Exploring Drought Responses in Mexican Soybeans: Plant Water Status, Shoot and Root Biomass, and Root System Architecture

Abstract

ABSTRACT

Drought stress is the primary abiotic threat to soybean production. Drought tolerance mechanisms in Mexican soybeans have not yet been fully studied. We examined the responses of three Mexican soybean genotypes, with varying maturities, to progressive drought stress and recovery irrigation, regarding plant water status, biomass traits, root system architecture, and their relationships. Genotypes I1240, I700 (intermediate maturity), and E2309 (early maturity) were examined since they showed drought tolerance characteristics under field conditions in previous studies. Drought stress was applied at the R2 growth stage for 17 days, after that irrigation was restored. Control plants were irrigated normally throughout the study. Responses to severe drought stress were different depending on the genotype. Early genotype E2309 exhibited a wider root system and tolerated drought stress maintaining shoot biomass and root characteristics at a low plant water potential. Intermediate genotypes, I1240 and I700, avoided drought stress achieving a greater root system at a higher plant water potential. Due to drought, genotype I1240 showed a more prominent increase in root diameter; meanwhile, root biomass was not decreased in genotype I700. Genotype I700 displayed a longer root system with the highest root‐to‐shoot ratio and recovered root growth more efficiently after recovery irrigation. General effects also indicated a longer root system in this genotype. Therefore, it could be used as a genetic source to improve root traits to cope with drought. Morphological and biomass root traits are suitable for selecting drought stress–tolerant soybean genotypes.

Introduction

Soybean [Glycine max (L.) Merr.] stands as an essential oilseed crop of global significance, holding profound implications for human and livestock sustenance. In 2023/2024, global soybean production was 398 million metric tons; Brazil, the United States, and Argentina are the foremost contributors, accounting for 39%, 28%, and 13% of the global output, respectively (USDA 2024). In the Mexican context, soybean production is mainly concentrated within states encompassing Tamaulipas, Chiapas, Campeche, Veracruz, and San Luis Potosi. On an annual basis, the average grain production was around 364 thousand metric tons in the 2015/2021 period (SIAP 2024). Nonetheless, a concerning trend has emerged in recent times wherein soybean production has faced a decline, and it was 176 thousand metric tons in 2022 (SIAP 2024), attributed primarily to diminished precipitation within the cultivating regions.

Climate change is a worldwide phenomenon, increasing the frequency of drought events in different agricultural regions. Drought, characterized by significantly reduced precipitation in a determined period, is the principal abiotic impediment to soybean production, limiting soil moisture content and hindering plant water-acquisition capabilities (Mishra and Singh 2010). The impacts of drought on plants depend on factors such as stress intensity, duration, crop growth stage, and genotype, as reported by Gray and Brady (2016), Anjum et al. (2017), and Kapoor et al. (2020). Soybean plants that undergo drought during the reproductive stage impose considerable limitations on plant growth and developmental processes (Du et al. 2020). Water deficiency in soil profoundly impacts the water status and the morphological features of plants, both below and above ground. This includes alterations in relative water content (Waqar, Bano, and Ajmal 2022); a decrease in plant height, leaf area (Dong et al. 2019), shoot dry weight (Stolf-Moreira et al. 2010; Du et al. 2020), and root fresh weight (Waqar, Bano, and Ajmal 2022); stunted root length (Hossain et al. 2014); and a decrease in fertile pod count (Mejaya et al. 2022). Therefore, soybean grain yield is compromised (Chandra et al. 2021).

Roots play a crucial role in the growth and yield of crop plants as they extract water and nutrients from the soil. They are also the first organs to sense and respond to drought stress, making them vital for plant survival. Consequently, root architecture significantly impacts crop growth and yield under water-deficit conditions (Fenta et al. 2014). Therefore, root phenotypes that penetrate the soil deeply and achieve greater root biomass (Ali et al. 2016; Lopes et al. 2011) or roots with wider xylem diameters and larger lateral root systems containing more root hairs (Tanaka et al. 2014; Vadez 2014) present advantages when faced with water scarcity situations. However, soybean breeding programs rarely incorporate root characteristics as a selection criterion to improve tolerance to drought (Falk et al. 2020). Because of that, persistent endeavors are necessary to employ phenomics tools to study variations in root system architecture and use the best root types in crop breeding and research programs (Mairhofer et al. 2013).

