Eighty cotton varieties from diverse geographical regions were chosen as the study’s experimental materials in this study ( Supplementary Table 1 ). The selection process took into account the geographical distribution and ecological adaptability of the varieties, with the objective of highlighting their broad distribution and rich diversity.

The experiment was conducted at Hebei Agricultural University’s Qingyuan Experimental Station in China (38.85°N, 115.30°E) during 2021 and 2022. Rain-out shelter were utilized in the field to minimize the impact of rainfall interference. The recorded average temperatures were 20.75°C with 2091.4 hours of sunshine in 2021, and 20.96°C with 2199.8 hours of sunshine in 2022 ( Supplementary Figure 1 ). The soil is sandy loam with organic matter 13.83 g·kg^-1, total nitrogen 0.93 g·kg^-1, available nitrogen 69.45 mg·kg^-1, available phosphorus 17.40 mg·kg^-1 and available potassium 121.36 mg·kg^-1. Details regarding soil bulk density and field water capacity are available in Supplementary Table 2 .

This experiment included two water treatments: well-watered (WW), maintained at 75 ± 5% soil relative water content; and drought stress (DS), maintained at 50 ± 5% soil relative water content. The seeds were planted in a randomized complete block design with three replicates per treatment. The drought stress treatment was initiated at the three-leaf stage and continued until maturity. The irrigation method was micro-sprinkler. The volume of irrigation water was monitored using a water meter (with an accuracy of 0.0001 m³). During the study, under well-watered and drought stress treatments detectors were used soil water content, including Sensoterra (Soil moisture monitoring system, Netherlands) and TDR (Trime Pico 64 Portable Soil Moisture Meter, EMIKO, GmbH, Bochum, Germany). Sensoterra monitored the 0-30 cm soil water content ( Supplementary Figure 2 ), and TDR monitored the 0-20 cm, 20-40 cm, and 40-60 cm soil water content over the entire growth period ( Supplementary Figure 3 ).

Cotton was sown using the hole-sowing method on April 24, 2021, and April 24, 2022. The planting density was 90,000 plants hm^-2 with a row spacing of 50 centimeters. Each plot received 450 kg ha^-1 of compound fertilizer containing 15% nitrogen, 15% P₂O₅ and 15% K₂O as base fertilizer. Additionally, 150 kg ha^-1 of urea (containing 46% nitrogen) was top-dressed at the flowering stage. Other field management practices, aligned with the methods employed in local high-yield cotton fields.

Microsoft Excel 2019 (Redmond, WA, USA) was utilized for data entry. The quantitative data was analyzed by a one-way analysis of variance (ANOVA) using R version 4.1.2. Cluster analysis were drawn using the Origin 2019b Software Program (OriginLab, Northampton, MA, USA). The graphs were generated using GraphPad Prism 9.0 (GraphPad Software, Inc, San Diego, USA). Adobe Illustrator 2020 (Adobe, San Jose, CA, USA) was used to compose the images.

Varieties with small LRA and large LRA were selected using Euclidean distance flattening and systematic cluster analysis. In 2021, the varieties were clustered into five classes with a Euclidean distance of 0.5. Class I was composed of 25 (32%) varieties characterized by a small LRA. Class II consisted of 14 (18%) varieties characterized by a lesser LRA. Class III included 35 varieties, representing 44% of all the varieties, with an intermediate LRA. Class IV represented one variety, 1% of all the varieties, with a larger LRA. Category V was composed of four varieties, accounting for 5% of all the varieties, characterized by the largest LRA ( Supplementary Figure 4A ). In 2022, the varieties were clustered into four classes with a Euclidean distance of 0.5. Category I included 15 varieties, which represented 19% of all the varieties, with a small LRA. Category II was composed of 43 varieties (54% of all the varieties) characterized by a lesser LRA. Class III was composed of 20 varieties (26% of all the varieties) with a larger LRA. Category IV included one variety (1% of all the varieties) with the largest LRA ( Supplementary Figure 4B ).

Based on the two-year experimental results, three varieties with small LRAs were identified, variety #38, variety #77, and variety #7; and three varieties with large LRAs were identified, variety #48, variety #47, and variety #75 ( Supplementary Figure 4 ). Further analysis will be conducted on these six varieties.

