Hormonal homeostasis and functional characterization of the strigolactone biosynthesis gene TpMAX1 during shoot branching in red clover (Trifolium pratense L.)

Branch number is a core agronomic trait determining plant architecture, regrowth capacity, and biomass in red clover ( Trifolium pratense L.). However, the underlying mechanisms of endogenous hormonal homeostasis and key gene regulation remain elusive. In this study, “Multi-branching” (MB) and “Few-branching” (FB) materials were used to systematically analyze the physiological and molecular basis of branching development. Results showed that the branch number and hay yield of MB were 1.74 and 1.85 times those of FB, respectively, characterizing a prominent “semi-dwarf and multi-branching” phenotype. Levels of ABA, GA3, and 5-deoxystrigol (5-DS) were significantly higher in MB tissues. Furthermore, tissue-specific analysis revealed that zeatin (ZT) was highly enriched in axillary buds, with a concentration 7.02-fold higher than that in leaves, suggesting a close association between ZT accumulation and bud outgrowth. Pearson correlation analysis showed that branch number was significantly and positively correlated with ABA, GA3, and specific hormonal ratios, including ABA/ZT and GA3/ZT ( r > 0.74, P < 0.01), indicating that hormonal homeostasis is closely associated with branching morphogenesis. At the molecular level, TpMAX1 , a key gene involved in strigolactone (SL) biosynthesis, was cloned and found to be expressed at relatively high levels in the leaves and axillary buds of MB plants. Exogenous hormone treatments demonstrated that TpMAX1 was strongly responsive to ABA treatment, exhibiting a 403.15-fold induction, and was significantly inhibited by IAA. Furthermore, exogenous rac-GR24 application significantly suppressed branching in MB and strongly induced the expression of the downstream signaling factor TpDELLA by 49.93-fold. Heterologous overexpression of TpMAX1 in Arabidopsis significantly reduced lateral branching and advanced flowering time, supporting its role in inhibiting branching and regulating floral transition. Collectively, these findings suggest that branch development in red clover is associated with a multi-hormone regulatory network involving TpMAX1 -mediated SL biosynthesis and ABA/SL/DELLA-related signaling, providing both a theoretical basis and candidate gene resources for the molecular breeding of high-yield legume forages.