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Summary Drought and salt stresses impose major constraints on soybean production worldwide. However, improving agronomically valuable soybean traits under drought conditions can be challenging due to trait complexity and multiple factors that influence yield. Here, we identified a nuclear factor Y C subunit (NF‐YC) family transcription factor member, GmNF‐YC14, which formed a heterotrimer with GmNF‐YA16 and GmNF‐YB2 to activate the GmPYR1‐mediated abscisic acid (ABA) signalling pathway to regulate stress tolerance in soybean. Notably, we found that CRISPR/Cas9‐generated GmNF‐YC14 knockout mutants were more sensitive to drought than wild‐type soybean plants. Furthermore, field trials showed that overexpression of GmNF‐YC14 or GmPYR1 could increase yield per plant, grain plumpness, and stem base circumference, thus indicating improved adaptation of soybean plants to drought conditions. Taken together, our findings expand the known functional scope of the NF‐Y transcription factor functions and raise important questions about the integration of ABA signalling pathways in plants. Moreover, GmNF‐YC14 and GmPYR1 have potential for application in the improvement of drought tolerance in soybean plants.

Background Abiotic stress severely influences plant growth and development. MYB transcription factors (TFs), which compose one of the largest TF families, play an important role in abiotic stress responses. Result We identified 139 soybean MYB-related genes; these genes were divided into six groups based on their conserved domain and were distributed among 20 chromosomes (Chrs). Quantitative real-time PCR (qRT-PCR) indicated that GmMYB118 highly responsive to drought, salt and high temperature stress; thus, this gene was selected for further analysis. Subcellular localization revealed that the GmMYB118 protein located in the nucleus. Ectopic expression (EX) of GmMYB118 increased tolerance to drought and salt stress and regulated the expression of several stress-associated genes in transgenic Arabidopsis plants. Similarly, GmMYB118 -overexpressing (OE) soybean plants generated via Agrobacterium rhizogenes ( A. rhizogenes )-mediated transformation of the hairy roots showed improved drought and salt tolerance. Furthermore, compared with the control (CK) plants, the clustered, regularly interspaced, short palindromic repeat (CRISPR)-transformed plants exhibited reduced drought and salt tolerance. The contents of proline and chlorophyll in the OE plants were significantly greater than those in the CK plants, whose contents were greater than those in the CRISPR plants under drought and salt stress conditions. In contrast, the reactive oxygen species (ROS) and malondialdehyde (MDA) contents were significantly lower in the OE plants than in the CK plants, whose contents were lower than those in the CRISPR plants under stress conditions. Conclusions These results indicated that GmMYB118 could improve tolerance to drought and salt stress by promoting expression of stress-associated genes and regulating osmotic and oxidizing substances to maintain cell homeostasis.

WRKYs are important regulators in plant development and stress responses. However, knowledge of this superfamily in soybean is limited. In this study, we characterized the drought- and salt-induced gene GmWRKY12 based on RNA-Seq and qRT-PCR. GmWRKY12, which is 714 bp in length, encoded 237 amino acids and grouped into WRKY II. The promoter region of GmWRKY12 included ABER4, MYB, MYC, GT-1, W-box and DPBF cis-elements, which possibly participate in abscisic acid (ABA), drought and salt stress responses. GmWRKY12 was minimally expressed in different tissues under normal conditions but highly expressed under drought and salt treatments. As a nucleus protein, GmWRKY12 was responsive to drought, salt, ABA and salicylic acid (SA) stresses. Using a transgenic hairy root assay, we further characterized the roles of GmWRKY12 in abiotic stress tolerance. Compared with control (Williams 82), overexpression of GmWRKY12 enhanced drought and salt tolerance, increased proline (Pro) content and decreased malondialdehyde (MDA) content under drought and salt treatment in transgenic soybean seedlings. These results may provide a basis to understand the functions of GmWRKY12 in abiotic stress responses in soybean.

Abiotic stresses, such as drought and salt, are major environmental stresses, affecting plant growth and crop productivity. Plant bZIP transcription factors (bZIPs) confer stress resistances in harsh environments and play important roles in each phase of plant growth processes. In this research, 15 soybean bZIP family members were identified from drought-induced de novo transcriptomic sequences of soybean, which were unevenly distributed across 12 soybean chromosomes. Promoter analysis showed that these 15 genes were rich in ABRE, MYB and MYC cis-acting elements which were reported to be involved in abiotic stress responses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that 15 GmbZIP genes could be induced by drought and salt stress. GmbZIP2 was significantly upregulated under stress conditions and thus was selected for further study. Subcellular localization analysis revealed that the GmbZIP2 protein was located in the cell nucleus. qRT-PCR results show that GmbZIP2 can be induced by multiple stresses. The overexpression of GmbZIP2 in Arabidopsis and soybean hairy roots could improve plant resistance to drought and salt stresses. The result of differential expression gene analysis shows that the overexpression of GmbZIP2 in soybean hairy roots could enhance the expression of the stress responsive genes GmMYB48, GmWD40, GmDHN15, GmGST1 and GmLEA. These results indicate that soybean bZIPs played pivotal roles in plant resistance to abiotic stresses.

