RsLBD3 regulates the secondary growth of taproot by integrating auxin and cytokinin signaling in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is a globally important root vegetable crop known for its diverse varieties and unique taproot characteristics. The LBD (LATERAL ORGAN BOUNDARIES DOMAIN) gene family, specific to plants, plays a pivotal role in the development of lateral plant organs. Nonetheless, the precise biological functions and molecular regulatory mechanisms of LBD genes in radish taproot development remain largely unexplored. In this study, the RsLBD3 gene was identified as a potential candidate affecting taproot size in radish through a genome-wide association study. Further investigation revealed two insertions in the C-terminal region of RsLBD3, with insertion 363 notably enhancing the transcriptional activation capability of RsLBD3. It was observed that radish taproots with RsLBD3 Ins-363 haplotype displayed significantly greater length and weight compared to those with RsLBD3 Del-363 haplotype. RNA in situ hybridization and reverse transcription quantitative polymerase chain reaction analysis revealed that the RsLBD3 gene exhibits high expression level in the vascular cambium and is induced by cytokinin treatment. Silencing the RsLBD3 gene resulted in the inhibition of vascular cambium activity in the taproot, thereby impeding thickening. Exogenous cytokinin treatment could partially rescue the small-taproot phenotypes caused by RsLBD3 silencing. Moreover, RsARF5 (AUXIN RESPONSE FACTOR 5), RsRR7b (RESPONSE REGULATOR 7), and RsCYCD3-1 (CYCLIN D3;1) were identified as target genes of RsLBD3. Notably, RsARF5 was found to directly regulate the expression of RsWOX4 (WUSCHEL-RELATED HOMEOBOX 4). Additionally, biochemical analysis demonstrated that RsTCP14 interacts with RsLBD3, contributing to the binding of RsLBD3 to its target genes. Collectively, these findings contribute to a better understanding of the regulatory mechanisms underlying taproot morphogenesis, and provide novel allelic variations for the genetic enhancement of taproot shape traits in radish.