There are genotypic differences among different LRA varieties under drought stress. Significant genotypic differences (p < 0.05) in the LRA were observed in both planting years ( Figure 1A ). In 2021, the range of the small LRA was 49.99 - 65.02 in varieties under drought stress and 59.65 - 79.33 for the large LRA. Drought stress significantly reduced the LRA in varieties with a small LRA and increased it in varieties with a large LRA compared to the well-watered ( Figure 1C ). Furthermore, under drought stress, the root dry weight and LRA were significantly reduced by 60% and increased by 22% in varieties with a large LRA compared to varieties with a small LRA ( Figures 1B, C ). In 2022, the range of the small LRA was 40.76 - 70.82 under drought stress and 63.16 - 79.17 for the large LRA ( Figure 1E ). Similarly, the root dry weight and LRA significantly increased by 26% and 32%, respectively, in the varieties with small LRAs under drought stress compared to the varieties with large LRAs ( Figures 1D, E ).

Changes in the stomatal opening, length, width, and density in the leaves of cotton varieties with different LRAs under well-watered and drought stress are shown in Figure 2A . Under well-watered, the differences in the stomatal opening ( Figures 2B, F ), width ( Figures 2C, G ), length ( Figures 2D, H ), and density ( Figures 2E, I ) among the cotton varieties with the different LRAs were insignificant. However, the stomatal width, length, and opening were suppressed, while the stomatal density was promoted in varieties with large and small LRAs under drought stress.

Moreover, the cotton varieties with different LRAs demonstrated varying abilities to regulate stomatal opening under drought stress. Cotton varieties with small LRAs exhibited a stronger regulatory capacity, and varieties with large LRAs exhibited the weakest ability to regulate the stomatal openings ( Figures 2B, F ). Under drought stress in 2021, the stomatal density of the small LRA varieties was significantly reduced compared to the large varieties of LRA. In addition, the stomatal opening and width were significantly increased by 15% and 47% in the small LRA compared to the large LRA, respectively ( Figures 2B, C ). However, the differences in stomatal length were insignificant ( Figure 2D ). The stomatal density also reduced by 10% ( Figure 2E ). In 2022, under drought stress, varieties with small LRAs exhibited a significant increase of 43% in stomatal opening and 47% in stomatal length compared to varieties with large LRAs ( Figures 2F, H ), with no significant difference in stomatal width ( Figure 2G ). Additionally, the stomatal density was significantly reduced by 21% ( Figure 2 ) for varieties with the small LRAs compared to those with the large LRAs.

There were significant differences in the canopy temperature between 2021 and 2022 under drought stress (p < 0.05, Figure 3 ). In 2021, the canopy temperatures under drought stress were significantly higher than those under well-watered in plants with large and small LRAs (p < 0.05). However, the canopy temperature in varieties with small LRAs was significantly reduced by 12% under drought stress compared to varieties with large LRAs ( Figure 3A ). The same pattern persisted in 2022, although the canopy temperature in the varieties with small LRAs was reduced by 9% under drought stress compared to the varieties with large LRAs ( Figure 3E ).

Drought stress had an inhibitory effect on the leaf water potential for both the large and small LRAs ( Figures 3B, F ). Nevertheless, notable distinctions emerged in the response of LRAs to drought stress. In particular, in 2021 and 2022, the leaf water potential of the small LRAs exhibited a significant increase of 13.68% and 25.77%, respectively, compared to their counterparts with large LRAs under drought stress. The trend in relative water content of leaves with large and small LRAs under drought stress mirrored the patterns observed in the leaf water potential ( Figures 3C, G ).

Drought stress inhibited the transpiration rate of both large and small LRAs ( Figures 3D, H ). However, there were significant genotypic differences in the response of LRAs to drought stress. Under drought stress in 2021 and 2022, the transpiration rate of small LRAs varieties increased significantly by 61.24% and 62.00% compared to that of large LRAs varieties ( Figures 3D, H ).

