Transcriptomic Analysis Uncovering Candidate Genes Associated with GH3 Mutant Seed Traits in Soybean

Soybean is the dominant source of plant-derived protein, yet improving seed protein content without reducing yield remains a breeding challenge. While the phytohormone auxin is well-established as a crucial regulator of seed development, its precise role in metabolic homeostasis governing protein accumulation remains largely unexplored. Here, we show that disrupting auxin conjugation via CRISPR-Cas9-generated gh3Q (GH3 quadruple) mutants elevates free auxin levels and is associated with increased seed size, hundred-seed weight, and protein content. Time-course transcriptome profiling of seed coats and embryos identified the R6-stage seed coat as a pivotal tissue for protein accumulation, where seven key seed storage protein genes (2S albumin and 11S glycinin family members) were significantly up-regulated. Weighted gene co-expression network analysis (WGCNA) further uncovered auxin polar transport modules and novel hub genes that are co-expressed with potentially link auxin signaling to protein storage pathways. These findings suggest that auxin homeostasis may link phytohormone signaling and metabolism allocation in soybean seed development. Our study provides genetic targets and a regulatory network for developing high-protein soybean varieties.