Soybean is a globally important leguminous crop, famous for its rich oil and protein contents. This crop is ecologically important and has a tendency to improve the soil’s fertility due to the assistance of symbiotically associated bacteria. In terms of cultivated area, soybean comes at fourth place after wheat, rice, and maize and at first place among legumes (Sedibe et al., 2023). In the year 2020, it was cultivated on a 127-million-hectare area of the world, with an annual yield of 354 million tonnes (Staniak et al., 2023). The yield of soybean varies from year to year due to various sorts of environmental factors such as drought, heat, and salinity (Wójcik-Gront et al., 2022). The stress conditions, particularly drought and salinity, induce disturbances at the cellular level causing secondary stresses, e.g., oxidative stress, owing to the generation of reactive oxygen species (ROS) (Hasanuzzaman et al., 2020). Besides that, ROS damage the biochemical structure of cell membranes due to lipid peroxidation and protein denaturation (Feng et al., 2023). Plants are naturally equipped with an antioxidant system to scavenge the ROS (Khan et al., 2009). In this regard, a speedy change in the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) is an indicator of plant tolerance to oxidative stress (Wang et al., 2018). Furthermore, the abiotic stresses interrupt the physiological processes (photosynthesis) due to the damage of enzymes involved in chlorophyll synthesis (Rajput et al., 2021). Like all living organisms, plants also have a tendency to counter the impacts of stress through different types of homeostatic mechanisms. In this context, plant increases the production of osmoprotectants like proline and glycine betaine (Wani et al., 2013; Xu et al., 2023). Besides that, the water status of plant changes due to abiotic stresses that further change the water potential and solute potential. Therefore, plants opt for some osmotic adjustments by producing some osmolytes (Wijewardana et al., 2019; Otie et al., 2021). These osmolytes play adaptive roles in plants in facilitating the osmotic adjustments and safeguarding the sub-cellular structures in stressed plants (Ding et al., 2024). On the other hand, salinity stress increases the accumulation of Na⁺ in plant cells (Jin et al., 2022). However, like all other living organisms, plants also respond, and as a homeostatic adjustment, plants enhance the efflux of Na⁺ due to the influx of K⁺ (Sun et al., 2021). It is essential for a plant to maintain a low Na⁺/K⁺ ratio under saline conditions to carry out normal physiological and biochemical processes (Sun et al., 2019). Furthermore, soybean is an ideal system to understand the genetic dynamics of drought and salinity tolerance in plants. Plants’ tolerance to drought and salinity is a complex trait regulated by various genes; therefore, it is important to understand various regulatory mechanisms in association with the expression of genes involved in regulatory networks—for instance, under oxidative stress, the overexpressed GmSOD1, GmCAT1, and GmPOD1 enhance the activities of SOD, CAT, and POD, respectively, that accelerate the detoxification of ROS such as H₂O₂ and O₂ ^.- (Li et al., 2020; Xu et al., 2023). The enzyme pyrroline-5-carboxylate synthase is involved in the synthesis of pyrroline-5-carboxylate (P5CS), a key precursor in proline synthesis exhibiting a protective role during drought and salinity stress (Xu et al., 2023). Besides that, the sodium and proton exchanger (NHX) mediates the efflux of Na⁺ through regulating the expression of genes, while AKT1 triggers the influx of K⁺ to balance the Na⁺/K⁺ ratio under saline conditions (Sun et al., 2019; Wang et al., 2021). Furthermore, Sun et al. (2019) have found that apart from regulating the efflux of Na⁺, GmNHX1 regulates the expression of a series of other genes including SKOR, SOS1, and AKT1 involved in salinity tolerance. Moreover, Wang et al. (2021) reported the essential role of GmAKT1 in the uptake of K⁺ to balance the Na⁺/K⁺ ratio under saline conditions. Furthermore, drought stress triggers the expression of dehydration-responsive element binding protein (DREB) that regulates the expression of other genes imparting drought tolerance in soybean (Zhou et al., 2022). The auxin-responsive factors (ARF) are also responsive to drought stress and regulate biochemical processes to enhance the plants’ drought tolerance potential (Ha et al., 2015). To date, various studies have been conducted to investigate the potential impact of drought and salt stress on soybean based on physiological and biochemical indicators; however, limited knowledge is available on physio-chemical changes, oxidative stress, osmolytic dynamics, and respective genetic control. The present study intended to elucidate the impacts of individual and combined treatments of drought and salt stress on the physiological, biochemical, and genetic indicators of stress tolerance in different soybean cultivars for a comparative understanding of the dynamics of stress tolerance.