Drought stress significantly impacted the plant height and aboveground dry matter mass across varieties with different LRAs ( Table 1 ). In particular, drought stress reduced the height of plants with small LRAs by 36% (2021) and 6% (2022) compared to plants under well-watered treatment. Compared with the well-watered, drought stress reduced the height in the plants with large LRAs by 54% (2021) and 45% (2022). Additionally, the aboveground biomass of varieties with the small LRAs was reduced by 32% and 12% under drought stress in 2021 and 2022, respectively, compared to well-watered conditions. Alternatively, the aboveground biomass in the varieties with large LRAs was reduced by 75% and 71% in 2021 and 2022, respectively. Furthermore, under drought stress, the plant height and aboveground biomass in varieties with small LRAs increased by 23% and 54% in 2021 compared to the large LRAs. Similarly, in 2022, the plant height and aboveground biomass of varieties with small LRAs increased by 39% and 64%, respectively, compared to the large LRAs ( Table 1 ).

Drought stress significantly reduced weight per plant, seed cotton yield and lint yield of cotton. Under drought stress in 2021, the seed cotton yields for varieties with small and large LRAs were 20% and 80% lower ( Figure 4B ); the lint yield was 36% and 80% lower ( Figure 4C ), and the weight per plant was 20% and 80% lower ( Figure 4A ), respectively, compared to under well-watered conditions. The same trend persisted in 2022, where the seed cotton yield was 24% and 71% lower ( Figure 4E ); the lint yield was 32% and 73% lower ( Figure 4F ), and the weight per plant was 24% and 71% lower ( Figure 4D ) under drought stress for varieties with small and large LRAs, respectively, compared to those under well-watered conditions.

There were genotypic differences in weight per plant, seed cotton yield and lint yield of cotton under drought stress. Under drought stress in 2021, the varieties with small LRAs recorded significantly increased seed cotton yield, lint yield and weight per plant by 79, 75 and 79%, respectively, compared to varieties with a large LRA ( Figures 4A-C ). Similarly, in 2022, varieties with smaller LRAs significantly increased seed cotton yield, lint yield, and weight per plant by 76%, 77%, and 76%, respectively, compared to varieties with larger LRAs ( Figures 4D-F ).

The LRA, stomatal opening and canopy temperature of the six varieties were significantly correlated with seed cotton yield under drought stress in 2021 and 2022. The findings revealed significant regressions (p < 0.05) between the LRA with stomatal opening, canopy temperature, and seed cotton yield in 2021 ( Figure 5 ). In particular, in 2021, the LRA significantly increased with the increasing canopy temperature (p < 0.05, Figure 5B ) and significantly decreased with the increasing stomatal opening (p < 0.05, Figure 5A ) and seed cotton yield (p < 0.05, Figure 5C ). Similarly, in 2022, the LRA significantly increased with the increasing canopy temperature ( Figure 5E ) and significantly decreased with the increasing stomatal opening ( Figure 5D ) and seed cotton yield (p < 0.05, Figure 5F ).

Under drought stress, the root system’s capacity to delve into the soil becomes crucial because it enables the plants to extract water from deeper layers, which directly influences their growth (Adcock et al., 2007). Thus, measuring the root angle is vital to assess the drought tolerance of crops (Lynch, 2013; Lynch and Wojciechowski, 2015; Colombi and Walter, 2017).

Notably, previous research demonstrated that the root angle in barley (Hordeum vulgare L.) (Robinson et al., 2018) and rice (Uga et al., 2013) directly impacting their final yield. Moreover, MansChadi et al. (2010) further illustrated that with narrower roots, more soil water was extracted, especially at profile depth. Maize also provides an excellent example where the root angle proves to be a significant factor in increase crop yield under drought stress (Ali et al., 2015). MansChadi et al. (2008) revealed that drought-tolerant wheat genotypes had a narrower angle of seminal axes. Furthermore, the wheat varieties cultivated in arid regions tend to exhibit narrower root angles and a more compact root structure, which enhances their ability to access deep soil water (MansChadi et al., 2008). This study was consistent with the results of these findings. These findings are consistent with the results of this study. In the context of this study, different cotton varieties exhibited genetic differences in their LRAs. In particular, drought stress significantly reduced the LRAs of varieties with smaller LRAs and increased those of varieties with larger LRAs.

This study explores the complex relationship between cotton LRA and stomatal traits under drought stress. Stomata are a vital component of the regulation of water by the plant under drought stress conditions. Generally, the leaf stomatal density increases under mild to moderate stress levels or at the onset of stress, such as drought stress (Fraser et al., 2009). This study was consistent with Fraser et al. (2009). We observed an increase in stomatal density in varieties with small and large LRAs under drought stress compared to well-watered conditions.