Melatonin (N-acetyl-5-methoxytryptamine) is involved in many developmental processes and responses to various abiotic stresses in plants. Most of the studies on melatonin focus on its functions and physiological responses in plants, while its regulation mechanism remains unknown. Caffeic acid 3-O-methyltransferase (COMT) functions at a key step of the biosynthesis process of melatonin. In this study, a COMT-like gene, TaCOMT (Traes_1AL_D9035D5E0.1) was identified in common wheat (Triticum aestivum L.). Transient transformation in wheat protoplasts determined that TaCOMT is localized in cytoplasm. TaCOMT in wheat was induced by drought stress, gibberellin (GA)3 and 3-Indoleacetic acid (IAA), but not by ABA. In TaCOMT transgenic Arabidopsis, melatonin contents were higher than that in wild type (WT) plants. Under D-Mannitol treatment, the fresh weight of the transgenic Arabidopsis was significantly higher than WT, and transgenic lines had a stronger root system compared to WT. Drought tolerance assays in pots showed that the survival rate of TaCOMT-overexpression lines was significantly higher than that of WT lines. this phenotype was similar to that the WT lines treated with melatonin under drought condition. In addition, the TaCOMT transgenic lines had higher proline content and lower malondialdehyde (MDA) content compared to WT lines after drought treatment. These results indicated that overexpression of the wheat TaCOMT gene enhances drought tolerance and increases the content of melatonin in transgenic Arabidopsis. It could be one of the potential genes for agricultural applications.

GRAS genes, which form a plant-specific transcription factor family, play an important role in plant growth and development and stress responses. However, the functions of GRAS genes in soybean ( Glycine max ) remain largely unknown. Here, 117 GRAS genes distributed on 20 chromosomes were identified in the soybean genome and were classified into 11 subfamilies. Of the soybean GRAS genes, 80.34% did not have intron insertions, and 54 pairs of genes accounted for 88.52% of duplication events (61 pairs). RNA-seq analysis demonstrated that most GmGRASs were expressed in 14 different soybean tissues examined and responded to multiple abiotic stresses. Results from quantitative real-time PCR analysis of six selected GmGRASs suggested that GmGRAS37 was significantly upregulated under drought and salt stress conditions and abscisic acid and brassinosteroid treatment; therefore, this gene was selected for further study. Subcellular localization analysis revealed that the GmGRAS37 protein was located in the plasma membrane, nucleus, and cytosol. Soybean hairy roots overexpressing GmGRAS37 had improved resistance to drought and salt stresses. In addition, these roots showed increased transcript levels of several drought‐ and salt-related genes. The results of this study provide the basis for comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in soybean.

GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus. GELPs are widely distributed in nature, from microbes to plants, and play crucial roles in growth and development, stress responses and pathogen defense. However, the identification and functional analysis of GELP genes are hardly explored in soybean. This study describes the identification of 194 GELP genes in the soybean genome and their phylogenetic classification into 11 subfamilies (A–K). GmGELP genes are disproportionally distributed on 20 soybean chromosomes. Large-scale WGD/segmental duplication events contribute greatly to the expansion of the soybean GDSL gene family. The Ka/Ks ratios of more than 70% of duplicated gene pairs ranged from 0.1–0.3, indicating that most GmGELP genes were under purifying selection pressure. Gene structure analysis indicate that more than 74% of GmGELP genes are interrupted by 4 introns and composed of 5 exons in their coding regions, and closer homologous genes in the phylogenetic tree often have similar exon-intron organization. Further statistics revealed that approximately 56% of subfamily K members contain more than 4 introns, and about 28% of subfamily I members consist of less than 4 introns. For this reason, the two subfamilies were used to simulate intron gain and loss events, respectively. Furthermore, a new model of intron position distribution was established in current study to explore whether the evolution of multi-gene families resulted from the diversity of gene structure. Finally, RNA-seq data were used to investigate the expression profiles of GmGELP gene under different tissues and multiple abiotic stress treatments. Subsequently, 7 stress-responsive GmGELP genes were selected to verify their expression levels by RT-qPCR, the results were consistent with RNA-seq data. Among 7 GmGELP genes, GmGELP28 was selected for further study owing to clear responses to drought, salt and ABA treatments. Transgenic Arabidopsis thaliana and soybean plants showed drought and salt tolerant phenotype. Overexpression of GmGELP28 resulted in the changes of several physiological indicators, which allowed plants to adapt adverse conditions. In all, GmGELP28 is a potential candidate gene for improving the salinity and drought tolerance of soybean.

