The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

WUSCHEL-related homeobox (WOX) is a family of transcription factors that are unique to plants and is characterized by the presence of a homeodomain. The WOX transcription factor plays an important role in regulating plant growth and development and the response to abiotic stress. Soybean is one of the most important oil crops worldwide. In this study, based on the available genome data of soybean, the WOX gene family was identified by bioinformatics analysis. The chromosome distribution, gene and protein structures, phylogenetic relationship and gene expression patterns of this family were comprehensively compared. The results showed that a total of 33 putative WOX genes in the soybean genome were found and then designated as GmWOX1- GmWOX33, which were distributed across 19 chromosomes except chromosome 16. Multiple sequence analysis of the GmWOX gene family revealed a highly conserved homeodomain. Phylogenetic tree analysis showed that 33 WOX genes could be divided into three major clades (modern/WUS, intermediate and ancient) in soybean. Of these 33 WOX genes, some showed differential expression patterns in the tested tissues (leaves, pods, unopen and open flowers, nodules, seed, roots, root hairs, stems, shoot apical meristems and shoot tips). In addition, the expression profile and qRT-PCR analysis showed that most of the GmWOX genes responded to different abiotic stress treatments (cold and drought). According to the expression pattern of GmWOX genes in the high regeneration capacity soybean material P3, overexpression of GmWOX18 was selected for function analysis. The overexpression of GmWOX18 increased the regeneration ability of clustered buds. The results will provide valuable information for further studies on the roles of WOX genes in regulating soybean growth, development and responses to abiotic stress, as well as a basis for the functional identification and analysis of WOX genes in soybean.

Chlorophyll plays an essential role in photosynthetic light harvesting and energy transduction in green tissues of higher plants and is closely related to photosynthesis and crop yield. Identification of transcription factors (TFs) involved in regulating chlorophyll biosynthesis is still limited in soybean (Glycine max), and the previously identified GmGATA58 is suggested to potentially modulate chlorophyll and nitrogen metabolisms, but its complete function is still unknown. In this study, subcellular localization assay showed that GmGATA58 was localized in the nucleus. Histochemical GUS assay and qPCR assay indicated that GmGATA58 was mainly expressed in leaves and responded to nitrogen, light and phytohormone treatments. Overexpression of GmGATA58 in the Arabidopsis thaliana ortholog AtGATA21 (gnc) mutant complemented the greening defect, while overexpression in Arabidopsis wild-type led to increasing chlorophyll content in leaves through up-regulating the expression levels of the large of chlorophyll biosynthetic pathway genes, but suppressing plant growth and yield, although the net photosynthetic rate was slightly improved. Dual-luciferase reporter assay also supported that GmGATA58 activated the transcription activities of three promoters of key chlorophyll biosynthetic genes of soybean in transformed protoplast of Arabidopsis. It is concluded that GmGATA58 played an important role in regulating chlorophyll biosynthesis, but suppressed plant growth and yield in transgenic Arabidopsis.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain–containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

Soybean (Glycine max L.) is a vital grain and oil crop, serving as a primary source of edible oil, plant-based protein, and livestock feed. Its production is crucial for ensuring global food security. However, soybean yields are severely impacted by various diseases, and the development of disease-resistant cultivars remains the most sustainable strategy for mitigating these losses. While stable genetic transformation is a common approach for studying gene function, virus-induced gene silencing (VIGS) offers a rapid and powerful alternative for functional genomics, enabling efficient screening of candidate genes. Nevertheless, the application of VIGS in soybean has been relatively limited. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for soybean, utilizing Agrobacterium tumefaciens-mediated infection. The TRV vector was delivered through cotyledon nodes, facilitating systemic spread and effective silencing of endogenous genes. Our results demonstrate that this TRV–VIGS system efficiently silences target genes in soybean, inducing significant phenotypic changes with a silencing efficiency ranging from 65% to 95%. Key genes, including phytoene desaturase (GmPDS), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4, were successfully silenced, confirming the system’s robustness. This work establishes a highly efficient TRV–VIGS platform for rapid gene function validation in soybean, providing a valuable tool for future genetic and disease resistance research.