Functional characterization and transcriptional regulation of the starch branching enzyme 1 in durian fruit

Starch metabolism is a key determinant of durian (Durio zibethinus L.) fruit quality, as it influences texture and sweetness during ripening. However, the molecular basis of starch branching during its accumulation remains poorly understood. In this study, we characterized a durian starch branching enzyme, DzSBE1, which is highly expressed at the immature-to-mature stage. Transcriptome and phylogenetic analyses revealed that DzSBE1 belongs to the plant SBE1 subfamily and contains all of the conserved catalytic features of the α-amylase superfamily. Recombinant DzSBE1, expressed as a soluble protein in Escherichia coli and likely dimeric, exhibited clear α-1,6-branching activity toward both amylose and amylopectin, with a preference for amylose. The enzyme showed optimal activity at pH 6.0-7.0 and 30-35 °C and does not require metal ions for catalysis. Product structural analyses revealed that DzSBE1 converts linear glucans into a more highly branched structure with enhanced crystallinity. Subcellular localization and transient overexpression in Nicotiana benthamiana leaves confirmed that DzSBE1 functions within plastids to promote α-1,6-branching. Furthermore, promoter analysis of DzSBE1 identified MYB and bHLH1 binding motifs. Dual-luciferase assays confirmed that DzMYB61-like, DzMYBETC3-like, and DzbHLH1 act as transcriptional activators, whereas DzMYB1 functions as a repressor. Co-expression assays further demonstrated that specific MYB-bHLH1 pairs cooperatively regulate DzSBE1 promoter activity. These findings indicate that DzSBE1 may act as an important enzyme facilitating starch branching during durian fruit maturation and provide comprehensive insights into the regulatory mechanisms linking starch synthesis to quality development in this economically important tropical crop.