The uORF-HsfA1a-WOX11 module controls crown root development in rice

OsWOX11 is a key essential determinant of crown root development in rice. However, either overexpression or downregulation of OsWOX11 results in pleiotropic developmental defects, including dwarfism and reduced yield. Therefore, it is necessary to ensure an optimal level of OsWOX11 expression for balancing the subterranean root system and aerial organ development. OsHsfA1a activates OsWOX11 expression by directly binding to heat stress element-like elements within its promoter. Genetic evidence demonstrated that OsHsfA1a overexpressing or knockout transgenic plants phenocopied the crown root growth in OsWOX11 transgenic plants. Additionally, increased expression of OsWOX11 in OsHsfA1a RNAi background could partially complement the defective crown root phenotypes. A uORF (uORF_HsfA1a) was identified within the 5'-untranslated region of OsHsfA1a. Transient expression assays coupled with ribosome profiling revealed that uORF_HsfA1a attenuated the translation efficiency of OsHsfA1a mRNA. Furthermore, HsfA1a_P:uORF_HsfA1a-HsfA1a-GFP plants exhibited wild-type crown root phenotypes, whereas uORF_HsfA1a knockout transgenic plants displayed similar crown root phenotypes to OsWOX11 overexpressing plants. These findings suggest that uORF_HsfA1a fine-tunes the crown root development by repressing OsHsfA1a translation, thereby indirectly modulating OsWOX11 transcript levels. Our study demonstrated a novel uORF_HsfA1a-HsfA1a-WOX11 regulatory module that coordinated transcriptional and translational control to maintain optimal OsWOX11 expression. This mechanism ensures the trade-off between root and shoot development. Importantly, targeting uORF_HsfA1a regulatory elements provided a new strategy for engineering robust root system architecture without compromising agronomic traits, thereby addressing a critical challenge in cereal crop improvement.