Certain studies found that drought-tolerant soybean genotypes showed substantial improvements in leaf expansion, water potential, root length, and biomass compared with susceptible genotypes when exposed to progressive drought stress and subsequent recovery irrigation (Hossain et al. 2014). In India, four soybean genotypes: EC538828, JS97-52, EC456548, and EC602288 presented greater drought avoidance (Jumrani and Bhatia 2019). This was evident from their deep root systems, high root-to-shoot ratios, and specific leaf weights under water-deficit conditions at the R5 growth stage. Another study carried out in South Africa indicated that the Jackson and Prima 2000 genotypes exhibited greater root length, surface area, volume, and number of root tips under drought stress for 30 days at the V3 growth stage compared with the A-5409RG genotype (Fenta et al. 2014). However, only Prima 2000 displayed superior shoot biomass and yield, indicating that it was the most drought-tolerant soybean genotype. In the meantime, of the 20 soybean accessions tested at the seedling stage with a 10% PEG 6000 concentration, nine demonstrated tolerance to simulated drought based on growth, physiological traits, and root system architecture (Esan, Obisesan, and Ogunbode 2023).

In Mexico, García-Rodríguez et al. (2017) evaluated the response to drought stress in soybean plants of 25 genotypes (with varying maturities) under field conditions. Drought stress was applied 30 days after planting. Under these conditions, drought significantly diminished the number of pods m⁻¹ and the weight of 100 seeds, reducing yield. However, later maturity genotypes experienced greater yield reductions due to drought than the earlier ones. Drought-tolerant cultivars, H02-2295 (early), H98-1240 (intermediate), and H10-0556 (late), showed the highest yield values due to the main effect of the genotype and consistent performance in water-deficit and well-watered conditions. Early genotypes such as H02-2309, H02-2348, Huasteca 300, and H98-1324 showed no significant change in plant height or chlorophyll content because of drought (García-Rodríguez 2014). The commercial soybean variety Huasteca 700 (intermediate), also known as genotype H02-1656, was highlighted in yield by the main effect of the genotype in the same study. Huasteca 700 is one of the major soybean varieties cultivated in Mexico, showing good growth and recovery after natural drought stress according to the farmers´ observations.

Little is known about the responses to drought in soybeans under a gradual water-deficit period, as in nature. Additionally, the contribution of phenotypic variations such as plant water status, shoot and root biomass, and root system architecture to drought tolerance in Mexican soybeans has not been fully studied. We suppose that drought-tolerant Mexican soybean genotypes can develop root system architectures capable of reaching water in deeper soil layers. According to the previous studies of García-Rodríguez (2014) and García-Rodríguez et al. (2017), in the present research work, the responses to progressive drought stress and recovery irrigation of three Mexican soybean genotypes were examined. The root system architecture, shoot and root biomass, plant water status, and their relationships were considered. This work also aimed to identify root system architecture traits suitable for screening drought-tolerant soybean genotypes.

Materials and Methods

Plant Materials, Growth Conditions, and Experimental Design

Seeds of three Mexican soybean genotypes, one with early maturity (H02-2309, named in this work as E2309) and two with intermediate maturity (H98-1240 and Huasteca 700, named in this work as I1240 and I700, respectively), were provided by The Soybean Breeding Program of the National Institute of Forestry, Agriculture, and Livestock Research (INIFAP), Mexico. In this study, the early and intermediate genotypes reached the R2 growth stage 40 and 50 days after planting, respectively. Genotypes E2309 and I1240 correspond to advanced lines and were studied under field conditions showing drought tolerance characteristics (García-Rodríguez 2014; García-Rodríguez et al. 2017). Genotype I700 is the newest soybean variety developed by INIFAP (Maldonado-Moreno, Ascencio-Luciano, and García-Rodríguez 2019), and it has become one of the most widely cultivated varieties under rainfed conditions in Tamaulipas and Chiapas, Mexico, showing better restoration of growth after natural water stress than other soybean commercial varieties, according to farmers' empirical evidence.

