The experiment was carried out in the intelligent greenhouse of the College of Agronomy, Hebei Agricultural University, in 2023 (Hebei, China). Upland cotton (Gossypium hirsutum L.) variety ‘Guoxin Cotton No. 9’ was used as the experimental material, and the seeds were provided by the General Union of Rural Technical Services of Guoxin (Hebei, China). The seeds were soaked in an incubator at 25°C for 24 h. After the seeds showed white tips, they were sown in white PVC culture pots (with a diameter of 10 cm, a height of 20 cm, and a volume of 1.6 L) filled with 2.5 kg of culture medium (with a volume ratio of soil to sand of 3:1), containing 16.93 g kg⁻¹ organic matter, 94.60 mg kg⁻¹ alkaline dissolved nitrogen, 25.33 mg kg⁻¹ effective phosphorus, and 202.07 mg kg⁻¹ effective potassium. Three seeds were sown in each culture pot, and only one cotton seedling was retained after one true leaf emerged.

The experimental design employed a randomized complete block design. When the third true leaves of the cotton seedlings were fully expanded, water treatments were initiated: control (CK), moderate drought (MD), and severe drought (SD), with relative water contents of 70–75%, 55–60%, and 40–45%, respectively (Song et al., 2023). Each treatment was replicated in 70 pots. The soil moisture content was monitored by the weighing method every day, and each pot was supplemented with water to reach the set moisture content. The relative humidity in the culture room was constant at (70 ± 5) %. The light intensity was 600 μmol·m⁻²·s⁻¹, and the photoperiod was 14/10 h, with day and night temperatures of 28°C/20°C.

The aboveground morphological traits and biomass were measured at 0, 7, 14, 21, and 28 days after the initiation of the water treatments. Three plants were selected for each treatment to determine the following parameters:

After 0, 7, 14, 21, and 28 days of drought treatment, the third leaf from the top was sampled. There were three replicates for each treatment. The leaf hydraulic conductivity was measured using a plant high-pressure flowmeter (HPFM-Gen, Dynamax, Houston, TX, USA) in transient mode on the leaf, retaining a petiole length of 2 cm, with the applied pressure ranging from 0 to 5 kPa s⁻¹ and the pressure and flow rate recorded every 2 seconds (Sack, 2002; Sack et al., 2005). The leaf hydraulic conductivity was calculated as follows:

The leaf area was measured using the leaf length and width method, which was calculated based on the leaf area correction factor method (Mao et al., 2014), as follows:

After 0, 7, 14, 21, and 28 days of drought treatment, the third leaf from the top was measured using a portable pressure chamber (PMS670, PMS Instrument Company, USA), with three replicates for each treatment. The leaves were first equilibrated in sealed black plastic bags for 20 min, then cut off at the base of the petiole and placed into a pressure chamber. Subsequently, pressure was slowly applied. The minimum pressure at which the first drop of water was observed exuding from the petiole under observation with a magnifying glass was regarded as the leaf water potential (Ψ_leaf) (Müllers et al., 2022).

On day 28 post-treatment, the third leaves from the top were collected from each treatment for leaf and petiole anatomical analyses. For leaves, 0.5 cm × 0.5 cm sections were cut perpendicular to the veins, while petioles were cut into 0.5–1 cm segments, starting 1 cm from the leaf. Three replicates per treatment were prepared using the paraffin section method. Samples were fixed with formaldehyde–alcohol–acetic acid (FAA), treated with xylene and absolute ethanol, and then embedded in paraffin (Zhang et al., 2022). After saffron and solid green staining, the sections were sealed and stored at 4°C. Images were captured using a digital microscope (BX53, Olympus, Monolith, Japan) and analyzed using NIS-Elements software. Leaf parameters included thickness, cross-sectional area, and xylem vessel area, number, and diameter. Petiole analyses focused on the cross-sectional area, diameter, and the area and number of xylem vessels.

On day 28 post-treatment, the third leaves from the top were scanned (Epson Perfection V39; Epson, Suwa, Japan) with three replicates per treatment. The major (VLA_major) and minor (VLA_minor) vein densities were measured. Primary veins were assessed in intact leaves, and secondary veins were assessed in half of them for VLA_major. Leaves were soaked in 95% ethanol for 1–2 days, stained with 1% saffron, and observed under a microscope (BX53, Olympus) to determine the vein length and field area (Lu et al., 2019). The vein density (VLA_minor) was calculated using NIS-Elements software, reflecting the length of veins per unit leaf area.

