Single-Cell Transcriptomics Reveals the FA9-VAP Module Regulating Fatty Acid Accumulation in Soybean Seeds

As a major commercial legume crop, soybean ranks among the world's most significant sources of edible oil and plant protein. We previously identified a SEIPIN homologue (FA9) at the fatty acid 9 locus that promotes fatty acid accumulation in soybean. To examine the detailed molecular mechanisms by which FA9 regulates lipid metabolism, we performed single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (stRNA-seq) of wild-type and FA9-knockout soybean seeds at the late maturity stage. scRNA-seq analysis identified 26 transcriptional clusters and revealed the spatial distribution of FA9 in seeds, in which the deletion of FA9 altered lipid and storage-related transcriptional programmes. On the basis of single-cell sequencing and immunoprecipitation-mass spectrometry (IP-MS), the vesicle-associated membrane protein (VAMP)-associated protein (VAP) was identified, and subsequent experiments demonstrated that FA9 interacts specifically with VAP via its N-terminal FFAT motif at the endoplasmic reticulum. Seeds of vap knockout (vap-KO1 and vap-KO2) and fa9 vap double knockout (fa9 vap-KO) lines, created by CRISPR-Cas9 gene editing, had higher protein contents and lower total fatty acid contents than wild-type soybean, whereas overexpression of FA9 and VAP enhanced lipid droplet formation in Nicotiana benthamiana. These findings reveal that FA9 interacts with VAP to promote lipid droplet biogenesis and lipid transport, thereby driving fatty acid accumulation in soybean seeds. This research provides new insight into the molecular mechanisms that regulate seed oil synthesis and identifies potential target genes for improvement of soybean oil quality through molecular breeding.