The present study was conducted in a glass house located at the experimental site of King Abdulaziz University, Jeddah, Saudi Arabia, 21°32′36″ N and 39°10′22″ E, at 12 m above sea level. Six different soybean cultivars—Rawal-1, Swat-84, NARC-1, and NARC-21 collected from National Agricultural Research Center (NARC), Islamabad, Pakistan, and G4620RX and DM45X61 collected from USA—were evaluated for salinity and drought tolerance. The tri-replicate experiment was conducted in a randomized complete block design (RCBD) using factorial arrangements with soybean cultivars as one factor and with drought and salinity treatments as second factor.

Soil drought and salinity are potential stresses restricting plant productivity. The impacts of drought and salinity on plants ranges from morpho-physiological adaptations to biochemical and molecular responses. The responses of plants to the twin abiotic stresses are a bit unique such that they are difficult to predict in isolation. The initial responses of plants to drought and salt stress are primarily similar as both perturb physiological processes and osmotic balances. The twin drought and salt stress directly affect Pn, Gs, and chl due to biochemical perturbances such that they produce intense secondary oxidative stress compared with the isolated application of stress. In short, combined drought and salt stress causes additional adverse effects on plants’ physiological and osmotic traits. Therefore, all soybean cultivars showed more drop in Pn, Gs, chl, RWC, and water potential and more rise in proline, GB, O₂ ^•−, H₂O₂, and antioxidant activities under the twin application of compared with the sole application of drought and salt stress. During stress, the enhanced levels of ROS show signaling function in addition to the synthesis of antioxidant enzymes. Plants are naturally equipped with protective responses including stomatal closure, activating ROS scavenging, stopping photosynthesis, and activating the expression of stress-related genes. Numerous research studies have found that coupled water and salt stress has more adverse effects on plants (Angon et al., 2022; Cao et al., 2023; Fu et al., 2023). Besides that, high Na⁺/K⁺ ratio was recorded at salt and combined drought–salt stress, illustrating that water deficiency does not increase the deposition of Na in plants. Moreover, plant experiences more oxidative stress under combined drought–salinity stress compared with the isolated application of stresses as indicated by the high production of O₂ ^•− and H₂O₂. Correspondingly, the high activities of antioxidant enzymes were recorded at the twin application of stress compared with sole stresses probably due to ROS scavenging mechanisms triggered due to the overproduction of ROS. In addition, the genes controlling the biochemical traits of stress tolerance were regulated differently under the sole and coupled applications of drought and salt stress. Overall, the integrated application of drought and salt stress depicted a high level of change in all physiological, biochemical, and indicators of stress tolerance.

Plants face oxidative stress due to the generation of reactive oxygen species such as hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^•−), which disrupts the structural integrity of membranes present in different organelles (Shah et al., 2017; Dos-Santos et al., 2022; Juan et al., 2021; Sachdev et al., 2021; Zhang et al., 2022). The abiotic stresses partially or wholly impede the vital physiological processes, including Gs and Pn, that are essential for plant survival (Ding et al., 2024). The coupled drought–salinity stress has an additional adverse effect on chlorophyll compared with individual stress, hence causing a high reduction in Pn and Gs compared to individual stress as reported by Otie et al. (2021) and Zhou et al. (2022) in soybean under salt and water stress, respectively. Similarly, the present study found a substantial reduction in chl, Gs, and Pn under combined drought–salt stress compared with individual stresses in all soybean genotypes ( Figure 1 ). Generally, the levels of compatible solutes glycine betaine and proline increase additionally when the plant is exposed to multiple stresses (Wani et al., 2013). The compatible solutes are non-toxic at a high concentration and serve as osmoprotectant to protect the plant from multiple stresses in different ways, such as cellular osmotic adjustment, retention of membrane integrity, and detoxification of ROS as reported by Xu et al. (2023). Figure 2 likewise illustrates the dynamic rise in the concentration of proline and GB under twin drought–salt stress in all soybean genotypes. Furthermore, the high proline and GB contents in soybean genotypes G4620RX, DM45X61, and NARC-21 compared with those in Rawal-1, NARC-1, and Swat-84 under coupled and individual drought and salinity treatment were an indicator of their strong molecular mechanism to counter the stress ( Figure 2 ). Moreover, the leakage of K⁺ and influx of Na⁺ is possibly