On day 28 post-treatment, the third leaf was selected from the main stems of cotton under each treatment (three replicates per treatment). The abaxial leaf surfaces were lightly coated with nail polish, allowed to dry for 3 min, and transferred onto clean slides using transparent tape. Observations and image acquisition were conducted using a digital microscope (BX53, Olympus, Monolith, Japan). The stomatal area and number were measured using NIS-Elements software, enabling the calculation of stomatal density (number of stomata per unit area).

On days 0, 7, 14, 21, and 28 post-treatment, the environmental control mode of a portable photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA) was adopted to measure the gas exchange parameters, including net photosynthetic rate (A_n), transpiration rate (E), stomatal conductance (g_s), and intercellular CO₂ concentration (C_i) in the third leaves from the top of cotton plants (Sáez et al., 2018). The light intensity of the red and blue light sources was 500 μmol·m⁻²·s⁻¹, and the CO₂ concentration was 400 μmol·mol⁻¹. There were three replicates for each treatment. The instantaneous water use efficiency of the leaves was calculated as follows (Li et al., 2021b):

After 0, 7, 14, 21, and 28 days of drought treatment, a PAM-2500 portable chlorophyll fluorometer (PAM-2500, WALZ, Effeltrich, Germany) was used to measure the maximum photochemical efficiency of photosystem II (PSII; Fv/Fm) and the actual photochemical quantum yield of PSII (ΦPSII) for each treatment. The measurement sites were the same as those for the photosynthesis measurements, and there were three replicates for each treatment.

To determine the aquaporin content, the third leaves from the top of cotton seedlings were sampled on days 0, 7, 14, 21, and 28 post-drought treatment (three replicates per treatment). Samples were labeled, wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and stored at –80°C. The aquaporin content was determined using an enzyme-linked immunosorbent assay (ELISA) kit (YJ191258) from Shanghai Yuanju Technology Center, following the manufacturer’s protocol.

To determine the expression of aquaporin genes, 50 mg of the same leaves was collected on day 28 post-treatment, frozen in liquid nitrogen, and stored at −80°C (three replicates per treatment). RNA was extracted, and its concentration and quality were assessed. RNA extraction and cDNA synthesis procedures are detailed in the appendix. All reagents were obtained from Sigma-Aldrich.

Data were recorded and organized using Microsoft Excel 2010. Statistical analyses were performed with IBM SPSS Statistics 25.0, employing one-way ANOVA followed by Duncan’s multiple comparison tests to assess significance. Graphs were plotted using GraphPad Prism 8.0, and correlation analyses were conducted with Origin 2023.

Drought stress exhibited progressive inhibitory effects on cotton seedling growth with increasing drought intensity and duration ( Figure 1A ). Quantitative analysis revealed significant reductions in key growth parameters, including plant height, stem diameter, leaf area, and aboveground dry matter mass. After 28 days of drought treatment, MD decreased these parameters by 28.52%, 15.32%, 43.51%, and 45.92%, respectively, compared with CK. SD induced more pronounced growth suppression, with corresponding reductions reaching 59.14% in plant height, 26.52% in stem diameter, 69.49% in leaf area, and 65.47% in aboveground dry matter mass ( Figures 1B–E ).

A_n and E of cotton seedlings that underwent drought treatment were substantially lower than those of the CK group starting after 7 days of treatment. This difference augmented progressively over time ( Figures 2A, B ). On day 28 of drought stress, g_s, A_n, E, and C_i in the MD treatment had diminished by 39.07%, 14.68%, 10.88%, and 9.88%, respectively. In the SD treatment, these values decreased by 48.11%, 26.39%, 26.42%, and 22.69%, respectively ( Figures 2A–D ). The Fv/Fm and ΦPSII values of the leaves under drought stress conditions were lower than those in CK. On day 28 of drought stress, Fv/Fm of the leaves in the MD and SD treatments was significantly reduced by 11.95% and 15.94%, respectively, compared with CK ( Figure 2E ). Similarly, ΦPSII decreased significantly by 12.67% and 20.72% in the MD and SD treatments, respectively ( Figure 2F ).

As the degree and duration of drought stress increased, K_leaf showed a decreasing trend compared with CK ( Figure 3A ), and by day 14 of drought treatment, a significant difference was observed, with K_leaf reduced by 7.17% and 18.77% in the MD and SD treatments, respectively, compared with CK. On days 21 and 28 of drought stress, K_leaf decreased by 6.54% and 9.18%, respectively, in the MD treatment and by 12.77% and 12.93%, respectively, in the SD treatment compared with CK ( Figure 3A ). Differences in Ψ_leaf were observed among treatments after 7 days of drought stress. Drought stress accelerated the decline in leaf water potential, and on day 28, leaf water potential in the SD treatment decreased significantly by 17.55% compared with CK ( Figure 3B ). On day 28 of drought stress, WUE_i increased significantly by 47.42% and 42.28% in the MD and SD treatments, respectively, compared with CK ( Figure 3C ).

