Brassinolide mitigates cadmium accumulation in Panax notoginseng via H₂O₂-PnBZR1-mediated pectin modification

Panax notoginseng is a valuable traditional Chinese medicinal herb; however, excessive cadmium (Cd) accumulation has long hindered the high-quality development of its industry. Brassinosteroid (BR) is known to enhance the P. notoginseng yield. However, whether it also confers resistance to Cd stress and its underlying mechanisms remain unclear. Here, we conducted field, pot, and hydroponic experiments to investigate the role of BR in regulating Cd distribution in P. notoginseng , and validated the function of PnBZR1 , a key transcription factor in BR signaling, by characterizing its activity in transgenic tobacco. Compared to untreated controls (CK), BR treatment for 30 days reduced Cd content in roots by 20.48 %, while increasing biomass and total saponin content by up to 11.43 %. Cd stress induced endogenous BR accumulation (17.08 % in roots, 35.71 % in leaves). Under Cd+BR treatment, Cd partitioning to the cell walls of rhizomes, main roots, and rootlets increased by 1.51-, 1.94-, and 1.37-fold compared to Cd treatment alone, whereas Cd distribution in the cytoplasmic supernatant and organelles significantly decreased. Mechanistically, BR upregulated the expression of PnRBOHA/B/C , leading to a 14.85 % increase in H₂O₂ content. BR and H₂O₂ synergistically promoted pectin biosynthesis and demethylesterification. Compared to the Cd+BRz+DPI+BR group, the Cd+BRz+DPI+BR+H₂O₂ group showed 19.23 % higher pectin uronic acid content and 36.24 % higher pectin methylesterase (PME) activity. Furthermore, H₂O₂ treatment increased the transcriptional activation of PnBZR1 by 10.78 %, which synergized with BR to upregulate PME expression, promoting Cd²⁺ binding to COO⁻ and -OH groups in the cell wall. In summary, BR enhances pectin synthesis and demethylesterification via the H₂O₂- PnBZR1 signaling cascade, facilitating Cd sequestration in the cell wall, limiting its entry into organelles, and thereby improving P. notoginseng tolerance to Cd stress. • BR altered the root cell wall component contents under Cd stress. • BR promoted pectin biosynthesis and demethylation in root cell wall. • H₂O₂ played a critical role in Cd accumulation in root cell wall. • Cd stress-induced H₂O₂ improved Cd accumulation in root cell wall. • BR- H₂O₂ interaction increased the number of carboxylic acid and hydroxyl groups.