Transcriptional regulatory network of WOX11 is involved in the control of crown root development, cytokinin signals, and redox in rice

The rice root system is mainly composed of post-embryonic shoot-borne roots called crown roots. WOX11, encoding a WUSCHEL-related homeobox domain transcription factor, is a key regulator of crown root growth and development in rice (Oryza sativa. L). In addition to specifically activating crown root development, WOX11 is also involved in lateral root initiation, root hair formation, and abiotic stresses. However, the gene regulatory network downstream of WOX11 remains largely unknown. Here, we studied the transcriptome of wox11 root tips by RNA-Seq and determined direct WOX11-binding targets by bioinformatic and biochemical analysis. The transcriptomic analysis revealed 664 differentially expressed genes, which covered a wide range of functions related to root development, cytokinin homeostasis/signaling, stress response, and redox metabolic processes. Bioinformatic analysis also revealed that the WOX11-binding motif was distributed over 41% (273/664) of the differentially expressed genes, and was mostly enriched in the promoter and intron regions. We used qRT-PCR and/or in situ hybridization to confirm co-expression of some of the WOX11-regulated genes in crown root development. We also used electrophoretic mobility shift assay and chromatin immunoprecipitation with anti-WOX11 antibody to validate direct regulation of these genes by WOX11. The analysis identified several genes that acted downstream of WOX11 in controlling crown root formation, cytokinin signaling, stress response, and redox metabolism. This work built a hierarchical regulatory model of WOX11 in rice crown root development.