After 7 days of drought stress, significant differences in aquaporin content were observed among the treatments ( Figure 4A ). By day 28 of drought stress, the aquaporin content in the MD and SD treatments had decreased by 31.07% and 36.73%, respectively, compared with the CK treatment ( Figure 4A ). Further analysis of the synthesized genes of water channel proteins showed that GhPIP2-1, GhPIP2-2, GhTIP1-2, and GhTIP1-3 were significantly downregulated under drought stress. The expression of GhPIP2-1, GhPIP2-2, and GhTIP1-2 was downregulated 2–2.5-fold ( Figure 4B ).

To identify the primary anatomical factors influencing K_leaf under drought stress, we conducted principal component analysis (PCA) on hydraulic conductivity and petiole anatomical traits ( Figure 8A ). The key factors influencing leaf hydraulic conductivity, including main vein density, secondary vein density, number of xylem vessels, xylem vessel diameter, xylem area, and epidermal cell thickness, accounted for 89.10% of the total variance. These factors were negatively correlated with primary vein density, and had extremely significant positive correlations with the number of xylem vessels, xylem vessel diameter, xylem area, and secondary vein density ( Supplementary Table S4 ). The main factors affecting K_leaf were xylem vessel diameter and xylem area of the petiole.

PCA was conducted on K_leaf and leaf anatomical traits ( Figure 8B ). Leaf hydraulic conductivity and leaf anatomical factors accounted for 94.50% of the total variance. K_leaf was mainly influenced by the number and diameter of xylem vessels in the leaf, upper epidermal cell thickness in the leaf, spongy tissue thickness, and the number of xylem vessels in the petiole. Moreover, these indicators had extremely significant positive correlations with K_leaf ( Supplementary Table S4 ). In conclusion, K_leaf was mainly affected by the diameter and number of xylem vessels in the leaf and petiole as well as secondary leaf vein density.

To better understand the relationships between leaf hydraulic conductivity and stomatal traits, we conducted PCA on the relevant indicators under three water treatments ( Figure 9 ). K_leaf was significantly negatively correlated with stomatal density and had extremely significant positive correlations with stomatal area, stomatal width, and stomatal aperture. K_leaf mainly influenced stomatal width and aperture.

Correlation analysis was carried out between leaf hydraulic conductivity and photosynthetic parameters. As shown in Figure 10 , there were significant positive correlations between K_leaf and g_s, A_n, E, and C_i. With the increase in drought stress severity, K_leaf decreased, and g_s, A_n, E, and C_i decreased with the decline in K_leaf ( Figures 10A–D ). In conclusion, the decrease in K_leaf under drought stress led to the decline in stomatal aperture and width, decreasing g_s, A_n, E, and C_i, attenuating photosynthetic function.

There was a positive correlation between K_leaf and leaf water potential ( Figure 10E ) and a negative correlation between K_leaf and instantaneous leaf water use efficiency ( Figure 10F ). Therefore, drought stress decreased leaf xylem vessel diameter, number, and K_leaf, leading to a decrease in leaf water potential and an increase in instantaneous leaf water use efficiency.

Leaf hydraulic dysfunction is a pivotal adaptive response when plants face drought stress, critically influencing plant fitness under water stress conditions (Blackman et al., 2010). Hydraulic traits act as multifaceted functional indices, governing water transport efficiency and stomatal regulation (Creek et al., 2018), and shaping plant ecological strategies in growth dynamics and resource competition (Cosme et al., 2017; Poorter et al., 2017). Previous studies have revealed species-specific drought adaptation mechanisms: rice enhances water-use efficiency by reducing leaf water potential (Yang et al., 2024), while maize prioritizes drought tolerance by decreasing leaf hydraulic conductivity (K_leaf) (Qiao et al., 2020). These findings align with current experimental evidence suggesting that progressive drought intensification reduces vascular water supply capacity, with synchronously decreasing K_leaf ( Figure 3A ) and leaf water potential ( Figure 3B ). This indicates that cotton plants adapt to drought conditions by reducing K_leaf and leaf water potential, thereby maintaining normal growth.

