Biochemical characterization and metabolic reprogramming of amino acids in Soybean roots under drought stress.

Amino acids play important roles in stress resistance, plant growth, development, and quality, with roots serving as the primary organs for drought response. We conducted biochemical and multi-omics analyses to investigate the metabolic processes of root amino acids in drought-resistant (HN44) and drought-sensitive (HN65) soybean (Glycine max) varieties. Our analysis revealed an increase in total amino acid content in both varieties, with phenylalanine, proline, and methionine accumulating in both. Additionally, several amino acids exhibited significant decreases in HN65 but slight increases in HN44. Multi-omics association analysis identified 13 amino acid-related pathways. We thoroughly examined the changes in genes and metabolites involved in various amino acid metabolism/synthesis and determined core genes and metabolites through correlation networks. The phenylalanine, tyrosine, and tryptophan metabolic pathways and proline, glutamic acid and sulfur-containing amino acid pathways were particularly important for drought resistance. Some candidate genes, such as ProDH and P4HA family genes, and metabolites, such as O-acetyl-L-serine, directly affected up- and downstream metabolism to induce drought resistance. This study provided a basis for soybean drought resistance breeding.

Current views of drought research: experimental methods, adaptation mechanisms and regulatory strategies.

Drought stress is one of the most important abiotic stresses which causes many yield losses every year. This paper presents a comprehensive review of recent advances in international drought research. First, the main types of drought stress and the commonly used drought stress methods in the current experiment were introduced, and the advantages and disadvantages of each method were evaluated. Second, the response of plants to drought stress was reviewed from the aspects of morphology, physiology, biochemistry and molecular progression. Then, the potential methods to improve drought resistance and recent emerging technologies were introduced. Finally, the current research dilemma and future development direction were summarized. In summary, this review provides insights into drought stress research from different perspectives and provides a theoretical reference for scholars engaged in and about to engage in drought research.

The application potential of mepiquat chloride in soybean: improvement of yield characteristics and drought resistance.

BACKGROUND: Drought can result in yield losses, the application of plant growth regulators is an effective measure to improve drought resistance and yield. The objective of the study was to explore the application potential of mepiquat chloride (MC) in regulating soybean yield and drought resistance. METHODS: In this study, a three-year field experiment was designed and combined with drought experiments to measure the yield of popularized varieties during 2021-2022 and drought-resistant and drought-sensitive varieties were selected, and planted in the field in 2023. RESULTS: MC increased the yield of HN84 and HN87 for two consecutive years from 2021 to 2022 and improved their physiological characteristics under field conditions. Under M200 treatment, the yield of HN84 increased by 6.93% and 9.46%, and HN87 increased by 11.11% and 15.72%. Different concentrations of MC have different effects on soybeans. The maximum increase of SOD, POD and proline in HN84 under M400 treatment reached 71.92%, 63.26% and 71.54%, respectively; the maximum increase of SOD, POD and proline in HN87 under M200 treatment reached 21.96%, 93.49% and 40.45%, respectively. In 2023, the foliar application of MC improved the physiological characteristics of HN44 and HN65 under drought-stress conditions. On the eighth day of drought treatment, compared to the drought treatment, the leaf and root dry weight of HN44 under M100 treatment increased by 17.91% and 32.76%, respectively; the dry weight of leaves and roots of HN65 increased by 20.74% and 29.29% under M200 treatment, respectively. MC also reduced malondialdehyde (MDA) content, decreased antioxidant enzyme activity and proline content. In addition, different concentrations of MC increased the chlorophyll fluorescence parameters (Fs, Fv/Fm, YII, and SPAD). In the field, the plant height of the two varieties decreased significantly, the yield increased, the number of two-grain and three-grain pods increased, and the stem length at the bottom and middle decreased with MC induction. CONCLUSIONS: The application of 100-200 mg/L MC effectively improved drought resistance and increased yield. This study provided support for the rational application of MC in soybean production.

Molecular mechanism of drought resistance in soybean roots revealed using physiological and multi-omics analyses.

