The transcription factor NnNAC100 positively regulates amylopectin biosynthesis by activating NnSBEII in the rhizome of Nelumbo nucifera Gaertn.

NnNAC100-NnSBEII modules enhance starch content of the rhizome in Nelumbo nucifera Gaertn. Nelumbo nucifera Gaertn. is a popular aquatic vegetable and traditional Chinese medicine whose quality and taste are mainly determined by the starch. Although starch-related genes have been functionally characterized, the regulated mechanism of enzyme (SBE) remains unclear. In this study, we identified and functionally elucidated the functions of NnSBEII and NnNAC100 using transient overexpression of NnSBEII and NnNAC100 in rhizomes of lotus, and it significantly increased the amylopectin content and total starch content. Accordingly, functional complementation assay in defective Arabidopsis also showed that NnSBEII compensated for the low content of starch in the mutant sbe2.2. In addition, overexpression of NnSBEII and NnNAC100 significantly increased the content of starch in transgenic lines. Consistently, opposite results were observed under the background of repressed NnSBEII and NnNAC100 in rhizomes of lotus. Furthermore, yeast one-hybrid and dual-luciferase assays revealed that NnNAC100 could directly bind to the NnSBEII promoter and promote the expression of NnSBEII. Transient overexpression of NnNAC100 upregulated NnSBEII expression significantly, while the expression level of AtSBE2.2 in transgenic Arabidopsis overexpressing NnNAC100 was higher than that of WT, which indicated that NnNAC100 promoted the synthesis of amylopectin by enhancing the expression of NnSBEII. In addition, we found that NnSBEII could form a complex protein by interacting with soluble starch synthase (NnSS2) to increase the activity of the SBEII enzyme. These results reveal a novel mechanism that the NnNAC100-NnSBEII-NnSBEII/NnSS2 module regulates amylopectin biosynthesis and these will provide insights into the broader implications of the regulation mechanism of starch biosynthesis.