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.

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The formation of tissues and organs in multicellular organisms are tightly controlled by transcriptional programs determined by temporal and spatial patterns of gene expression. As an important regulator of rice crown root development, WOX11 is essential for crown root formation and its transcript level is positively correlated with crown roots biomass. However, how WOX11 is regulated during crown root primordium emergence and outgrowth still remains unknown. In this study, variations of WOX11 genomic sequence were analyzed, and the highest genetic diversity was found within its promoter, which contained a non-canonical miniature inverted-repeat transposable element (ncMITE) sequence. Analysis of WOX11 promoter driven reporter gene GUS (β-glucuronidase) transgenic plants pWOX11(ncMITE+):GUS and pWOX11(ncMITE-):GUS uncovered higher GUS expression level in crown roots of pWOX11(ncMITE+):GUS plants. Furthermore, pWOX11(ncMITE+):WOX11-FLAG in wox11 background could complement crown root number and length compared to those of wild type (WT), while pWOX11(ncMITE-):WOX11-FLAG could not. These results suggested that the ncMITE was positively associated WOX11 transcripts in rice crown roots. In addition, DNA methylation nearby ncMITE region attenuated the activation effect of the ncMITE on WOX11 expression, which might be also the cause conferred to the root-specific expression of WOX11. This work provides novel insight into WOX11 expression regulation and reveals a promising target for genetic improvement of root architecture in rice.