Soybeans are one of the most cultivated crops worldwide and drought can seriously affect their growth and development. Many studies have elucidated the mechanisms through which soybean leaves respond to drought; however, little is known about these mechanisms in roots. We used two soybean varieties with different drought tolerances to study the morphological, physiological, and molecular response mechanisms of the root system to drought stress in seedlings. We found that drought stress led to a significant decrease in the root traits and an increase in antioxidant enzyme activity in the two varieties. Drought-resistant varieties accumulate large amounts of flavonoids and phenolic acids at the metabolic level, which causes variations in drought resistance. Additionally, differences in gene expression and drought-resistance pathways between the two varieties were clarified using transcriptome analysis. Through a multi-omics joint analysis, phenylpropanoid and isoflavonoid biosynthesis were identified as the core drought resistance pathways in soybean roots. Candidate genes and marker metabolites affecting drought resistance were identified. The phenylpropanoid pathway confers drought tolerance to roots by maintaining a high level of POD activity and mediates the biosynthesis of various secondary drought-resistant metabolites to resist drought stress. This study provides useful data for investigating plant root drought responses and offers theoretical support for plant breeding for drought resistance.

Mechanism of Mepiquat Chloride Regulating Soybean Response to Drought Stress Revealed by Proteomics.

Soybeans are the main sources of oil and protein for most of the global population. As the population grows, so does the demand for soybeans. However, drought is a major factor that limits soybean production. Regulating soybean response to drought stress using mepiquat chloride (MC) is a feasible method; however, its mechanism is still unclear. This study used PEG-6000 to simulate drought stress and quantitative proteomic techniques to reveal changes in Heinong44 (HN44) and Heinong65 (HN65) subjected to drought following the application of 100 mg/L of MC. The results showed that SOD in HN44 did not change significantly but decreased by 22.61% in HN65 after MC pretreatment, and MDA content decreased by 22.75% and 21.54% in HN44 and HN65, respectively. Furthermore, MC improved the GSH-ASA cycle and simultaneously promoted the Calvin cycle process to enable the plant to maintain a certain carbon assimilation rate under osmotic stress. In addition, MC upregulated some proteins during gluconeogenesis and starch metabolism and increased soluble sugar content by 8.41% in HN44. MC also reduced ribosomal protein abundance, affecting translation and amino acid metabolism. In summary, MC improved GSH-ASA cycle and Calvin cycle under stress to alleviate oxidative damage and maintain crop growth. Our study is the first to report the mechanism of MC regulation in soybean under osmotic stress, providing new insights for the rational application of MC in soybean.

Comparing the Salt Tolerance of Different Spring Soybean Varieties at the Germination Stage.

Salinization is a global agricultural problem with many negative effects on crops, including delaying germination, inhibiting growth, and reducing crop yield and quality. This study compared the salt tolerance of 20 soybean varieties at the germination stage to identify soybean germplasm with a high salt tolerance. Germination tests were conducted in Petri dishes containing 0, 50, 100, 150, and 200 mmol L-1 NaCl. Each Petri dish contained 20 soybean seeds, and each treatment was repeated five times. The indicators of germination potential, germination rate, hypocotyl length, and radicle length were measured. The salt tolerance of 20 soybean varieties was graded, and the theoretical identification concentration was determined by cluster analysis, the membership function method, one-way analysis of variance, and quadratic equation analysis. The relative germination rate, relative germination potential, relative root length, and relative bud length of the 20 soybean germplasms decreased when the salt concentration was >50 mmol L-1, compared with that of the Ctrl. The half-lethal salt concentration of soybean was 164.50 mmol L-1, and the coefficient of variation was 18.90%. Twenty soybean varieties were divided into three salt tolerance levels following cluster analysis: Dongnong 254, Heike 123, Heike 58, Heihe 49, and Heike 68 were salt-tolerant varieties, and Xihai 2, Suinong 94, Kenfeng 16, and Heinong 84 were salt-sensitive varieties, respectively. This study identified suitable soybean varieties for planting in areas severely affected by salt and provided materials for screening and extracting parents or genes to breed salt-tolerant varieties in areas where direct planting is impossible. It assists crop breeding at the molecular level to cope with increasingly serious salt stress.

SNP application improves drought tolerance in soybean.