Background Crop productivity is challenged by abiotic stresses, among which drought stress is the most common. NF-Y genes, especially NF-YA genes, regulate tolerance to abiotic stress. Results Soybean NF-Y gene GmNFYA5 was identified to have the highest transcript level among all 21 NF-YA genes in soybean ( Glycine max L.) under drought stress. Drought-induced transcript of GmNFYA5 was suppressed by the ABA synthesis inhibitor naproxen (NAP). GmNFYA5 transcript was detected in various tissues at vegetative and reproductive growth stages with higher levels in roots and leaves than in other tissues, which was consist with the GmNFYA5 promoter: GUS fusion assay. Overexpression of GmNFYA5 in transgenic Arabidopsis plants caused enhanced drought tolerance in seedlings by decreasing stomatal aperture and water loss from leaves. Overexpression and suppression of GmNFYA5 in soybean resulted in increased and decreased drought tolerance, respectively, relative to plants with an empty vector (EV). Transcript levels of ABA-dependent genes ( ABI2 , ABI3 , NCED3 , LEA3 , RD29A , P5CS1 , GmWRKY46 , GmNCED2 and GmbZIP1 ) and ABA-independent genes ( DREB1A , DREB2A , DREB2B , GmDREB1 , GmDREB2 and GmDREB3 ) in transgenic plants overexpressing GmNFYA5 were higher than those of wild-type plants under drought stress; suppression of GmNFYA5 transcript produced opposite results. GmNFYA5 probably regulated the transcript abundance of GmDREB2 and GmbZIP1 by binding to the promoters in vivo. Conclusions Our results suggested that overexpression of GmNFYA5 improved drought tolerance in soybean via both ABA-dependent and ABA-independent pathways.

Summary Sterols have long been associated with diverse fields, such as cancer treatment, drug development, and plant growth; however, their underlying mechanisms and functions remain enigmatic. Here, we unveil a critical role played by a GmNF‐YC9‐mediated CCAAT‐box transcription complex in modulating the steroid metabolism pathway within soybeans. Specifically, this complex directly activates squalene monooxygenase (GmSQE1), which is a rate‐limiting enzyme in steroid synthesis. Our findings demonstrate that overexpression of either GmNF‐YC9 or GmSQE1 significantly enhances soybean stress tolerance, while the inhibition of SQE weakens this tolerance. Field experiments conducted over two seasons further reveal increased yields per plant in both GmNF‐YC9 and GmSQE1 overexpressing plants under drought stress conditions. This enhanced stress tolerance is attributed to the reduction of abiotic stress‐induced cell oxidative damage. Transcriptome and metabolome analyses shed light on the upregulation of multiple sterol compounds, including fucosterol and soyasaponin II, in GmNF‐YC9 and GmSQE1 overexpressing soybean plants under stress conditions. Intriguingly, the application of soybean steroids, including fucosterol and soyasaponin II, significantly improves drought tolerance in soybean, wheat, foxtail millet, and maize. These findings underscore the pivotal role of soybean steroids in countering oxidative stress in plants and offer a new research strategy for enhancing crop stress tolerance and quality from gene regulation to chemical intervention.

Ankyrin repeat (ANK) proteins are essential in cell growth, development, and response to hormones and environmental stresses. In the present study, 226 genes were identified and classified into nine subfamilies according to conserved domains in the soybean genome ( L.). Among them, the was highly induced by drought, salt, and abscisic acid. The encodes a protein that belongs to the ANK-RF subfamily containing a RING finger (RF) domain in addition to the ankyrin repeats. Heterologous overexpression of in transgenic improved the germination rate under drought and salt treatments compared to wild-type. Homologous overexpression of improved the survival rate under drought and salt stresses in transgenic soybean hairy roots. In response to drought or salt stress, overexpression in soybean hairy root showed higher proline and lower malondialdehyde contents, and lower H O and O contents compared control plants. Besides, activated transcription of several abiotic stress-related genes, including , , , , and under drought and salt stresses in soybean. These results provide new insights for functional analysis of soybean ANK proteins and will be helpful for further understanding how ANK proteins in plants adapt to abiotic stress.