The sole active enzyme-encoding gene MlTPS1 in the trehalose biosynthesis pathway plays a crucial role in reproductive regulation of Magnolia liliiflora Desr.

Magnolia species hold significant economic value in pharmaceutical and cosmetic industries, yet their complex genetic backgrounds have hindered progress in understanding reproductive regulation mechanisms. This study reported a novel trehalose metabolism-mediated reproductive regulation mechanism identified in M. liliiflora ‘Hongyuanbao’, a twice-annually flowering variety. Metabolomic analysis revealed dynamic trehalose metabolic shifts during two flower bud differentiation phases, with molecular characterization identifying four Trehalose-6-Phosphate Synthase (TPS) proteins. Only the Class Ⅰ member, MlTPS1, exhibited catalytic activity, which was enhanced by N-terminal truncation. Distinct from the nuclear-cytoplasmic localization of Arabidopsis ( Arabidopsis thaliana ) AtTPS1, MlTPS1 was exclusively localized to the cytoplasm. Functional characterization demonstrated that MlTPS1 possesses a reproductive-promoting function independent of trehalose metabolism and mediates downstream pathways different from AtTPS1 . Additionally, the promoter of MlTPS1 was specifically responsive to sugar and light signaling. Yeast Two-Hybrid (Y2H) screening identified 241 potential MlTPS1-interacting proteins, including diverse Transcription Factors (TFs) and Protein Kinases (PKs), with 13 directly linked to reproductive development. MlTPS1 could regulate its own activity or directly modulate downstream target genes by forming protein complexes through these interactions. These new findings highlight the critical regulatory roles of the non-catalytic functions of MlTPS1. Collectively, we innovatively proposed a regulatory model in which MlTPS1 integrates sugar signaling with reproductive development through metabolic regulation and a protein interaction network. This study provides key insights into the pleiotropic roles of TPS1 proteins in plant reproductive regulation. • Trehalose metabolism played a critical role in reproductive development. • MlTPS1 was the only class I TPS protein with confirmed enzymatic activity. • MlTPS1 was responsible for trehalose synthesis and also promoted flowering. • 241 MlTPS1-interacting proteins were identified, forming an interaction network. • MlTPS1 is a key node linking trehalose metabolism with reproductive regulation.