As an important bioactive molecule, nitric oxide (NO) can effectively alleviate the effects of drought stress on crops. However, it is still unclear whether it can increase the stress resistance of soybean. Therefore, in this study, our objective was to explore the effect of exogenous NO application on the physiological characteristics of soybean seedlings under drought stress. As test material, two soybean varieties, HN65 and HN44, were used, while sodium nitroprusside (SNP) of 100 µmol L-1, 200 µmol L-1, 500 µmol L-1, 1000 µmol L-1 served as an exogenous NO donor, and PEG-6000 as an osmotic regulator to simulate drought stress. The effects of irrigation with different SNP concentrations for different days on the physiological characteristics of the soybean seedlings under drought conditions were then investigated. The results obtained showed that the activities of antioxidant enzymes, osmotic regulator contents, as well as the abscisic acid and salicylic acid contents of the plant leaves increased with increasing SNP concentration and treatment time. However, we observed that excessively high SNP concentrations decreased the activities of key nitrogen metabolism enzymes significantly. This study provides a theoretical basis for determining a suitable exogenous NO concentration and application duration. It also highlights strategies for exploring the mechanism by which exogenous NO regulates crop drought resistance.

Regulation of soybean drought response by mepiquat chloride pretreatment.

Introduction: Soybean is the world's most important cultivated crop, and drought can affect their growth and, eventually, yields. Foliar application of mepiquat chloride (MC) can potentially alleviate the damage caused by drought stress in plants; however, the mechanism of MC regulation of soybean drought response has not been studied. Methods: This study investigated the mechanism of soybean drought response regulation by mepiquat chloride in two varieties of soybean, sensitive Heinong 65 (HN65) and drought-tolerant Heinong44 (HN44), under three treatment scenarios, normal, drought stress, and drought stress + MC conditions. Results and discussion: MC promoted dry matter accumulation under drought stress, reduced plant height, decreased antioxidant enzyme activity, and significantly decreased malondialdehyde content. The light capture processes, photosystems I and II, were inhibited; however, accumulation and upregulation of several amino acids and flavonoids by MC was observed. Multi-omics joint analysis indicated 2-oxocarboxylic acid metabolism and isoflavone biosynthetic pathways to be the core pathways by which MC regulated soybean drought response. Candidate genes such as LOC100816177, SOMT-2, LOC100784120, LOC100797504, LOC100794610, and LOC100819853 were identified to be crucial for the drought resistance of soybeans. Finally, a model was constructed to systematically describe the regulatory mechanism of MC application in soybean under drought stress. This study fills the research gap of MC in the field of soybean resistance.

Effects of Exogenous Sodium Nitroprusside Spraying on Physiological Characteristics of Soybean Leaves at the Flowering Stage under Drought Stress.

Nitric oxide (NO) plays a significant role in plant drought resistance. However, the effects of the exogenous application of NO to crops under drought stress vary within and among species. In this study, we explored the influence of exogenous sodium nitroprusside (SNP) on the drought resistance of soybean leaves in the full flowering stage using two varieties: drought-tolerant HN44 and non-drought-tolerant HN65. Spraying SNP on soybean leaves at the full flowering period under drought stress improved the NO content in soybean leaves. The activities of nitrite reductase (NiR) and nitrate reductase (NR) in leaves were affected by NO inhibition. The activity of antioxidant enzymes in leaves increased with the extension of SNP application time. Contents of osmomodulatory substances, including proline (Pro), soluble sugar (SS), and soluble protein (SP) increased gradually with the extension of SNP application time. The malondialdehyde (MDA) content decreased as the NO content increased, thus reducing membrane system damage. Overall, spraying SNP reduced damage and improved the ability of soybean to cope with drought. This study explored the physiological changes of SNP soybean under drought stress and provided theoretical basis for improving drought-resistant cultivation of soybean.

Corrigendum: Mepiquat chloride inhibits soybean growth but improves drought resistance.

[This corrects the article DOI: 10.3389/fpls.2022.982415.].

Effects of Weak and Strong Drought Conditions on Physiological Stability of Flowering Soybean.

Soybean is an important food crop in the world. Drought can seriously affect the yield and quality of soybean; however, studies on extreme drought-weak and strong-are absent. In this study, drought-tolerant soybean Heinong 44 (HN44) and sensitive soybean Heinong 65 (HN65) were used as the test varieties, and the effects of strong and weak droughts on the physiological stability of soybean were explored through the drought treatment of soybean at the early flowering stage. The results showed that the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anions (O2·-) increased with the increase in the degree of drought. The plant height and relative water content decreased, and photosynthesis was inhibited. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and the total antioxidant capacity (T-AOC) showed a trend of first increasing and then decreasing. Through contribution analysis, CAT changed the most, and the role of SOD gradually increased with the aggravation of drought. With the aggravation of drought, the contents of soluble sugar (SSC) and proline (Pro) increased gradually, and the content of soluble protein (SP) increased initially and then decreased. According to contribution analysis, SSC had the highest contribution to osmotic adjustment. SSC and Pro showed an upward trend with the aggravation of drought, indicating that their role in drought was gradually enhanced.