The extent of the increase in stomatal density varies among types of crops. In this study, we found that under drought stress, varieties with smaller LRAs had a 10% and 21% reduction in stomatal density compared to those with larger LRAs in 2021 and 2022, respectively. Additionally, we observed that under drought stress, as the stomatal density increased, the stomatal width, length, and aperture decreased. This is consistent with the results of Hetherington et al. (2003), who indicated that the stomatal size decreases with increasing stomatal density. Plants exchange gases, including water vapor, through stomata. This is particularly important for plants grown under limited water resources (Gray and Hetherington, 2004; Shimazaki et al., 2008; Yoo et al., 2009; Zhang et al., 2018). Drought stress reduced the stomatal opening in metatarsal leaves (Grantz and Schwartz, 1988), soybean (Ohashi et al., 2006), and winter wheat (Li et al., 2004), which is consistent with the results of this study. Our research showed that the stomata of varieties with large and small LRAs reduced their degree of stomatal opening under drought stress. However, the extent of the reduction of stomatal opening varied among crop types. We found that compared to cotton varieties with larger LRAs, those with smaller LRAs increased their stomatal opening by 15% and 43% under drought stress in 2021 and 2022, respectively. Our results are in agreement with Vinarao et al. (2023) who showed that the narrow root cone angle rice genotypes have higher stomatal conductance compared to the wide root cone angle rice genotypes.

Overall, drought-tolerant varieties can more easily access deep water, and the amount of water accessed can be greater to compensate for the loss of large transpiration caused by stomatal opening, which can improve the drought tolerance of plants, leading to better survival and growth of plants under drought stress conditions.

The canopy temperature is affected by genetic, environmental, and field management factors (Balota et al., 2007; Reynolds et al., 2009). The exchange of energy between the canopy and the external environment happens through radiation, conduction, convection, transpiration, and various metabolic processes, including enzymatic reactions, respiration, and photosynthesis (Yoshimoto et al., 2005). In various crops, such as upland rice, sugar beet (Beta vulgaris subsp. vulgaris Altissima Group), and potato (Solanum tuberosum L.), the difference between the canopy and air temperatures indicate drought tolerance (Fukuoka et al., 2003). Therefore, the canopy temperature is a crucial indicator to monitor the responses of plants to drought stress.

This study delved into the effects of drought stress on cotton canopy temperature, and it is closely related to the LRA. During drought stress, elevated canopy temperatures may increase the rates of plant transpiration and accelerate the evaporation of soil water, which increases the plant’s water needs (Carvalho et al., 2020). However, excessively high canopy temperatures can trigger heat stress responses in plants, which disrupts their normal growth and development (Mohammed et al., 2018). Moller et al. (2006) also suggested a model to estimate the water status of grape (Vitis vinifera L.) using the canopy temperature. Different plant varieties exhibit varying adaptations to temperature. This study shows that, under drought stress conditions, cotton plants experienced a notable increase in canopy temperature. However, in the years 2021 and 2022, cotton varieties with smaller LRAs exhibited a significant reduction in the canopy temperature by 12% and 9%, respectively, when compared to varieties with larger LRAs under drought stress. This results is consistent with Vinarao et al. (2023), who showed that compared to rice genotypes with wide root cone angle, the narrow root cone angle group had a 0.3-1.0° C decrease in canopy temperature below 20 centimeters.

The reduction in canopy temperature among in cotton varieties with smaller LRAs suggests an improved ability to regulate water use under drought stress. This adaptation enables these plants to adapt to limited water availability more effectively, which ultimately results in improved drought tolerance and potentially higher yields. These findings underscore the intricate relationship between the root system traits, canopy temperature, and drought tolerance in cotton.

Our research unveiled a compelling correlation between the LRA and leaf stomatal opening, canopy temperature, and yield. Research has shown that cotton varieties with larger LRAs may lead to elevated leaf temperatures during drought stress. Conversely, under drought stress conditions, cotton varieties with smaller LRAs consistently displayed lower leaf canopy temperatures. This phenomenon arises from their enhanced ability to efficiently access water deep within the soil, which potentially induces greater transpiration in plants, thus, ameliorating water stress. These findings align closely with the research by Kato and Okami (2010) in which deeper-rooted varieties maintained open stomata and sustained lower leaf temperatures even when soil water potential dropped below -50 kPa, which illustrated the varietal adaptation of stomatal traits to drought stress. This discovery emphatically underscores the pivotal role played by the LRAs in facilitating the access of plants to deeper soil layers, thereby alleviating water deficits in the topsoil. The resultant reduction in leaf canopy temperatures suggests the potential of smaller LRAs to mitigate the heat stress induced by drought.