K_leaf is a critical hydraulic signal modulated by multifaceted anatomical and molecular factors (Sack and Scoffoni, 2013). Water transport in leaves operates through two sequential pathways: xylem vessels in petioles and vascular bundles, and the post-xylem pathway involving aquaporin-mediated membrane transport. These pathways contribute substantially to total leaf hydraulic resistance, with their coordinated regulation directly determining K_leaf (Prado and Maurel, 2013; Kaack et al., 2021). Our findings revealed that drought stress disrupted these pathways synergistically—reducing vein density, xylem vessel diameters and numbers, and aquaporin expression—collectively impairing hydraulic efficiency. This dual pathway suppression provides mechanistic insights into drought-induced K_leaf decline.

VLA_minor plays a key role in leaf pulp hydraulic transport, especially under drought stress, and its regulatory role significantly affects leaf hydraulic efficiency and photosynthetic capacity. Unlike the VLA_major, which mainly provides mechanical support and water redundancy, VLA_minor is directly involved in water transport between leaf pulp cells, thereby affecting K_leaf and gas exchange efficiency (Baird et al., 2021). In this study, drought stress significantly reduced VLA_minor (40.31% and 41.86% decrease in MD and SD treatments, respectively), which was significantly and positively correlated with the decrease in K_leaf ( Figure 7B ; Supplementary Figure S1B ). This finding contrasts with the findings in rice, where there was no correlation between VLA_minor and K_leaf under drought conditions (Xiong et al., 2015; Caringella et al., 2015; Scoffoni et al., 2011), whereas cotton exhibited a significant decrease in VLA_minor, leading to a decrease in K_leaf. This difference may stem from the differences in leaf vein structure and water utilization strategies between cotton and rice. Rice, as a monocotyledon, has a parallel leaf vein structure with higher hydraulic redundancy under drought conditions, whereas the reticulate leaf vein structure of cotton is more susceptible to drought-induced embolism and cell wall thickening (Zou et al., 2022). In addition, the decrease in VLA_minor may also be related to anatomical changes in cotton chloroplasts, such as a reduction in the thickness of spongy tissue ( Table 2 ), which further limits the efficiency of water transport between chloroplasts (Sack and Frole, 2006). Thus, VLA_minor is not only a key regulator of cotton leaf hydraulic efficiency, but also an important component of its drought adaptation strategy. Future studies should further explore the potential of increasing VLA_minor through genetic improvement or agronomic measures to enhance the hydraulic efficiency and photosynthetic performance of cotton under drought conditions.

Plant water transport relies on the axial xylem vessel system (Cochard et al., 2004). This hydraulic pathway initiates soil water absorption via roots, progressing through root-to-stem xylem networks, petiolar vessels, and leaf vein vessels, ultimately delivering water to mesophyll cells for transpirational loss through stomata. Xylem vessel morphology directly regulates water transport efficiency from stems to foliar tissues (Kaack et al., 2021). Our investigation revealed marked anatomical alterations under drought conditions, including a diminished xylem vessel diameter, frequency, and cross-sectional area in both leaves and petioles ( Figure 5 ; Tables 1 , 2 ), which confirmed that stomatal change served as an adaptive strategy for mitigating water loss while maintaining plant viability. These structural modifications highlight the physiological acclimation mechanism in cotton under water deficit.

Previous studies have shown that the morphology of xylem vessels affects K_leaf. The larger the vessel diameters and the greater their number, the larger the K_leaf, resulting in enhanced water transport capacity. Conversely, smaller vessel diameters, reduced vessel areas, and fewer vessels increase the hydraulic resistance of the leaf, thereby reducing K_leaf (Aasamaa et al., 2005; Jafarikouhini and Sinclair, 2023). Our study identified four key anatomical determinants, namely, upper epidermal cell thickness, xylem area in leaf/petiole tissues, vessel diameter, and vascular bundle frequency, that exhibited significant positive correlations with K_leaf ( Figures 8 , 9 ; Supplementary Table S4 ). Notably, xylem vascular architecture emerges as the principal modulator of leaf hydraulic vulnerability under mild-to-moderate drought (Bouche et al., 2015; Trifiló et al., 2016). Under severe drought conditions, xylem embolism can lead to irreversible damage, significantly affecting water transport and leaf hydraulic conductivity (Knipfer et al., 2015). The phenomenon of embolism severely impedes water transport, leading to an increase in hydraulic resistance (Trifiló et al., 2016). Our study observed that under drought stress, both the number and diameter of xylem vessels in the leaves and petioles decreased under the MD and SD treatments ( Tables 1 , 2 ), indicating the occurrence of changes in the xylem vessels that resulted in a significant reduction in K_leaf ( Figure 3A ). Although embolism quantification remains technically challenging in herbaceous species, the observed structural degradation under SD conditions suggests its detrimental role in K_leaf reduction. Future studies should prioritize non-destructive embolism detection techniques to elucidate their long-term impacts on hydraulic performance and refine our understanding of drought adaptation strategies in crops.

