Spatiotemporal Transcriptomics Reveals That a ZF-HD-GmCWINV4 Module Controls Carbon Partitioning and Seed Size in...

Seed size and oil content are key determinants of agronomic productivity in soybean (Glycine max). Although the ultrastructure and cellular composition of soybean seeds have been well characterised, the spatiotemporal expression patterns of genes governing sucrose transport and catabolism across distinct cell types remain poorly understood. Here, we present a high-resolution spatiotemporal transcriptomic atlas of early soybean seed development, spanning the globular, heart, and cotyledon stages. We delineated 17 distinct cell types and identified 249 cell-type marker genes. We further characterised the phloem region of the seed coat, a key compartment associated with sucrose transport and metabolism. Systematic profiling of sugar-related genes revealed cluster-specific and developmental stage-dependent transcriptional patterns. We functionally characterised GmCWINV4, a phloem-specific cell wall invertase that hydrolyses sucrose into glucose and fructose. Overexpression of GmCWINV4 substantially increased seed size, weight, and oil content in soybean. We also identified two zinc finger homeodomain (ZF-HD) transcription factors, GmZF-HD27 and GmZF-HD36, that directly activate GmCWINV4 transcription by binding to 'ATTA' cis-elements in its promoter. Moreover, we demonstrated that sucrose acts as an upstream signal that induces GmZF-HD36 transcription in a dose-dependent manner. Collectively, these findings provide a comprehensive spatiotemporal transcriptomic resource for studying soybean seed development and reveal a previously unreported ZF-HD-GmCWINV4 regulatory module that modulates post-phloem sucrose metabolism, thereby contributing to increased seed size and oil content. These findings offer mechanistic insights and potential targets for improving seed yield and nutritional quality in soybean.