Identification and functional verification of key iron homeostasis genes in soybean roots and nodules through integrated transcriptome and proteome analysis

Iron (Fe) is an essential nutrient for soybean [Glycine max (L.) Merr.] growth and symbiotic nitrogen fixation. However, the mechanisms underlying Fe homeostasis in nodules remain poorly understood. In this study, we conducted integrated transcriptome and proteome analyses of soybean roots and nodules under Fe deficiency to identify distinct Fe regulatory networks. Notably, nodules retained 42% of Fe levels under Fe-deficient conditions, despite severe depletion in roots (85% loss) and leaves (71% loss), suggesting a prioritized Fe allocation mechanism. Transcriptome and proteome sequencing of roots and nodules under Fe-sufficient and -deficient conditions revealed significant differences, confirming distinct expression profiles in nodules compared with roots. Among the differentially expressed genes, those encoding vacuolar Fe transporter-like protein 1a (GmVTL1a), yellow-strip like 7 (GmYSL7), and natural resistance-associated macrophage protein 3a (GmNRAMP3a) were highly expressed in nodules, emerging as key candidates. Transgenic soybeans expressing promoter:GUS fusion constructs for GmVTL1a, GmYSL7, and GmNRAMP3a confirmed their expression in nodules. Functional studies demonstrated that GmVTL1a mediates Fe transport across the symbiosome membrane, while GmYSL7 is critical for nodule development. Knockout of either gene impaired nitrogen fixation and ureide synthesis. Co-expression analysis of GmVTL1a and GmVTL1b identified 19 putative transcription factors potentially regulating GmVTL1a. An immunoprecipitation-mass spectrometry assay on nodule protein extracts from the pGmVTL1a-3Flag-gGmVTL1a plants yielded 55 candidate interactors, including 26 nodule-expressed proteins and 17 that overlapped with known symbiosome membrane proteins. Taken together, our study reveals nodule-specific adaptations in Fe homeostasis, highlighting GmVTL1a and GmYSL7 as central players.