Soybean seeds were disinfected with a 10% sodium hypochlorite solution for 3 min and thoroughly rinsed thrice with deionized water. After that, seeds were germinated in 72 cell-plastic trays using a general mix of 37.5% peat moss Sunshine mix 3, 25% leaf soil, 12.5% silt soil, 12.5% vermiculite, and 12.5% perlite. Ten days after planting, soybean seedlings were transplanted into 5-L plastic pots filled with a substrate composed of 60% sand soil, 30% silt soil, and 10% vermiculite. A total of 90 plants, 30 for each genotype, were grown under glasshouse conditions (average temperature of 23°C ± 1°C, relative humidity of 60% ± 4%, and natural photoperiod) during the summer growing season in Irapuato, Mexico (20°43′10.2″ N, 101°19′44.2″ W).

A factorial arrangement random block design with three replicates was used to evaluate the effects of water conditions on three Mexican soybean genotypes throughout the five sampling dates. The two water conditions were well-watered, regarded as a control treatment, and water deficit. Water deficit was imposed on half of the plants at the R2 growth stage (Fehr and Caviness 1977) by gradually reducing irrigation for 17 days, trying to simulate real drought conditions in the field. In Mexico, most droughts in the main soybean production region occur during the reproductive growth stages of crops (mid-summer drought). Later, recovery irrigation was applied for 8 days. Plants under well-watered conditions received regular irrigation every third day. The variables were measured at four sampling dates during the stress period: 0, 7, 13, and 17 days of water deficit and one at 8 days after recovery irrigation. A weekly dose of 300 mL plant⁻¹ of Hoagland solution (Hoagland and Arnon 1950) was administered as a nutrient source.

Determination of Soil and Plant Water Status

Soil water status was measured by calculating the gravimetric soil moisture. Soil samples were collected from the middle of each pot and weighed immediately. The samples were then dried in an oven at 37°C until constant weight was achieved. Gravimetric soil moisture was calculated by using the formula “mass of moist soil (g)-mass of dried soil (g)/mass of dried soil (g) × 100” (Voroney 2019). Plant water status was assessed by measuring water potential at the stem center of plants before sunrise, using a Scholander chamber (Soilmoisture Equipment Corp, Santa Barbara, CA, USA).

Evaluation of Shoot and Root Characteristics

Phenotypic characteristics such as shoot dry weight, root dry weight, root length, and root-to-shoot ratio were measured. Soybean plants were extracted from the pots and roots were separated from the shoots. Then, roots were washed thoroughly with deionized water to remove soil particles and dried with a paper towel to measure root length. Samples of roots and shoots were then dried at 60°C for 48 h, and shoot dry weight and root dry weight were determined. Root dry weight divided by shoot dry weight was used to calculate the root-to-shoot ratio. Before drying, root samples were photographed for root system architecture characterization.

Characterization of Root System Architecture

The washed root system of the plants was placed in a tray filled with water and photographed with a digital camera Realme 7 5G. The resultant images were converted to black and white, the background was removed, and the contrast was increased using Adobe Photoshop CS5 (Version 12.0) software (Ramírez-Flores et al. 2019). The processed images were scaled and then analyzed using a custom-made ImageJ plugin, Root Image Analysis-J (or RIA-J), within ImageJ/FIJI (Version 2.9.0) according to the procedure proposed by Lobet et al. (2017). We extracted a set of morphological root image variables comprising total root length, average root diameter, root projected area, and the number of root tips for each image. Additionally, we incorporated geometric variables including root width, root depth, width-to-depth ratio, and convex hull area.