Effects of Different Drought Degrees on Physiological Characteristics and Endogenous Hormones of Soybean.

Drought affects crop developmentnand growth. To explore the physiological effects of drought stress on soybean, HeiNong44 (HN44) and HeiNong65 (HN65) varieties were used as experimental materials and PEG-6000 was used as the osmotic medium. The antioxidant enzyme activity, osmotic adjustment substance content, antioxidant capacity, and endogenous hormone content of the two soybean varieties were studied under different drought degrees and different treatment durations. Drought stress caused significant physiological changes in soybean. The antioxidant enzyme activities, osmoregulation substance content, and total antioxidant capacity (T-AOC) of HN65 and HN44 showed an increasing trend under mild and moderate drought, however, they first increased and then decreased under severe drought conditions. Following the extension of treatment time, malondialdehyde (MDA) showed an increasing trend. As drought increased, gibberellin (GA) content showed a decreasing trend, while abscisic acid (ABA), salicylic acid (SA), and zeatin nucleoside (ZA) content showed an increasing trend. The auxin (IAA) content of the two varieties showed opposite change trends. In short, drought had a significant impact on the physiology of these two soybean varieties; however, overall, the drought resistance of HN65 was lower than that of HN44. This study provides a research theoretical basis for addressing the drought resistance mechanism and the breeding of drought resistant soybean varieties.

Mepiquat chloride inhibits soybean growth but improves drought resistance.

Soybeans are an important economic crop. As the most widely used growth regulator globally, the molecular mechanism of mepiquat chloride (DPC) in soybean remains unknown. In this study, RNA sequencing technology combined with ultra-performance liquid chromatography and tandem mass spectrometry were used to analyze the changes in the leaf transcriptome and metabolomics of soybean leaves at the seedling stage under DPC stress. The results showed that differentially expressed genes related to photosynthesis and cell wall synthesis were significantly downregulated at the transcriptional level. In addition, the syntheses of gibberellin, zeatin, brassinolide, and other plant hormones were inhibited in the signal transduction pathway of plant hormones, thereby inhibiting plant growth. In contrast, at the metabolic level, the expression levels of flavonoid differential metabolites were significantly increased, and the proportions of flavonoids in the two varieties were 61.5 and 66%, respectively. The combined analysis of transcriptome and metabolomics showed that the differential expressed genes and metabolites were mainly enriched in the isoflavonoid biosynthesis and flavonoid biosynthesis pathways. Principally, DPC inhibited plant growth but improved drought resistance. Our study is the first to report the molecular mechanism of DPC regulation in soybean, providing useful insights into the rational application of DPC in soybean.

Transcriptomic and Metabolomic Analysis of Seedling-Stage Soybean Responses to PEG-Simulated Drought Stress.

Soybean is an important crop grown worldwide, and drought stress seriously affects the yield and quality of soybean. Therefore, it is necessary to elucidate the molecular mechanisms underlying soybean resistance to drought stress. In this study, RNA-seq technology and ultra-performance liquid chromatography-tandem mass spectrometry were used to analyze the transcriptome and metabolome changes in soybean leaves at the seedling stage under drought stress. The results showed that there were 4790 and 3483 DEGs (differentially expressed genes) and 156 and 124 DAMs (differentially expressed metabolites), respectively, in the HN65CK vs. HN65S0 and HN44CK vs. HN44S0 comparison groups. Comprehensive analysis of transcriptomic and metabolomic data reveals metabolic regulation of seedling soybean in response to drought stress. Some candidate genes such as LOC100802571, LOC100814585, LOC100777350 and LOC100787920, LOC100800547, and LOC100785313 showed different expression trends between the two cultivars, which may cause differences in drought resistance. Secondly, a large number of flavonoids were identified, and the expression of Monohydroxy-trimethoxyflavone-O-(6″-malonyl)glucoside was upregulated between the two varieties. Finally, several key candidate genes and metabolites involved in isoflavone biosynthesis and the TCA cycle were identified, suggesting that these metabolic pathways play important roles in soybean response to drought. Our study deepens the understanding of soybean drought resistance mechanisms and provides references for soybean drought resistance breeding.

Physiology and metabonomics reveal differences in drought resistance among soybean varieties.

