 and wox11 mutants. Furthermore, cytokinin can promote WOX11 protein accumulation in root meristem. Together, these findings show that cytokinin accumulation is tightly regulated by the WOX11-CRL1 complex during crown root elongation by counter-acting on the negative regulatory effects of cytokinin on root development. Importantly, these results reveal an intrinsic link between WOX11 protein accumulation and cytokinin to maintain crown root growth.