Data Analysis

All statistical analyses were performed in the R statistical package (Version 4.3.2) (R Core Team 2023). A three-way ANOVA analysis using the basic functions of the package was conducted to determine the main effects of the factors and their interactions on each variable. Tukey's HSD test with a 95% confidence interval was applied using the “agricolae” package to distinguish the statistically significant differences between means. Significant sampling date × water deficit interactions were plotted. These interactions were considered to know the variables responding to different levels of drought stress throughout the study. A one-way ANOVA analysis was used to deduce the genotype response to water deficit when a significant sampling date × water condition interaction was observed, using Tukey's HSD test to compare the means of each treatment. The contribution of each variable to the variance under water deficit during progressive drought stress and after recovery irrigation was determined through a Principal Component Analysis using the “FactoMineR” and “factoextra” packages. The Pearson's correlation coefficient was calculated using the “cor” function to determine the degree of association between variables. In both cases, data were plotted using the “ggplot2” package.

Results

Soil and Plant Water Status Decreased by Irrigation Reduction

The significant sampling date × water condition interaction on the gravimetric soil moisture (Table S1) confirmed progressive drought stress throughout the experiment. Irrigation reduction in the water-deficit conditions led to a gradual decrease in the gravimetric soil moisture recording 11.5%, 8.5%, 5.1%, and 3.5% at 0, 7, 13, and 17 days of water deficit, respectively (Figure 1A). This allowed us to categorize mild, moderate, and severe drought stress at 7, 13, and 17 days of water deficit. The gravimetric soil moisture in the control treatment was maintained at around 11.0% during the study. Soil moisture under water-deficit conditions was restored after recovery irrigation reaching a gravimetric soil moisture of 11.5%.

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As shown in Figure 1B and according to significant sampling date × water condition interaction for plant water potential (Table S1), there is no response to mild drought stress. However, plant water potential was reduced by 90.0%, 129.1%, and 40.1% under moderate and severe drought stress and after recovery irrigation, respectively. We analyzed the effect of water deficit on the plant water potential of soybean genotypes at 13 and 17 days of water deficit and 8 days after recovery irrigation. All three genotypes showed similar reductions in plant water potential during moderate drought stress (Figure 2A). However, during severe drought stress, plant water potential was lower in genotype E2309 than in the intermediate genotypes (Figure 2B). Eight days after recovery irrigation, soybean genotypes recovered partially plant water potential. Plant water potential restoration was more efficient in genotype I1240 than in E2309 and I700 genotypes (Figure 2C), since the differences between well-watered and water-deficit conditions were 16.0%, 36.9%, and 100.0%, respectively.

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Shoot and Root Biomass Responded to Severe Drought Stress

Sampling date × water condition interaction was significant for biomass traits (Table S1). Severe drought stress triggered detrimental effects on shoot and root dry weight, which were not recovered after re-irrigation. Stressed plants under water-deficit conditions increased shoot dry weight from 0 to 13 days of water deficit but stopped growing between moderate and severe drought stress (Figure 3A). A rise in shoot dry weight was observed after recovery irrigation. On the other hand, root dry weight accumulation stopped at mild drought stress, restoring after re-irrigation (Figure 3B). Under well-watered conditions, shoot and root dry weight displayed a linear increase throughout the study, they reached higher values than under water-deficit conditions during severe drought stress and after re-irrigation (Figure 3A,B). Interestingly, the root-to-shoot ratio resulted in similar scores in both water conditions during the study (Figure 3C).

