Seed germination initiates the plant life cycle, but it exhibits high sensitivity to salt stress, which is a significant environmental factor limiting rice production. Brassinosteroid (BR) is a growth-promoting phytohormone that mitigates various stresses in rice including salt, drought, and extreme temperatures. However, the mechanisms by which BR alleviates salt stress during seed germination remain inadequately characterized. This study demonstrates that seed-specific overexpression of OsDWF4, a rate-limiting gene in BR biosynthesis, enhances rice germination. The DWF4-OX lines, which have greater endogenous BR content in the seeds, showed better germination under salt stress, corroborating the results obtained through exogenous BR application. Antioxidant enzyme analyses demonstrated that BR enhances the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Metabolomic analysis revealed that BR mitigates salt stress primarily through the biosynthesis of phenylpropanoids and secondary metabolites. Transcriptomic analysis indicated that both endogenous and exogenous BR share five co-regulated target genes and utilize a common biosynthetic pathway for stilbenoids, diarylheptanoids, and gingerols. These findings confirm the capacity of BR to enhance seed germination under salt stress and identified several BR-mediated targets for developing salt-tolerant rice varieties that are suitable for direct seeding cultivation.

Seed germination is the beginning of a new lifecycle, and involves many complex physiological and biochemical reactions including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo. Although glutelin is a dominant storage protein in rice, its contribution to seed germination and its regulatory mechanisms are mostly unknown. Gibberellin (GA) and brassinosteroid (BR), two major growth-promoting phytohormones, also play positive roles in controlling seed germination. However, how GA and BR interact and coordinate seed germination and facilitate glutelin mobilization remains unclear. In the present study, biochemical and physiological analyses of seed germination indicated that both GA and BR promote seed germination and post-germination growth. Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity. Proteomic and qRT-PCR results showed that the expression of several glutelin proteins and their encoding genes was induced by BR and GA in the embryo. Expression assays suggested that the increased accumulation of glutelin protein in the embryo was due to the accelerated degradation of glutelin by a cysteine proteinase (REP-1) in the endosperm. The breakdown of glutelin in the endosperm showed a strict positive correspondence with the length of the shoot. The GluA2 mutation led to reduced degradation rate of glutelin and defects in seed germination, and the promotion effect of GA on seed germination was weakened in the glua2 mutant. In vitro culture assay of rice embryos showed that glutelin mobilization functioned downstream of the GA and BR pathways to promote shoot elongation. These findings suggest a mechanism that mediates crosstalk between BR and GA in co-regulating rice seed germination and embryo growth.