Zn Supplementation Mitigates Drought Effects on Cotton by Improving Photosynthetic Performance and Antioxidant Defense Mechanisms

Drought is recognized as a paramount threat to sustainable agricultural productivity. This threat has grown more severe in the age of global climate change. As a result, finding a long-term solution to increase plants’ tolerance to drought stress has been a key research focus. Applications of chemicals such as zinc (Zn) may provide a simpler, less time-consuming, and effective technique for boosting the plant’s resilience to drought. The present study gathers persuasive evidence on the potential roles of zinc sulphate (ZnSO4·7H2O; 1.0 g Kg−1 soil) and zinc oxide (ZnO; 1.0 g Kg−1 soil) in promoting tolerance of cotton plants exposed to drought at the first square stage, by exploring various physiological, morphological, and biochemical features. Soil supplementation of ZnSO4 or ZnO to cotton plants improved their shoot biomass, root dry weight, leaf area, photosynthetic performance, and water-use efficiency under drought stress. Zn application further reduced the drought-induced accumulations of H2O2 and malondialdehyde, and electrolyte leakage in stressed plants. Antioxidant assays revealed that Zn supplements, particularly ZnSO4, reduced reactive oxygen species (ROS) accumulation by increasing the activities of a range of ROS quenchers, such as catalase, ascorbate peroxidase, glutathione S-transferase, and guaiacol peroxidase, to protect the plants against ROS-induced oxidative damage during drought stress. Increased leaf relative water contents along with increased water-soluble protein contents may indicate the role of Zn in improving the plant’s water status under water-deficient conditions. The results of the current study also suggested that, in general, ZnSO4 supplementation more effectively increased cotton drought tolerance than ZnO supplementation, thereby suggesting ZnSO4 as a potential chemical to curtail drought-induced detrimental effects in water-limited soil conditions.


Introduction
Droughts are one of the most significant challenges in world agriculture, diminishing crop productivity and sustainability [1]. The situation has worsened in recent decades due

Plant Materials and Experimental Design
A high-yielding cotton (G. hirsutum) variety, cv. CB-12, was used to evaluate the effects of Zn supplementations on mitigating the damaging effects of drought stress. Cotton Research Training and Seed Multiplication Farm, Cotton Development Board, Gazipur, Bangladesh, provided the cotton seeds that were utilized in the study. During the experimental period, the temperatures varied from 10 • C to 35 • C, with humidity ranging from 51% to 98%. First, healthy seeds were treated for 20 min with a 5% (v/v) sodium hypochlorite solution prepared with 0.2% (v/v) Tween-20 for surface sterilization, and subsequently rinsed three times with distilled water (dH 2 O). The seeds were pre-soaked in dH 2 O for 8 h at room temperature under dark conditions to accelerate the germination process. Ten pre-soaked seeds were then sown in 2.5-L plastic pots holding 2.6 Kg of soil each. As a means of preparing the soil, the topsoil was combined with cow manure and sand in a weight-based ratio of 2:1:0.5. The pH of the soil was 6.7. The soil was treated with the well-known pesticide furadan (3.0 g Kg −1 ) to stop the spread of diseases that originate there. Prior to seed sowing, 1.0 g ZnSO 4 (ZnSO 4 ·7H 2 O, CAS Number: 7446-20-0, Merck, Germany) or ZnO (CAS Number: 1314-13-2, Merck, Germany) was mixed with per Kg of the potting soil. Appropriate doses of ZnSO 4 and ZnO were selected based on a small-scale experiment (Supplementary Figure S1). Seven equally germinated seeds were kept in each pot. A second dose of Zn supplementation was directly applied to the potted soil after 30 days of seed sowing (five-leaf stage). Forty-day plants at the first-square stage were subjected to drought stress using the methodology of Rahman et al. [32]. Plants were subjected to drought stress by having the irrigation cut off for 6 days, whereas the control plants were provided with adequate water during the experiment. In brief, the whole experimental setup was consisted of six treatment groups: (i) well-watered control plants (WW), (ii) ZnSO 4 -supplemented well-watered control plants (ZnSO 4 ), (iii) ZnO-supplemented well-watered control plants (ZnO), (iv) drought-stressed plants (DS), (v) ZnSO 4 -supplemented drought-stressed plants (ZnSO 4 + DS), and (vi) ZnOsupplemented drought-stressed plants (ZnO + DS). All morphological, physiological, and biochemical analyses were executed on the 3rd leaves (from the bottom), which were freshly harvested from 46-day-old plants. The experiment was performed three times to confirm the precision of the findings.

