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

Crown root (CR) 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 CR development. Despite their importance, whether and how the two regulators coordinate CR formation remains unclear. Electrophoretic mobility shift assays, transient expression, and chromatin immunoprecipitation qPCR suggested that WOX11 and CRL1 directly bind to OsCKX4 to regulate its expression during CR development. CRL1 enhances OsCKX4 activation through direct interaction with WOX11 at root emergence and elongation stages. Genetic dissection showed that the wox11/crl1 double mutant exhibits a more severe root phenotype. OsCKX4 knockout plants generated by CRISPR/Cas9 exhibited fewer CRs and higher cytokinin levels in the root meristem. Increased expression of OsCKX4 could partially complement the CR phenotypes of both crl1 and wox11 mutants. Furthermore, cytokinin can promote WOX11 protein accumulation in the root meristem. Together, these findings show that cytokinin accumulation is tightly regulated by the WOX11-CRL1 complex during CR elongation by counteracting the negative regulatory effects of cytokinin on root development. Importantly, these results reveal an intrinsic link between WOX11 protein accumulation and cytokinin to maintain CR growth.