Furthermore, since the plant leaf temperature is typically influenced by the canopy temperature and controlled by the stomatal opening, our study highlights the significant correlation between the LRA and stomatal traits. The LRA affects the internal water transport in plants by influencing the stomata opening. We found that under drought stress, cotton varieties with smaller LRAs had relatively increased stomatal openings compared to varieties with larger LRAs. This could be because cotton plants with smaller LRAs can typically access deeper soil water, maintain a higher water supply, alleviate drought stress, and enable sufficient stomatal openings to support transpiration losses. In contrast, cotton plants with larger LRAs may face a lower water supply and be unable to sustain transpiration losses. Thus, they respond to drought stress by closing their stomata. In particular, under drought stress, cotton varieties with smaller LRAs exhibited a 15%-43% increase in their stomatal opening compared to varieties with larger LRAs. These results are consistent with those of Kato and Okami (2010) and Vinarao et al. (2023), who demonstrated that, varieties with deeper or narrower root systems maintained larger stomatal openings and higher stomatal conductance under drought stress. Therefore, this response is crucial for water resource conservation since it is sufficient to maintain a higher water supply, alleviate drought stress, and improve the plant’s resistance to water deficits.

Importantly, these findings significantly impact seed cotton yield. Under drought stress conditions, cotton varieties with smaller LRAs consistently outperform other varieties, primarily owing to their ability to access more water resources, thereby increasing seed cotton yield. However, cotton varieties with larger LRAs may be subject to water limitations, which results in reduced yields. Our research indicates that the seed cotton yield, lint yield, and individual plant weight of varieties with smaller LRAs increased significantly by 76%-79%, 75%-77%, and 76%-79%, respectively, compared to varieties with larger LRAs. These results are consistent with those of MansChadi et al. (2010), who demonstrated that wheat genotypes with narrow root angles exhibit 6%-28% higher yields than conventional varieties.

This correlation between the LRA and canopy temperature, stomatal opening, and yield strongly suggests the potential to use the LRA as a key trait in breeding programs aimed at developing drought-resistant cotton varieties. Recent studies provide support for such interrelationships. Vinarao et al. (2023) published research that illustrated a similar relationship, albeit in a different crop context. In particular, the RCA had significant negative correlations with the total root length (r = -0.70), leaf area (r = -0.67), proportion of deep roots below 20 cm (r = -0.65), and stomatal conductance (r = -0.71). Conversely, it had a significant positive correlation with canopy temperature (r = 0.66). These findings align closely with our results where we identified a negative correlation between the LRA and stomatal opening and seed cotton yield along with a positive correlation with canopy temperature.

In summary, the reduction in stomatal aperture reduces water loss, but also weakens photosynthesis and transpiration, and blocks assimilate transfer and heat dissipation in the plant body, a large increase in temperature will cause irreversible damage to plant organs and the yield loss of cotton will be greater (Sánchez et al., 2014). This understanding provides valuable insights into the mechanisms by which cotton adapts to water scarcity, thus, offering a promising avenue for precision breeding efforts. By selecting cotton varieties with smaller LRAs, breeders can facilitate the development of more robust cotton crops, ensuring sustainable production in water-limited environments and addressing the challenges posed by evolving climate patterns.

The evaluation of drought tolerance using physiological mechanisms has made little progress. There are three main issues at the heart of this dilemma. First, the specific plant traits that enhance drought tolerance remain uncertain. Secondly, whether plant traits represent mutually exclusive substitutes, trade-offs, or orthogonal processes is unclear. Third, it is unclear whether some traits should be included in the drought stress treatment group. This study has addressed the first and second challenges. Our results demonstrate that the LRA, leaf canopy temperature, and stomatal opening are important factors in improving plant water relations and achieving higher seed cotton yield under drought stress. Despite the progress in linking LRA phenotypes and performance, the genetic basis of LRA remains poorly understood and merits further investigation.