Background: INDETERMINATE DOMAIN proteins (IDDs) are a plant-specific transcription factor family, and members of this family play crucial roles in regulating growth and development as well as environmental adaptation. However, a comprehensive analysis of the IDD family in soybean [Glycine max (L.) Merrill] is limited. Methods and Results: A total of 27 GmIDD genes were identified in the soybean genome, unevenly distributed across 14 chromosomes, and their encoded proteins all harbor a conserved INDETERMINATE (ID) domain with two Cys2His2 (C2H2) and two Cys2HisCys (C2HC) zinc finger motifs. Phylogenetic analysis classified these GmIDD genes into three subgroups. Soybean GmIDD genes exhibit high homology with their Arabidopsis thaliana IDD counterparts. Cis-acting element analysis indicated that the promoters of GmIDD genes are enriched in light-responsive elements (such as Box4), hormone-responsive elements (such as ABRE and AuxRR-core), and abiotic stress-responsive elements (such as MBS and LTR). The qRT-PCR results showed that GmIDD3/5/14/22/26 were upregulated under salt stress, while GmIDD8/9/10/12/16/17/19/20/23/24/25/27 were obviously downregulated during treatment. Under drought stress, the expression levels of GmIDD4/6/7/10/14/16/19/22/24/25/26/27 were upregulated during the treatment. The expression levels of GmIDD1/2/3/4/12/14/15/16/17/18/22/23/25/26 were induced by short-day conditions, whereas GmIDD9/13/19/21 were induced by long-day conditions in soybean leaves. Conclusions: This study provides a theoretical basis for further understanding the functions of the soybean IDD gene family in abiotic stress tolerance and photoperiod adaptability.

Seed weight represents a critical yield-associated quantitative trait in soybean (Glycine max) and is governed by polygenic regulatory networks. Although several seed-weight-related genes have been functionally characterized, the interactome of the major seed-weight determining gene SW16.1 remains largely unexplored. In this study, we constructed a yeast two-hybrid (Y2H) cDNA library using pooled total RNA isolated from developing seeds of the soybean cultivar Nannong 1138-2 covering five key seed-development stages. Using GmSW16.1 (cultivated Nannong 1138-2 allelic) as bait, Y2H screening was performed. Following multi-round screening, Sanger sequencing, and filtering based on functional annotation, 11 candidate interactors assigned to nine distinct functional categories were recovered. Interactions were further verified through point-to-point Y2H assays, quantitative α-galactosidase (α-Gal) activity measurements, and bimolecular fluorescence complementation (BiFC). We confirmed that metallothionein-like protein 4A-related Glyma.18G157800 (GmMT4) and the papain-like cysteine protease Glyma.08G116300 physically interact with both GmSW16.1 and GsSW16.1 (wild soybean N24852). A two-glutamine polymorphism within the PolyQ (polyglutamine) repeat tract of these two allelic proteins did not abolish protein–protein interactions, yet significantly altered their interaction strength toward the above-mentioned partners. Glyma.08G116300 displayed preferential transcript accumulation during soybean seed development, and its transcript abundance differed dynamically between the chromosome segment substitution line CSSL3068 and its cultivated parent Nannong 1138-2 across developmental stages. Heterologous overexpression of this gene in Arabidopsis thaliana significantly increased the 1000-seed weight (by 12–15%) and increased seed length and width. This study delivers a seed-development-specific Y2H library resource for soybean research. We put forward a testable working hypothesis describing a dual-layer regulatory module integrating transcriptional control and direct protein–protein interaction for the GmSW16.1-GmMT4 regulatory node. These findings advance our mechanistic understanding of how natural PolyQ allelic variation shapes soybean seed-weight performance and provides candidate gene materials for soybean molecular design breeding.