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Water-deficit conditions slightly reduced shoot dry weight at severe drought stress and after recovery irrigation in intermediate genotypes (Figure 4A,D). However, well-watered and water-deficit conditions resulted in similar root dry weight for genotype I700 (Figure 4B,E). Conversely, water deficit had a greater impact on root dry weight in the I1240 genotype. The E2309 genotype exhibited lower biomass at severe drought stress but shoot and root dry weight were similar between water conditions (Figure 4A,B). In the meantime, it reached lower shoot and root biomass due to water deficit after recovery irrigation (Figure 4D,E). No significant differences were observed in the root-to-shoot ratio during severe drought stress (Figure 4C). After re-irrigation, genotype I700 reached the highest root-to-shoot ratio under water deficit, indicated by a 5.4% increase related to the control plants (Figure 4E). Conversely, the root-to-shoot ratio was reduced by 12.9% in genotype E2309.

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The Average Root Diameter Increased But Root Growth Decreased Under Severe Drought Stress

It was found that the interaction between the sampling date and water condition was significant for average root diameter, root projected area, and number of root tips (Table S1). Root thickness and root growth behaved distinctively throughout the experiment. As depicted in Figure 3D, the average root diameter was similar in both water conditions throughout the study except for 17 days of water deficit. At that sampling date, the average root diameter increased by 29.7%. Conversely, the root projected area and the number of root tips displayed a similar increase in both water conditions from 0 to 13 days of water deficit (Figure 3E,F). Nonetheless, these traits were reduced by 35.8% and 28.3% during the severe drought stress. The root projected area and the number of root tips tended to recover after re-irrigation.

At 17 days of drought stress, the average root diameter increased in all three soybean genotypes under water-deficit conditions (Figure 5A). The average root diameter improved by 34.2% and 30.3% in the I1240 and I700 genotypes, respectively, which reached the highest scores. Conversely, the root projected area was reduced by 40.7% and 36.9%, respectively (Figure 5B). The number of root tips dropped by around 31.0% in both genotypes (Figure 5C). Although intermediate genotypes recorded greater root projected area and number of root tips than the E2309 genotype under water-deficit conditions, these characteristics were less reduced in the early genotype between well-watered and water-deficit conditions (Figure 5B,C), showing decreases of 21.6% and 16.3% for root projected area and number of root tips, respectively.

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Biomass and Root System Architecture Strongly Contributed to the Variance

We conducted a Principal Component Analysis to explore structure and relationships within our data under water-deficit conditions throughout the study. The first two principal components explained 67.2% of the variance (Figure 6 and Table S2). Principal Component 1 accounted for 51.2% of the variance and was influenced by shoot dry weight, root dry weight, total root length, root projected area, number of root tips, root width, and root depth (Figure 6 and Table S3). Principal Component 2 accounted for 16.0% of the variance and was influenced by gravimetric soil moisture, plant water potential, root-to-shoot ratio, average root diameter, and width-to-depth ratio (Figure 6 and Table S3). Intermediate genotypes were associated with Principal Component 1. They exhibited higher biomass and notable root system architecture characteristics at higher plant water potential. Meanwhile, the E2309 genotype was associated with the Principal Component 2. It was mainly characterized by higher width-to-depth and root-to-shoot ratios, and lower plant water potential. The I1240 and I700 genotypes overlapped when grouped by ellipses at a 95% confidence interval (Figure 6).

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Biomass and Morphological Root System Architecture Characteristics Were Strongly Correlated

As shown in Figure 7, the shoot and root biomass and morphological root system architecture characteristics showed the most significant correlations (p ≤ 0.001). The strongest positive correlations were displayed between the root projected area with root dry weight and the number of root tips (r ≥ 0.90).

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Other direct significant correlations (p ≤ 0.001) with an r = 0.64–0.86 were observed between root length with total root length and root depth; shoot dry weight with root dry weight, total root length, root projected area, number of root tips, and root width; root dry weight with total root length, number of root tips, root width, and root depth; total root length with root projected area, number of root tips, root width, and root depth; root projected area with root width and root depth; root width with number of root tips and root depth; and average root diameter with root depth. In contrast, inverse correlations with an r value between −0.69 and −0.88 were detected between the width-to-depth ratio with root length, average root diameter, and root depth (p ≤ 0.001).