BACKGROUND: The soybean is an important food crop worldwide. Drought during the first pod stage significantly affects soybean yield, and understanding the metabolomic and physiological changes in soybeans under drought stress is crucial. This study identified the differential metabolites in initial pod stage soybean leaves under polyethylene glycol-simulated drought stress, using ultra performance liquid chromatography and tandem mass spectrometry, and the physiological indexes related to drought resistance. RESULTS: Physiologically, drought resistance also generates enzyme and antioxidant activity; levels of superoxide dismutase, peroxidase, and catalase first increased and subsequently decreased, while those of soluble sugar, soluble protein, malondialdehyde, and proline content increased in both varieties. The contents of CAT, proline and soluble sugar in Heinong 44 (HN44) were higher than those in Heinong 65 (HN65), and the contents of MDA were lower than those in HN65. In metabolomics, the OPLS-DA model was used to screen different metabolites. KEGG analysis showed that the two varieties resisted drought through different ways. Amino acid metabolism and lipid metabolism play a key role in drought resistance of the two varieties, respectively. TCA cycle was one of the core pathways of drought resistance in two varieties. Changes in the content of L-Asparagine and citric acid may be one of the reasons for the difference in drought resistance between the two varieties. CONCLUSIONS: We think that the reasons of drought resistance among soybean varieties are as follows: the main metabolic pathways are different under drought stress; the contents of metabolites in these metabolic pathways are different; some physiological indexes are different, such as MDA, CAT, proline content and so on. Our study enhances the understanding of the metabolomic soybean drought stress response and provides a reference for soybean drought resistance breeding.

Study on the Regulatory Effects of GA3 on Soybean Internode Elongation.

Excessive plant height is an important factor that can lead to lodging, which is closely related to soybean yield. Gibberellins are widely used as plant growth regulators in agricultural production. Gibberellic acid (GA3), one of the most effective active gibberellins, has been used to regulate plant height and increase yields. The mechanism through which GA3 regulates internode elongation has been extensively investigated. In 2019 and 2020, we applied GA3 to the stems, leaves, and roots of two soybean cultivars, Heinong 48 (a high-stalk cultivar) and Henong 60 (a dwarf cultivar), and GA3 was also applied to plants whose apical meristem was removed or to girded plants to compare the internode length and stem GA3 content of soybean plants under different treatments. These results suggested that the application of GA3 to the stems, leaves, and roots of soybean increased the internode length and GA3 content in the stems. Application of GA3 decreased the proportion of the pith in the soybean stems and primary xylem while increasing the proportion of secondary xylem. The apical meristem is an important site of GA3 synthesis in soybean stems and is involved in the regulation of stem elongation. GA3 was shown to be transported acropetally through the xylem and laterally between the xylem and phloem in soybean stems. We conclude that the GA3 level in stems is an important factor affecting internode elongation.

Nitrate Transport and Distribution in Soybean Plants With Dual-Root Systems.

Nitrate absorbed by soybean (Glycine max L. Merr.) roots from the soil can promote plant growth, while nitrate transported to nodules inhibits nodulation and nodule nitrogen fixation activity. The aim of this study was to provide new insights into the inhibition of nodule nitrogen (N) fixation by characterizing the transport and distribution of nitrate in soybean plants. In this research, pot culture experiments were conducted using a dual root system of soybeans. In the first experiment, the distribution of 15N derived from nitrate was observed. In the second experiment, nitrate was supplied-withdrawal-resupplied to one side of dual-root system for nine consecutive days, and the other side was supplied with N-free solution. Nitrate contents in leaves, stems, petioles, the basal root of pealed skin and woody part at the grafting site were measured. Nitrate transport and distribution in soybean were analyzed combining the results of two experiments. The results showed that nitrate supplied to the N-supply side of the dual-root system was transported to the shoots immediately through the basal root pealed skin (the main transport route was via the phloem) and woody part (transport was chiefly related to the xylem). There was a transient storage of nitrate in the stems. After the distribution of nitrate, a proportion of the nitrate absorbed by the roots on the N-supply side was translocated to the roots and nodules on the N-free side with a combination of the basal root pealed skin and woody part. In conclusion, the basal root pealed skin and woody part are the main transport routes for nitrate up and down in soybean plants. Nitrate absorbed by roots can be transported to the shoots and then retranslocated to the roots again. The transport flux of nitrate to the N-free side was regulated by transient storage of nitrate in the stems.

