Legumes can utilize atmospheric nitrogen via symbiotic nitrogen fixation, but this process is inhibited by high soil inorganic nitrogen. So far, how high nitrogen inhibits N₂ fixation in mature nodules is still poorly understood. Here we construct a co-expression network in soybean nodule and find that a dynamic and reversible transcriptional network underlies the high N inhibition of N₂ fixation. Intriguingly, several NAC transcription factors (TFs), designated as Soybean Nitrogen Associated NAPs (SNAPs), are amongst the most connected hub TFs. The nodules of snap1/2/3/4 quadruple mutants show less sensitivity to the high nitrogen inhibition of nitrogenase activity and acceleration of senescence. Integrative analysis shows that these SNAP TFs largely influence the high nitrogen transcriptional response through direct regulation of a subnetwork of senescence-associated genes and transcriptional regulators. We propose that the SNAP-mediated transcriptional network may trigger nodule senescence in response to high nitrogen.

Fructose-1,6-bisphosphate aldolase (FBA) is an important catalytic enzyme in carbon metabolism and plays an important role in plant growth and development. Currently, the biological functions of FBA in soybean (Glycine max) remain unknown. In this study, we conducted research on FBA in soybean and identified 14 GmFBA genes. Among them, GmFBAc1 and GmFBAc2 are broadly expressed in different tissues. Double mutant lines of GmFBAc1 and GmFBAc2 were obtained by CRISPR-Cas9 gene editing technology. Compared with the wild type, the double-gene homozygous mutant gmfbac1gmfbac2 exhibited dwarf seedlings and narrow leaflets, indicating that GmFBAc1 and GmFBAc2 are critical for soybean growth and development. The gmfbac1gmfbac2 metabolomic analysis revealed that compared to the wild type, carbohydrate metabolism was reduced and amino acid metabolism was enhanced in gmfbac1gmfbac2 mutant leaves. Transcriptomic analysis showed that genes in IAA signaling and JA signaling were downregulated and upregulated, respectively. Our study demonstrates an important role of GmFBAc1 and GmFBAc2 in modulating carbon metabolism and phytohormone homeostasis.

Legumes have the capability to form nodules that facilitate symbiotic nitrogen fixation (SNF) with rhizobia. Given the substantial energy consumption during the process of SNF, legumes need to optimize nodule number in response to everchanging environmental scenarios. The TGACG BINDING FACTOR1/4 (TGA1/4) are key players in the basal immune response of plants. In this study, both β-glucuronidase staining and quantitative reverse transcription PCR (qRT-PCR) demonstrated that both GmbZIP4a and GmbZIP4b are inducible upon rhizobial inoculation. To investigate their roles further, we constructed gmbzip4a/b double mutants using CRISPR/Cas9 system. Nodulation assessments revealed that these double mutants displayed a reduction in the number of infection threads, which subsequently resulted in a decreased nodule number. However, the processes associated with nodule development including nodule fresh weight, structural characteristics, and nitrogenase activity, remained unaffected in the double mutants. Subsequent transcriptome analyses revealed that zeatin biosynthesis was downregulated in gmbzip4a/b mutants post rhizobial inoculation. Supporting these findings, genes associated with cytokinin (CTK) signaling pathway were upregulated in Williams 82 (Wm82), but this upregulation was not observed in the double mutants after rhizobial treatment. These results suggest that GmbZIP4a/b positively influences nodule formation by promoting the activation of the CTK signaling pathway during the early stages of nodule formation.

Soybean serves as a primary global source of high-quality protein and oil, and improving its yield and quality is critical for global food security. Despite advances in gene editing and molecular breeding, the regulatory mechanisms underlying leaf senescence remain poorly understood, limiting yield gains in soybean. Building on our previous identification of SNAP1/2/3/4—a cluster of NAC transcription factors regulating nodule senescence—we reported their pivotal role in modulating aboveground agronomic traits. The knockout mutants of SNAP1/2/3/4 exhibited delayed leaf senescence and prolonged maturation period. These changes led to concurrent improvements in grain yield, seed size, and oil content. Our study reveals a dual regulatory function of SNAP1/2/3/4 in both nodule and leaf senescence and uncovers a molecular target to coordinately enhance soybean yield and quality by fine-tuning of developmental timing.

Soybean (Glycine max L.) is gaining in importance due to its many uses, including as a food crop and a source of industrial products, among others. Increasing efforts are made to accelerate soybean research and develop new soybean varieties to meet global demands. Soybean research, breeding, identification, and variety protection all rely on precise genomic information. While DNA markers are invaluable tools for these purposes, the older generations, especially those developed before the advent of genome sequencing, lack precision and specificity. Thankfully, advancements in genome sequencing technologies have generated vast amounts of sequence data over the past decade, allowing precise and high-resolution analyses. However, making sense of the genomic information requires a certain level of professional training and computational power, which are not universally available to researchers. To address this, we generated a set of PCR-based DNA markers out of the existing genomic data from 228 popular soybean varieties that offer precise, unambiguous genomic information and can be easily adapted in various applications. A standard operating procedure (SOP) was also designed for these markers and validated on diverse soybean varieties to ensure their reproducibility. This user-friendly universal panel of DNA markers, along with the SOP, will facilitate soybean research and breeding programs through simple applications.

Triacylglycerol (TAG) serves as the primary storage lipid in plants and is essential for seed germination and early seedling development. Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step of TAG biosynthesis and is a key determinant of seed oil content and composition. Brassinosteroid (BR) signaling plays a pivotal role in coordinating seed development, oil biosynthesis, and filling duration. Here, we uncover a previously unrecognized function of the soybean (Glycine max) NAM, ATAF1,2, and CUC2 (NAC) transcription factor soybean nitrogen associated NAP (SNAP), a member of the SNAC-B/NAC-like, activated by AP3/PI (NAP) subfamily, in seed development, showing that SNAP directly represses DGAT1 and BR signaling's core component BES1/BZR1 homolog 4-1 (BEH4-1), thereby constraining oil accumulation and developmental progression. Integrative RNA sequencing and chromatin immunoprecipitation sequencing analyses demonstrate that SNAP orchestrates a broad transcriptional program encompassing oil biosynthesis genes (e.g., GmWRI1, GmKAS), sugar transporters (GmSWEET), and seed development networks, while also acting as a direct transcriptional repressor of DGAT1 and BEH4-1 by binding to their promoters. Notably, the SNAP family has previously been characterized exclusively as a regulator of the nodule nitrogen response and senescence. Our findings reveal a conserved yet distinct function of SNAP in mediating seed development and crosstalk with BR signaling, uncovering its pleiotropic roles beyond nodulation and providing key genetic targets for molecular breeding of high-yield, high-oil soybean materials.