Measurement of Growth-Related Attributes and Leaf Succulence
Three cotton plants were randomly chosen from each treatment to measure shoot length, shoot dry weight (SDW), and root dry weight (RDW). Shoot length (cm) was measured using a measuring scale from the soil contact point to the tip of the plant. To measure SDW and RDW (g), samples were placed into an envelope, followed by oven drying at 72 • C until the weight became constant before recording the weights using an electric balance. Leaf area (cm 2 ) and leaf succulence were estimated using the 3rd leaf (counted from the bottom) following the formulae of Carleton and Foote [33] and Silveira et al. [34], respectively.

Estimation of Electrolyte Leakage and Leaf Relative Water Contents
Leaf electrolyte leakage and RWCs were measured following the methods described by Das et al. [35] and Rahman et al. [36], respectively.

Evaluation of the Gas Exchange Parameters
Under full sunlight, between 11:30 A.M. and 1:00 P.M., a transportable infrared gas analyzer system (LI-6400XT, LI-COR Inc., Lincoln, NE, USA) was employed to measure net photosynthetic rate (P n ), leaf temperature (LT), stomatal conductance to H 2 O (g sw ), and transpiration rate (E). Thereafter, intrinsic water-use efficiency (WUE int ) was calculated by dividing P n by g sw , while instantaneous water-use efficiency (WUE ins ) was calculated by dividing P n by E.

Determination of the Levels of Chlorophylls and Carotenoids
The contents of chlorophylls (Chl a, Chl b, and total Chls) and carotenoids (Cars) were determined in leaf extracts collected using 80% acetone, following the methods described by Lichtenthaler and Wellburn [37].

Measurement of Malondialdehyde and Hydrogen Peroxide Levels
Malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) levels in cotton leaf tissues were measured using the methods outlined by Kim et al. [38] and Yu et al. [39], respectively.

Measurement of Proline and Water-Soluble Proteins Contents
The content of proline was measured using the acid-ninhydrin method as outlined by Bates et al. [41] with a slight modification. First, acid-ninhydrin was prepared by mixing 0.625 g of ninhydrin in 15 mL of glacial acetic acid and 10 mL of 6 M orthophosphoric acid. Around 0.1 g of fresh cotton leaves was homogenized in 2.5 mL of a 3% sulfosalicylic acid aqueous solution (extraction buffer), and then centrifuged at 12,000 rpm for 10 min. Afterward, 0.5 mL of the supernatant was mixed with 0.5 mL of acid-ninhydrin and 0.5 mL of glacial acetic in a glass tube and incubated in a water bath at 100 • C for 1 h, and subsequently kept in ice to terminate the reaction. The reactant solution was then mixed with 1.5 mL of toluene, vortexed for 10 s, and incubated at room temperature for 10 min. The absorbance of the upper light orange layer separated from the aqueous phase was measured at 520 nm. Toluene was used as a blank. The concentration of proline was estimated from a standard curve using the following formula provided by Bates et al. [41].
On the other hand, the Bradford protein assay was employed to quantify the watersoluble proteins (WSPs) content of the cotton leaf samples [42]. Total free amino acids content were determined following the protocol of Lee and Takahashi [43].

Statistical Analysis
Statistix version 10.0.0.9 software (Analytical Software, Tallahassee, FL, USA) was used to perform one-way analysis of variance (ANOVA) and a mean comparison test on the acquired data using the least significant difference (LSD) test at a significance level of p < 0.05. Significant differences among the treatments are represented by alphabetic letters. The experiment was performed three times, and each of them had three biological replicates per treatment, and similar results were obtained. Means and standard deviations (SDs) of three biological replicates for each treatment from one independent experiment are shown in the figures.

Selection of the Best Dose of ZnSO 4 and ZnO for Drought Alleviation
A noteworthy improvement in phenotypic appearance, such as less wilting, yellowing, and the drying of leaves, in ZnSO 4 (1 and 1.5 g Kg −1 soil) and ZnO (1 and 1.5 g Kg −1 soil)-supplemented drought-stressed cotton plants was observed as compared with that of 'DS' plants (Supplementary Figure S1A In case of ZnO, all shoot biomass-related parameters under drought stress conditions tended to gradually increase up to 1.0 g Kg −1 soil supplementation, and afterwards, they showed a decreasing trend (Supplementary Figure S1E,F). Considering the above findings, the 1.0 g Kg −1 soil dose for both chemicals was chosen to carry out further physiological and biochemical studies.