due to ROS production under water-deficit and salt-elevated conditions as confirmed through various studies (Feng et al., 2023; Ali and Rab, 2017; Demidchik and Maathuis, 2007; Wu, 2018). Besides that, salinity triggers the influx of Na⁺ and leakage of K⁺ that lead toward a high Na⁺/K⁺ ratio causing leaf necrosis, pigment degradation, and disruption of water and osmotic potentials (Otie et al., 2021). Parallel with these findings, the current study reported a significant increase in Na⁺/K⁺ in all soybean genotypes due to saline and drought conditions ( Figure 2 ). Conversely, the soybean genotypes G4620RX, DM45X61, and NARC-21 illustrated a lesser increase in Na⁺/K⁺ ratio even under saline stress, which is attributed to their high salt tolerance and speedy influx of K⁺ as explained by Ali and Rab (2017). Furthermore, plants possess various enzymatic and non-enzymatic processes to detoxify the harmful effect of oxidative stress imposed by ROS produced as a consequence of sole and combined drought–salinity stress (Liu et al., 2017; Hasanuzzaman et al., 2020). As the coupled drought–salt stress poses an additional oxidative stress, it therefore necessitates a comparatively high catalytic activity of antioxidant enzymes as reviewed by Cao et al. (2023). Hence, an increase in the catalytic activity of antioxidant enzymes CAT, SOD, and POD indicates the activation of the ROS scavenging mechanism (Rajput et al., 2021; Kesawat et al., 2023). The reduction in H₂O₂ and superoxide (O₂ ^•−) concentration owing to the enhanced activity of antioxidant enzymes is an indicator of tolerance to oxidative stress as reported by Khan et al. (2009) and Liu et al. (2017) in soybean. Similarly, in the present study, the soybean genotypes G4620RX, DM45X61, and NARC-21 manifested a high activity of SOD, POD, and CAT for ROS (H₂O₂ and O₂ ^•−) scavenging under coupled drought–salt stress compared with their individual application ( Figure 3 ). This proves the high biochemical tolerance of these genotypes compared with Rawal-1, NARC-1, and Swat-84. Under abiotic stresses, plants can likewise alter water relation in order to retain various cellular functions (Zhang et al., 2023)—for instance, plants undergo osmotic adjustment through the accumulation of compatible osmolytes such as glycine betaine (GB) and proline (Doğan, 2011; Akitha et al., 2015; Ghosh et al., 2021). These osmotic adjustments intimate the plant to keep the cell volume at low water potential (ψ_w), which is vital to maintain the metabolic functions (Ghosh et al., 2021). Besides that, the high RWC, increasing osmotic potential (ψ_s), and decreasing water potential (ψ_w) under drought and salinity stress illustrate the crop’s high tolerance to drought and salinity stress as reported by Wijewardana et al. (2019) and Otie et al. (2021), respectively, in soybean. The present study has further confirmed their findings and reported high RWC, high osmotic potential (ψ_s), and low water potential (ψ_w) in soybean genotypes G4620RX, DM45X61, and NARC-21, exhibiting comparatively high physiological and biochemical tolerance under drought and salinity stress ( Figure 4 ). In plants, the physiological and biochemical traits are strongly associated, and their correlation varies according to the type of genotype and nature of stress (Liu et al., 2017; Vital et al., 2022; Ding et al., 2024). Moreover, the extent of physiological and biochemical responses under drought and salinity varies due to the varying tolerance tendency of soybean genotypes as shown in Figures 5 – 7 . Besides that, the heatmap analysis has further confirmed the varying intensity of expression of each trait in all soybean genotypes under individual and integrated levels of drought and salinity stress ( Figure 8 ). To date, various studies have been conducted to investigate the impacts of drought and salinity on the physiological and biochemical processes of soybean, but studies investigating the impacts on oxidative stress, physio-chemical changes, osmolytic dynamics, and genetic indices are limited. Although stress tolerance mechanisms vary from crop to crop, basic cellular responses to abiotic stresses are almost conserved in most of the plant species (Zhang et al., 2023)—for example, different abiotic stress elements trigger oxidative stress, protein denaturation, and osmotic stress in plants, which result in identical adaptive strategies such as production of stress proteins, induction of ROS-scavenging mechanisms, and accumulation of compatible osmolytes (Sachdev et al., 2021). Therefore, it is necessary to understand the physiological and biochemical markers of stresses with respect to their genetic determinants. Besides that, the genes GmCAT1, GmSOD, and GmPOD1 detoxify ROS through regulating the activities of antioxidant enzymes CAT, SOD, and POD, respectively. The antioxidant enzyme SOD converts superoxide (O₂ ^•−) to H₂O₂, which is further detoxified to O₂ and H₂O due to the catalytic activities of CAT and POD (Wang et al., 2018). Li et