It is worth noting that soil moisture was not correlated with shoot and root biomass and root system architecture. However, a moderate direct correlation between gravimetric soil moisture and plant water potential was observed (r = 0.61, p ≤ 0.001). On the other hand, the plant water potential was poorly correlated with root projected area, root width, and root depth (r = 0.35–0.43; p ≤ 0.05).

Discussion

This study revealed that Mexican soybean genotypes were tolerant to mild and moderate drought stress. Meanwhile, plant water status, shoot and root biomass, and morphological root system architecture characteristics such as average root diameter, root projected area, and the number of root tips performed differently among the early and intermediate soybean genotypes at severe drought stress, where the most significant impacts were observed. Shoot and root biomass also differed between soybean genotypes after recovery irrigation.

Results suggested that the E2309 genotype exhibited a drought tolerance mechanism, indicating the ability to maintain its metabolic processes at a low water status under deficiencies of soil moisture (Manavalan et al. 2009; Hossain et al. 2014; Zhao, Aleem, and Sharmin 2017). The plant water potential deeply decreased in this genotype during most drought-stressed conditions. However, no notable detrimental effects in shoot and root biomass, root-to-shoot ratio, and morphological root system architecture characteristics were observed, perhaps due to osmotic adjustments under soil moisture scarcity (Nguyen, Babu, and Blum 1997; Zhao, Aleem, and Sharmin 2017). Similar results were reported in other drought-tolerant soybean genotypes such as AGS383 (Fatema et al. 2023) and several wild accessions (Nguyen et al. 2024). Curiously, stressed plants of the E2309 genotype tended to recover plant water status after re-irrigation but shoot and root biomass slightly decreased. Therefore, the effects of water deficiency were observed post-severe drought stress concerning plant growth. In this way, Dong et al. (2019) concluded that prolonged drought stress at the reproductive stage affected the growth and development of soybean plants, which were not compensated after 5 and 10 days of soil rehydration.

The main effects revealed that the early genotype had a greater root-to-shoot ratio (Table S4), suggesting greater root growth than shoot growth. This was not associated with the maturity of genotype E2309 since the intermediate genotype I700 also had a greater root-to-shoot ratio (Table S4), as addressed below. The association between a higher root-to-shoot ratio and drought-tolerant soybean genotypes has been well-established (Mwenye et al. 2018). Plants are assumed to stimulate or maintain root growth while reducing shoot growth to use the available water more efficiently (Li et al. 2021). Such strategies to tolerate drought conditions in soybean plants have been reported in several studies (Djekoun and Planchon 1991; Sinclair, Zwieniecki, and Holbrook 2008; Wijewardana et al. 2019). Also, the largest width-to-depth ratio in the early genotype (Table S6) demonstrated a wider root system architecture (Figure 8A). A wider root system maximizes the distribution of lateral roots in the upper soil layers to forage for nutrients at a low metabolic cost (Falk et al. 2020). This probably influenced the lower plant water potential in genotype E2309 under stress conditions, because of reduced water absorption from the deeper layers of the soil. The superior performance of root-to-shoot and width-to-depth ratios in this genotype was also confirmed by the Principal Component Analysis.

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We found that intermediate genotypes displayed similar responses to severe drought stress, as evidenced by the grouping observed in the Principal Component Analysis. They avoided the extreme effects of soil moisture deficiencies, exhibiting a higher plant water status because of a longer root system architecture, which allows efficient water absorption from the deeper soil layers (Manavalan et al. 2009; Zhao, Aleem, and Sharmin 2017). Moreover, drought avoidance also involves the control of stomatal opening, transpiration rate, and water loss reductions from tissues (Nguyen, Babu, and Blum 1997). Such physiological characteristics will need to be addressed in subsequent evaluations.