Both ZnSO 4 and ZnO Improved the Phenotypes, Growth, and Biomass of Cotton Plants Subjected to Drought Stress
Water deprivation for 6 days caused remarkable disturbances in the phenotypic appearance of 'DS' plants as compared with that of 'WW' plants ( Figure 1A,B). Typical drought-stressed symptoms such as wilting, the yellowing of leaves, and the drying of the bottom leaves were evidently observed in 'DS' plants ( Figure 1A,B). Contrariwise, a noteworthy improvement in phenotypes such as less wilting and the yellowing of leaves were observed in 'ZnSO 4 + DS' and 'ZnO + DS' plants ( Figure 1A Figure 1G). In general, plants supplemented with ZnSO 4 had a better phenotypic appearance, as well as better growth and biomass outputs, than those of ZnO-supplemented plants in both stressed and non-stressed circumstances. 'ZnSO4', and 'ZnO' plants were comparable ( Figure 1G). In general, plants supplemented with ZnSO4 had a better phenotypic appearance, as well as better growth and biomass outputs, than those of ZnO-supplemented plants in both stressed and non-stressed circumstances. Significant changes (p < 0.05) among the treatments are denoted by different letters (a-e) above the bars as calculated using the least significant difference test. Here, the treatments are well-watered

Both ZnSO 4 and ZnO Improved Gas Exchange Attributes of Cotton Plants Subjected to Drought Stress
Significant reductions of P n (by 85.5%), g sw (by 98.0%), and E (by 95.0%) were observed in 'DS' plants, which contrast with the respective values of 'WW' plants ( Figure

Both ZnSO4 and ZnO Improved Gas Exchange Attributes of Cotton Plants Subjected to Drought Stress
Significant reductions of Pn (by 85.5%), gsw (by 98.0%), and E (by 95.0%) were observed in 'DS' plants, which contrast with the respective values of 'WW' plants (

Both ZnSO 4 and ZnO Improved Photosynthetic Pigment Contents of Cotton Plants Subjected to Drought Stress
Expectedly, significantly reduced levels of Chl a, Chl b, total Chls, and Cars were recorded in the leaves of 'DS' plants relative to those of 'WW' plants ( Figure  the bars as calculated using the least significant difference test. Here, the treatments are well-watered plants (WW), ZnSO4-supplemented well-watered plants (ZnSO4), ZnO-supplemented wellwatered plants (ZnO), drought-stressed plants (DS), ZnSO4-supplemented drought-stressed plants (ZnSO4 + DS), and ZnO-supplemented drought-stressed plants (ZnO + DS). FW, fresh weight; WUE, water-use efficiency.

Both ZnSO4 and ZnO Improved Photosynthetic Pigment Contents of Cotton Plants Subjected to Drought Stress
Expectedly, significantly reduced levels of Chl a, Chl b, total Chls, and Cars were recorded in the leaves of 'DS' plants relative to those of 'WW' plants ( Figure

Both ZnSO4 and ZnO Protected Cotton Plants from Drought-Induced Oxidative Damage
In comparison with 'WW' plants, water withholding for 6 days caused significant increases in the levels of H2O2 (by 135.        Significant changes (p < 0.05) among the treatments are denoted by different letters (a-d) above the bars as calculated using the least significant difference test. Here, the treatments are well-watered

Both ZnSO 4 and ZnO Improved Water Balance and Osmoprotectant Levels
Under drought stress, 'DS' plants showed remarkable increases in the content of proline (by 12,250.8%), total free amino acids (166.3%), and WSPs (34.6%) as compared with those of 'WW' plants ( Figure 6A-C). Contrariwise, RWCs were found to be significantly reduced in 'DS' plants in comparison with those in 'WW' plants ( Figure 6D). On the other hand, the Zn application significantly decreased the proline (by 64.0 and 57.7%) and total free amino acid (by 28.6 and 15.9%) levels, but increased the WSP (by 29.7 and 64.0%, respectively) contents in 'ZnSO 4 + DS' and 'ZnO + DS' plants relative to those of 'WW' plants ( Figure 6A-C). Interestingly, the RWCs level were found to be significantly increased in 'ZnSO 4 + DS' and 'ZnO + DS' plants compared with the respective values in 'DS' plants ( Figure 6D). Although the proline and WSP contents remained comparable among 'WW', 'ZnSO 4 + DS', and 'ZnO + DS' plants, the total free amino acid contents (by 43.0 and 42.8%) and RWCs (by 7.1%) significantly increased in 'ZnSO 4 ' and 'ZnO' plants, respectively, compared with the respective values in 'WW' plants ( Figure 6A-D).

