Vegetable and grain soybeans are typically distinguished by harvest time and pod size, yet their nutritional differences are often overlooked in breeding programs. This study compared 10 varieties each of vegetable and grain soybeans to find key nutritional markers distinguishing them. Results showed that vegetable soybeans have higher concentrations of sucrose, total soluble sugar, and crude protein, along with lower concentrations of crude oil and total fatty acid. Specifically, vegetable soybeans contain a relatively higher amount of unsaturated fatty acids, particularly oleic acid, at green edible stages. Principal component analysis of 12 nutritional components revealed clear distinctions between vegetable and grain soybeans. Additionally, machine learning algorithms identified sucrose as the most critical nutritional marker for distinguishing these two types. Dynamic RNA-seq analysis combined with weighted gene co-expression network analysis identified a sucrose-related module, highlighting GmSPS17 as a predominant sucrose phosphate synthase encoding gene involved in sucrose accumulation in soybean seeds. Furthermore, we identified GmZF-HD1 as an upstream transcription factor regulating GmSPS17. Yeast one-hybrid, luciferase, and electrophoretic mobility shift assays confirmed that GmZF-HD1 directly activates GmSPS17 transcription. Overexpression experiments in hairy roots validated that GmZF-HD1 enhances GmSPS17 expression, thereby increasing sucrose accumulation. In summary, this study establishes sucrose as a key nutritional marker for distinguishing vegetable soybeans from grain soybeans and elucidates the GmZF-HD1–GmSPS17 regulatory pathway, providing valuable insights into sugar accumulation mechanisms and offering guidance for breeding high-sugar vegetable soybean varieties.

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