At the most drought-stressed sampling date, plant water status was higher in intermediate genotypes than in the E2309 genotype. Similarly, among four soybean genotypes, genotype BMG2026 reached higher leaf water potential under drought conditions at the pod development stage (Chowdhury et al. 2017). Curiously, restoring plant water potential was less efficient in genotype I700, displaying a significant difference between both water conditions after recovery irrigation. The decrease in plant water potential was associated with a higher root-to-shoot ratio in this genotype, which indicates that more water is allocated to the roots (Li et al. 2021). Shoot and root biomass and morphological root system architecture characteristics were also greater in the intermediate genotypes. However, shoot biomass was reduced by water deficit compared to well-watered conditions. In concordance, a 24% reduction in shoot dry mass was observed in the soybean commercial variety OAC Bayfield subjected to water deficit (Gebre and Earl 2020). Our results also showed that the root projected area and the number of root tips were lower due to water-deficit conditions in the intermediate genotypes. The decrease in root growth because of drought stress in soybean cultivars has been reported in previous studies. Xiong et al. (2021) exposed that under gradual drought conditions, roots grew best in mild drought and worse in severe drought. Drought stress at the R2 growth stage resulted in decreased root area at different soil depths in several Brazilian soybean genotypes (Franchini et al. 2017). In the meantime, reduction in root surface area and root tips were found in genotype AG5409RG, which was subjected to drought stress 30 days after planting (Fenta et al. 2014). The decrease of shoot biomass at severe drought stress in the intermediate genotypes was maintained after soil rehydration. Likewise, other investigations concluded that reductions in soybean shoot traits due to drought, such as plant height and leaf area, exhibited partial compensation and did not return to the control level after re-irrigation (Dong et al. 2019, 2024).

According to the main effects, the total root length (Table S5) and geometrical root system architecture characteristics such as root width, root depth, and convex hull area (Table S6) were similar in both intermediate genotypes, indicating an extensive and robust root system to explore efficiently the soil surface for resources acquisition (Comas et al. 2013; Lynch 2013; Postma, Dathe, and Lynch 2014). Soybean genotype PI89134 is another example of a root phenotype that maximized the total root length, while genotypes PI507488 and PI578364 displayed root phenotypes with larger root width and convex hull area (Falk et al. 2020).

Notably, the I700 genotype had similar root dry weight under well-watered and water-deficit conditions at severe drought stress, as the drought-tolerant soybean genotypes Embrapa 48 and BRS 284 with superior growth under different water availability conditions (Franchini et al. 2017). After recovery irrigation, root dry weight in genotype I700 showed a slight increase. Higher root biomass is a trait highly correlated with drought tolerance in soybeans, maintaining plant productivity under water stress conditions (Liu et al. 2005). The main effects analysis also exhibited that genotype I700 excelled in root growth, as reflected by a higher root length, root-to-shoot ratio (Table S4), and root projected area (Table S5), demonstrating a better root system architectureto cope with drought stress (Figure 8C) than genotype I1240 (Figure 8B). Research in other groups has indicated that drought-tolerant soybean genotypes produce longer roots with higher dry mass (Hossain et al. 2014). Furthermore, it was demonstrated that root system architectures that penetrate deeper into the soil, with wider diameters, larger lateral roots, and more root hairs exhibit greater tolerance to water stress (Lopes et al. 2011; Ali et al. 2016; Tanaka et al. 2014; Vadez 2014).

A relative increase in average root diameter was observed because of severe drought conditions in all three genotypes. This reflected the thickening of the older roots, not the new ones since the number of root tips was reduced by water deficit. Kodadinne Narayana et al. (2024) stated that root diameter confers drought tolerance in soybeans at different growth stages. Larger root diameters facilitate water uptake by increasing the surface area available for absorption (Carvalho and Foulkes 2013). Moreover, increasing average root diameter improves the ability of roots to penetrate hard soil layers (Lynch 2013). The increase in average root diameter was more prominent in the I1240 genotype. Little is known about root diameter responses to drought stress in soybeans at the reproductive growth stages. However, at the vegetative growth stage, a higher root diameter under water deficit was observed in 20 Chinese soybean varieties (Yan et al. 2020); meanwhile, soybean genotypes S49LL34 and DG 5170RR2/STS produced larger root diameters than genotype AG4632 (Kodadinne Narayana et al. 2024). In contrast, Wijewardana et al. (2019) reported that severe drought conditions reduced root diameter by 21% and 14% in the AG5332 and P533RY soybean cultivars, respectively, when drought stress was applied at the same growth stage. Similarly, the PEG-simulated drought stress reduced the root diameter of 20 soybean accessions at the seedling stage (Esan, Obisesan, and Ogunbode 2023).