Aquaporins, which are pivotal regulators of plant water homeostasis, mediate critical physiological processes, including transmembrane water transport and stress responses. Specifically, the decrease in aquaporins gene expression directly affected K_leaf, as aquaporins play a key role in water transport in chloroplasts and vascular sheath cells (Maurel et al., 2016). It is generally believed that under drought stress conditions, plants maintain their internal water by downregulating aquaporin synthesis gene expression levels and reducing aquaporin content (Afzal et al., 2016). In this study, the aquaporin content under the CK treatment was significantly higher than under the MD and SD treatments ( Figure 4A ). This aligns with the results of Šurbanovski et al. (2013), who demonstrated drought intensity-dependent suppression of PIP isoforms, and Xue et al. (2021), who reported coordinated PIP/TIP depression in drought-stressed strawberry. In this study, under the MD treatment, the expression of PIP-related synthesis genes in cotton leaves were down-regulated by 1.0–1.5-fold, while under the SD treatment, it decreased by 2.0–2.5-fold. Furthermore, under both drought treatments, the expression of TIP-related synthesis genes also decreased by 1.0–1.5-fold ( Figure 4B ). These results suggest that cotton adjusts aquaporin gene expression in its leaves to regulate the aquaporin content, thereby controlling the water transport efficiency. Thus, changes in aquaporins genes expression are not only an important molecular marker of cotton’s response to drought stress, but also a key driver of its reduced hydraulic efficiency and photosynthetic performance. Future studies could regulate the expression of aquaporins through gene editing or transgenic techniques to explore their potential in improving drought tolerance in cotton.

Green plants are confronted with a contradictory challenge—maximizing absorption of carbon dioxide for photosynthesis while minimizing water loss. Stomatal regulation plays a crucial role in this process (Harayama et al., 2019). Stomata not only regulate the entry of carbon dioxide but also control water evaporation. Therefore, stomatal behavior is vital for plant responses to water stress. Typically, plants regulate g_s by changing stomatal size or stomatal density, which allows them to maintain growth and physiological activities under constantly changing environmental conditions (Fang et al., 2019). This experiment demonstrated that g_s was significantly and positively correlated with stomatal width, stomatal area, and stomatal aperture, but it showed a highly significant negative correlation with stomatal density ( Supplementary Figure S4A–D ). These results suggest that changes in stomatal traits directly affect stomatal conductance, thereby influencing the plant’s photosynthetic capacity. Under drought conditions, leaves with smaller stomatal sizes and higher densities are more sensitive to changes in the external environment, with faster opening and closing speeds, which effectively reduce water loss and enhance water use efficiency (Raven, 2014; Kardiman and Ræbild, 2017). In this study, MD and SD treatments decreased stomatal width by 6.83% and 13.37%, respectively, and stomatal aperture by 20.59% and 27.58%, respectively, and K_leaf significantly decreased under both treatments ( Figure 3A ). These findings suggest that plants reduce water loss and maintain growth by decreasing stomatal size and increasing stomatal density.

Previous studies across various species have shown that under short-term environmental changes, g_s and K_leaf exhibit a positive correlation, suggesting that K_leaf is a potential trigger for the decrease in g_s (Theroux Rancourt et al., 2015; Xiong et al., 2018; Wang et al., 2018). Gleason et al. (2017) found that the plant’s water supply capacity weakened as drought stress intensified, decreasing K_leaf, which triggered stomatal closure, reduced the photosynthetic rate, and improved the water use efficiency in the leaves. This further confirms that K_leaf is significantly positively correlated with stomatal aperture and width ( Figure 9 ; Supplementary Table S4 ), indicating that as K_leaf decreases, the stomatal width and aperture also decrease, directly affecting the plant’s photosynthetic function. In addition, K_leaf had an extremely significant positive correlation with g_s and A_n under different drought treatments ( Figures 10A, B ). These findings suggested that drought stress affected hydraulic signaling by reducing K_leaf, which triggers rapid stomatal closure and a decrease in aperture, ultimately reducing water loss. However, this adaptive mechanism also decreases photosynthesis.

While our study provides valuable insights into the physiological and anatomical responses of cotton leaves to drought stress using potted plants in a greenhouse, it is important to acknowledge some limitations. The experimental setup, while allowing precise control of conditions, may not fully replicate field complexities. Consequently, yield and quality traits, which are critical for assessing the long-term impact of drought stress on cotton production, were not measured. Future research should extend these findings to field trials and explore the impacts of prolonged drought.