BZR2-GRF5 acts as a hub module in regulating in vitro regeneration through brassinosteroid-auxin crosstalk in hybrid sweetgum.

In vitro plant regeneration is critical for germplasm multiplication, conservation, and genetic improvement in woody plants, primarily via two pathways: somatic embryogenesis (SE) and organogenesis. Brassinosteroids (BR) and auxin are key hormones regulating plant regeneration. However, the synergistic mechanism between these hormones in hybrid sweetgum remains poorly understood. We demonstrated that LsfBZR1/2 in the BR signaling pathway interacts with LsfGRF2/3/5. Functional characterization revealed that overexpression of either LsfBZR2 or LsfGRF5 significantly promoted callus proliferation while suppressing somatic embryo formation during SE, and enhanced shoot regeneration efficiency in organogenesis. LsfBZR2-LsfGRF5 module was found to differentially regulate the auxin efflux gene LsfWAG2 in a tissue-specific manner. During SE, LsfBZR2 overexpression suppresses LsfWAG2 expression, leading to auxin accumulation that promotes embryogenic callus (EC) proliferation. Conversely, in the organogenesis pathway, LsfBZR2 overexpression enhanced LsfWAG2 expression, which reduced auxin levels and consequently accelerated shoot regeneration. This study represents the first identification of the LsfBZR2-LsfGRF5 module as a central regulator with dual functions in different tissues of hybrid sweetgum. Through the crosstalk between BR and auxin, this module influences both SE and organogenesis. Our study provides evidence supporting the elucidation of the molecular mechanism of in vitro regeneration in hybrid sweetgum, which is of great significance for breeding and large-scale production.