Interestingly, we observed no stronger correlations between the gravimetric soil moisture and the plant water potential with shoot and root biomass and the root system architecture, suggesting that the influence of the genetic background on the morphology of root system and biomass traits, mainly at mild and moderate drought stress, does not change significantly depending on water conditions, which is desirable in new cultivars focused on climate change (Lopez, Freitas Moreira, and Rainey 2021). The correlation study also confirmed that greater geometrical root traits influenced an increase in morphological root traits, which led to an increase in root and shoot biomass. For example, an increase in the depth and width of roots was associated with an increase in total root length and root projected area. In turn, an increase in total root length and number of root tips was associated with an increase in both root and shoot dry weight. This was associated with the intermediate genotypes in the Principal Component Analysis. On the contrary, the early genotype showed a higher width-to-depth ratio but displayed lesser geometrical and morphological root traits.

The outcomes of this work indicated that morphological root system architecture characteristics could be used to screen soybean genotypes that are tolerant to drought stress since they were influenced by geometrical root traits and improved root and shoot biomass. Besides, these characteristics explained most of the variance in the Principal Component Analysis. Nevertheless, it would be beneficial to assay the root system architecture of Mexican soybean genotypes under field conditions. In agreement with Khan et al. (2023), root morphological traits profoundly influence plant growth, tolerance to abiotic stresses, and grain yield of soybeans. Such root traits have been suggested as suitable for selecting drought-tolerant soybean genotypes in previous research (Fenta et al. 2011, 2014; Jumrani and Bhatia 2019; Falk et al. 2020; Xiong et al. 2021; Contreras-Soto et al. 2022; Esan, Obisesan, and Ogunbode 2023; Fatema et al. 2023). Further physiological, biochemical, molecular, and agronomical studies are necessary to fully elucidate the drought tolerance mechanisms in these Mexican soybean genotypes.

In summary, Mexican soybean genotypes were tolerant to mild and moderate drought stress. Plant water status did not respond to mild drought stress. Severe drought stress increased root thickness. On the contrary, plant water status, shoot and root biomass, and morphological root system architecture characteristics were reduced. Among them, biomass traits were most impacted by severe drought stress, which was not restored following subsequent irrigation. Notwithstanding, it would be interesting to examine the growth recovery of soybean plants over an extended soil reirrigation period to ascertain whether the effects of drought stress are long-lasting. Responses to severe drought stress were different depending on the genotype. The early genotype E2309 exhibited a wider root system and tolerated severe drought stress maintaining shoot biomass and root characteristics at low plant water potential, possibly due to osmotic adjustments. Meanwhile, intermediate genotypes displayed severe drought stress avoidance reaching higher plant water potential and greater root system characteristics. Drought increased the average root diameter in genotype I1240, and root biomass was not reduced in genotype I700, which was higher than that observed in genotype I1240. Also, general effects analysis indicated a longer root system in the I700 genotype, suggesting more efficient water use from deeper soil layers. Interestingly, this genotype showed a higher root-to-shoot ratio after soil rehydration, recovering root growth more efficiently than the other genotypes. Therefore, we considered that the I700 genotype could be used in breeding programs to improve the root system architecture. Root biomass and morphological root traits are suitable for screening drought-tolerant soybean genotypes under controlled conditions.

Acknowledgments

We thank the National Council for the Humanities, Sciences, and Technologies for supporting García-Rodríguez through scholarship No. 778860, and the valuable help of Roxana Silerio-Espinosa (Instituto Nacional de México-Celaya) during the data collection.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

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