Identification, functional and interaction proteins analysis of BjuBBX25b involved in development regulation and Cd responsiveness in Brassica juncea.

B. juncea (Brassica juncea var. latipa Li) is an important leafy vegetable in China, which is suitable for both fresh consumption and processing. In this study, 71 BjuBBXs were identified and categorized into five subfamilies with segmental duplication driving family expansion. Expression profiling by RNA-seq and qRT-PCR revealed that BjuBBX25b exhibits distinct organ-specific expression patterns and was significantly up-regulated in roots under cadmium (Cd) stress, prompting further functional investigation. To elucidate its role in development and heavy metal response, three BjuBBX25b overexpression (OE) lines were generated in Arabidopsis. These lines displayed reduced rosette leaf number and shortened siliques (pod length) compared to wild-type plants. qRT-PCR analysis revealed significant down-regulation of auxin-related genes (AtYUC6, AtPIN4), developmental regulators (AtCUC2, AtDPA4), and Cd-responsive transporters (AtHMA3, AtPAMP4) under heavy metal stresses (Mn, Zn, Mg, Cd). Yeast two-hybrid and BiFC assays identified six BjuBBX25b-interacting proteins, including BjuPRMT1.1. Expression analysis showed that while BjuBBX25b and BjuPRMT1.1 were down-regulated in flower buds, the genes encoding its six interacting partners were up-regulated during early reproductive stages (squaring stage). Subcellular localization revealed that BjuBBX25b localizes to the nucleus and its interactors predominantly in the cytoplasm. Collectively, these findings demonstrate that BjuBBX25b plays a multifaceted role in regulating leaf development, reproductive processes, and Cd stress response, likely through negative modulation of auxin signaling components, developmental regulators, and heavy metal-related transporters, with its interacting proteins (BjuPRMT1.1) potentially contributing to these regulatory processes.