Both ZnSO4 and ZnO Improved Water Balance and Osmoprotectant Levels
Under drought stress, 'DS' plants showed remarkable increases in the content of proline (by 12,250.8%), total free amino acids (166.3%), and WSPs (34.6%) as compared with those of 'WW' plants ( Figure 6A-C). Contrariwise, RWCs were found to be significantly reduced in 'DS' plants in comparison with those in 'WW' plants ( Figure 6D). On the other hand, the Zn application significantly decreased the proline (by 64.0 and 57.7%) and total free amino acid (by 28.6 and 15.9%) levels, but increased the WSP (by 29.7 and 64.0%, respectively) contents in 'ZnSO4 + DS' and 'ZnO + DS' plants relative to those of 'WW' plants ( Figure 6A-C). Interestingly, the RWCs level were found to be significantly increased in 'ZnSO4 + DS' and 'ZnO + DS' plants compared with the respective values in 'DS' plants ( Figure 6D). Although the proline and WSP contents remained comparable among 'WW', 'ZnSO4 + DS', and 'ZnO + DS' plants, the total free amino acid contents (by 43.0 and 42.8%) and RWCs (by 7.1%) significantly increased in 'ZnSO4' and 'ZnO' plants, respectively, compared with the respective values in 'WW' plants ( Figure 6A-D).

Discussion
The current study provides a picture of how Zn application, either in the form of ZnSO4 or ZnO, confronted the deleterious effects of drought in cotton plants applied at the first square stage. To understand Zn-mediated drought tolerance mechanisms, we explored various morphological and biochemical features that are crucial for cotton survival in water-limited circumstances.
Drought imposition induced severe damage to cotton plants, as seen by the withering, yellowing, and drying of leaves, as well as the considerable slowing of growth and biomass gain ( Figure 1A-F). Zn supplementations, on the other hand, reduced leaf wilting and yellowing symptoms and increased growth and biomass production, thus helping cotton plants overcome the drought-induced negative effects ( Figure 1A-F). Importantly, the increased root biomass in Zn-supplemented plants indicated that Zn enabled cotton plants to forage more water and nutrients, which ultimately resulted in enhanced