al. (2020) noticed the higher transcript of GmCAT1, GmSOD, and GmPOD1 in tolerant soybean genotypes under oxidative stress imposed by high aluminum content that substantially lowered the concentration of ROS, including H₂O₂ and superoxide radical (O₂ ^•−). Correspondingly, the current study reported the significant upregulation of GmCAT1, GmPOD1, and GmSOD in soybean genotypes G4620RX, DM45X61, and NARC-21 along with the increased catalytic activities of antioxidant enzymes CAT, POD, and SOD that caused a significant decline in ROS (H₂O₂ and O₂ ^•−) under oxidative stress imposed by sole and twin drought and salt stress ( Figure 9 ). This has confirmed the potential role of genetic determinants in regulating the antioxidant potential of soybean genotypes through regulating the biochemical mechanisms. Xu et al. (2023) confirmed that GmP5CS is a downstream gene of GmCOL1a whose overexpression improves the salt tolerance and drought resistance in soybean due to an increase in proline content, RWC, and CAT, POD, and SOD catalytic activities. Similar to these findings, the current study recorded a parallel increase in the expression of GmP5CS along with RWC, proline concentration, and enzymatic (SOD, POD, and CAT) activities in soybean genotypes G4620RX, DM45X61, and NARC-21, showing more physiological and biochemical tolerance under isolated and combined versions of drought and salinity stresses ( Figure 9 ). Furthermore, Jin et al. (2022) and Sun et al. (2021) found that NHX gene improves the salt tolerance tendency of soybean by decreasing the Na⁺ content in the cytoplasm or by keeping a low Na⁺/K⁺ ratio. Besides that, Sun et al. (2019) has further confirmed that, in addition to mediating the efflux of Na⁺, GmNHX1 regulates the expression of a series of other genes, including SKOR, SOS1, and AKT1, involved in salinity tolerance. Moreover, Wang et al. (2021) reported the essential role of GmAKT1 in the uptake of K⁺ to balance the Na⁺/K⁺ ratio under saline conditions. Complementary to these findings, the current study has recorded a significant increase in the uptake of K due to the increased expression of GmNHX1 and GmAKT1 in the genotypes G4620RX, DM45X61, and NARC-21 as evidenced by their decreased Na⁺/K⁺ ratio compared with Rawal-1, Swat-84, and NARC-1 ( Figure 9 ). In fact, the molecular crosstalk of GmAKT1 with plant phytohormones triggers the essential physiological mechanisms providing soybean with tolerance against abiotic stress (Wang et al., 2021). The overexpression of GmDREB1 confers soybean with tolerance against drought stress as reported by Zhou et al. (2022) and Chen et al. (2022). The current study likewise recorded a consistent increase in the expression of GmDREB1 in soybean cultivars G4620RX, DM45X61, and NARC-21, showing high physiological and biochemical tolerance to drought and salt stress ( Figure 9 ). This can be attributed to the tendency of GmDREB1 to regulate the expression of osmotic and oxidative stress-related protein that reduces the cell injury caused by salt and drought stress, thus improving the stress tolerance of soybean (Jiang et al., 2014; Kidokoro et al., 2015). Besides that, Ha et al. (2015) recorded the potential role of GmARFs in inducing drought tolerance in soybean through modulating the interaction of auxins with other hormones. Correspondingly, the current study confirmed their findings and found a dynamically increasing expression of GmARF1 in genotypes G4620RX, DM45X61, and NARC-21 showing physiologically and biochemically high tolerance against drought stress ( Figure 9 ).

In the present study, combined drought and salt stress altered soybean’s physiological, biochemical, genetic, and osmotic responses more intensively rather than their individual application. This proves that simultaneous exposure of plants to multiple abiotic stresses causes a severe manipulation in plant traits; however, the extent of plant trait manipulation strictly adheres with the plant’s ability to withstand stress. Furthermore, the present study also revealed that tolerance to abiotic stresses is undoubtedly an intricate phenomenon at the cellular and whole plant levels. In fact, this is due to the complex interaction between stress elements and different physiochemical and molecular mechanisms determining the plant’s growth and developmental processes. Overall, the soybean genotypes G4620RX, DM45X61, and NARC-21 depicted a high tolerance to the combined and individual applications of salt and drought stresses based upon physiological and molecular mechanisms. As the development of stress-tolerant crops requires knowledge on the contributing physiological and biochemical processes and the genetic control of participating traits, the present study will hence provide a considerable insight to elucidate the molecular dynamics of abiotic stress tolerance in soybean. Besides that, it will further contribute in devising soybean breeding strategies against drought and salt stresses by focusing on physio-chemical and genetic dynamics in unison.