Physiological Response of Soybean Plants to Water Deficit.

Soybean is an important cash crop in the world, and drought is the main reason for the loss of soybean plants productivity, with drought stress during the most water-sensitive flowering period of soybeans. In this article, drought-tolerant variety Heinong 44 (HN44) and drought-sensitive variety Heinong 65 (HN65) were used as experimental materials. Drought treatment was carried out at the early flowering stage. The method of controlling soil moisture content was used to simulate different degrees of drought, and the physiological changes of these two varieties of soybean under different soil moisture contents were studied. The results showed that with a decrease in soil moisture content, the content of malondialdehyde (MDA) in soybean leaves increased significantly; the activities of peroxidase (POD), catalase (CAT), and ascorbic acid peroxidase (APX) increased first and then decreased; the content of proline, soluble sugar, and soluble protein increased; and the total antioxidant capacity (T-AOC) increased significantly. When the soil moisture content was 15.5%, the degree of membrane lipid peroxidation, osmotic regulatory substances, antioxidant enzyme activity, and T-AOC increased the most, and the decrease in drought-tolerant variety HN44 was significantly less than that of drought-sensitive variety HN65. Our research reveals the response law of soybean crops to physiological characteristics under water deficit and provides theoretical basis and guiding significance for drought-resistant cultivation and breeding of soybean.

A study on soybean responses to drought stress and rehydration.

To investigate soybean responses to drought stress and growth through metabolism compensation after rehydration, and for the establishment of an optimal water-saving irrigation model, we used the soybean variety Suinong 14 as experimental material and adopted a weighing method for water control in potted plants. We exposed soybean plants to stress treatments at different growth stages using different stress levels and durations. We then studied the effects of drought stress and rehydration on soybean growth and development, osmoregulation, and endogenous hormonal regulations, as well as antioxidant systems. The results showed that drought stress inhibited increases in the soybean plant height and leaf area. This inhibition became more significant as the level, duration, and frequency of the drought stress increased. After rehydration, the soybean plant heights and leaf areas exhibited rapid increases and partial compensation for their decreased sizes. As the level, duration, and frequency of drought stress increased, the compensation effect decreased, but it did not return to the control level. Drought stress reduced the chlorophyll content and relative water content in the soybean leaves and increased the osmolyte contents, antioxidant potential, and peroxidation of the membrane lipids. In addition, the changes mentioned above became more dramatic as the drought stress level, duration, and frequency increased. Upon rehydration, various levels of growth compensation were observed in each physio-biochemical parameter. As the drought stress level, duration, and frequency increased, the compensation effect also increased. Overall, the compensation effect for drought stress that occurred at the early growth stages was higher than that at the later growth stages. Drought stress led to decreases in the ZR/IAA and ZR/ABA ratios in soybean leaves and an increase in the ABA/(IAA + GA + ZR) ratio; thus, the plant growth was inhibited. These hormone ratios exhibited more dramatic changes when the drought stress level became more severe and the stress duration was prolonged. After rehydration, these hormone ratios produced equal compensation effects. Therefore, the compensatory effect of rewatering after drought stress is conditional. Severe stress, especially long-term severe stress, will reduce the compensatory effect. At the same time, drought resistance treatment at seedling stage can improve the adaptability and compensatory effect of re-drought at grain filling stage.

Transcriptome profilling analysis characterized the gene expression patterns responded to combined drought and heat stresses in soybean.

Heat and drought are the two major abiotic stress limiting soybean growth and output worldwide. Knowledge of the molecular mechanisms underlying the responses to heat, drought, and combined stress is essential for soybean molecular breeding. In this study, RNA-sequencing was used to determine the transcriptional responses of soybean to heat, drought and combined stress. RNA-sequencing analysis demonstrated that many genes involved in the defense response, photosynthesis, metabolic process, etc. are differentially expressed in response to drought and heat. However, 1468 and 1220 up-regulated and 1146 and 686 down-regulated genes were confirmed as overlapping differentially expressed genes at 8 h and 24 h after treatment, and these genes are mainly involved in transport, binding and defense response. Furthermore, we compared the heat, drought and the combined stress-responsive genes and identified potential new targets for enhancing stress tolerance of soybean. Comparison of single and combined stress suggests the combined stress did not result in a simple additive response, and that there may be a synergistic response to the combination of drought and heat in soybean.