Discussion
The current study provides a picture of how Zn application, either in the form of ZnSO 4 or ZnO, confronted the deleterious effects of drought in cotton plants applied at the first square stage. To understand Zn-mediated drought tolerance mechanisms, we explored various morphological and biochemical features that are crucial for cotton survival in water-limited circumstances.
Drought imposition induced severe damage to cotton plants, as seen by the withering, yellowing, and drying of leaves, as well as the considerable slowing of growth and biomass gain ( Figure 1A-F). Zn supplementations, on the other hand, reduced leaf wilting and yellowing symptoms and increased growth and biomass production, thus helping cotton plants overcome the drought-induced negative effects ( Figure 1A-F). Importantly, the increased root biomass in Zn-supplemented plants indicated that Zn enabled cotton plants to forage more water and nutrients, which ultimately resulted in enhanced photosynthetic activity and improved growth characteristics in drought-stricken environments ( Figure 1E) [44,45]. The notable improvements in cotton growth and the shoot and root biomass of Zn-supplemented plants supported that exogenous Zn played a cru-cial role in enhancing cotton's resilience to drought stress. Improvements of the plant's growth and biomass under water-stress conditions upon the application of ZnO [27] and ZnSO 4 [21] were also reported in H. annuus. Interestingly, the current study found that ZnSO 4 -supplemented plants thrived and generated more shoot and root biomass than ZnO-supplemented plants did in both stress and non-stress situations ( Figure 1A-F). A widespread agreement exists that soil pH is a major determinant of Zn availability, especially when it is applied in a non-soluble form such as ZnO [46][47][48]. In general, soil acidity increases ZnO solubility and availability [48]. On the other hand, a previous study showed that when Zn was administered as ZnSO 4 (the ionic form), it could be dissolved across a wide range of pH values (4.10-7.98) [49]. The soil used in the current study could be classified as neutral because the soil pH was 6.7. This could be a limiting factor that reduced Zn availability to the plants supplemented with ZnO. It could be likely that the higher solubility and better distribution nature of ZnSO 4 compared to those of ZnO might ensure increased availability of Zn to soil; thus, ZnSO 4 performed better in improving cotton's growth performance and drought tolerance ( Figure 1A-F).
Water stress undermines crop growth and productivity by impairing the leaf water status and gas exchange ability of plants [50]. Drought stress reduces the stomatal conductance and transpiration rates, resulting in higher leaf temperature and wilting [51], as also demonstrated in the current study ( Figure 1A,B, and Figure 2D). It is well known that Zn improves stomatal conductance by maintaining K + influx in guard cells, resulting in higher WUE in water-stressed conditions [33]. In line with this statement, noteworthy improvements in stomatal conductance, the transpiration rate, and WUE were observed in 'ZnSO 4 + DS' and 'ZnO + DS' plants, indicating a better drought acclimation strategy in Zn-supplied cotton plants ( Figure 2B,C,E,F).
Reduced growth and biomass production ( Figure 1A-F) can be attributed partly to drought-induced impairment of the photosynthetic performance (Figure 2A), possibly through the degradation of the photosynthetic pigments ( Figure 3A-D), as also observed in Glycine max and Oudeneya africana under water-shortage conditions [52,53]. Conversely, Zn-supplemented cotton plants retained higher levels of photosynthetic pigments (e.g., Chl a, Chl b, total Chls, and Cars), along with a heightened photosynthetic rate, upon their exposure to water-withholding-induced drought stress (Figures 2A and 3A-D). These findings suggested that Zn might contribute to the synthesis and/or slowing of the destruction of photosynthetic pigments, thereby aiding in the maintenance of optimal photosynthetic performance under water-stressed conditions (Figures 2A and 3A-D). Indeed, Zn acts as a co-factor for many proteins and enzymes involved in plant pigment biosynthesis, which is required to keep the chlorophyll biosynthesis pathway active, particularly in stressful environments [54]. The beneficial effects of Zn application in augmenting photosynthetic pigments and photosynthetic efficiency have also been well documented in a range of plant species, including S. lycopersicum, T. aestivum, and Cicer arietinum [55][56][57]. Previous reports suggest that there is a direct relationship between higher net photosynthesis and higher leaf area [33], which was also evidently observed in Zn-supplemented cotton plants ( Figure 1F). Leaf area expansion directly regulates the light acquisition rate of plants, and thus, the overall photosynthetic performance and carbon assimilation and distribution in different plant parts [58][59][60]. Together, Zn supplementation improved the gas exchange characteristics and safeguarded photosynthetic pigments, which enhanced WUE and photosynthetic efficiency to support better growth and survival in a water-scarce environment.
Drought-induced ROS accumulation is thought to have a correlation with photooxidative damage and impairment of the plant's growth [61,62], as was also clearly found in drought-stressed cotton plants ( Figure 1A,B and Figure 4A). Our analyses showed that both 'ZnSO 4 + DS' and 'ZnO + DS' plants generated less H 2 O 2 , and consequently, reduced levels of MDA and EL under water-deficit conditions ( Figure 4A-C). Plants experiencing drought are more prone to lipid peroxidation, which results in the membrane damage responsible for a number metabolic disturbance at the cellular level [63]. Zn is special because it does not go through redox cycling while it is in the divalent state, which allows it to survive and be stable in biological media [64]. These characteristics allow Zn to shield membrane lipids from ROS toxicity, which in turn prevents ion leakage through the membranes [17]. Moreover, Zn addition remarkably enhanced the activities of several key antioxidant enzymes, namely CAT, GST, GPOD, and APX, in drought-stressed cotton plants ( Figure 5A-D), suggesting a crucial role of Zn in protecting cells from oxidative damage by eliminating excessive H 2 O 2 ( Figures 4A and 5A,C,D). The enhanced activity of GST in Zn-supplemented drought-stressed cotton plants ( Figure 5B) might activate the GSH-dependent peroxide quenching mechanism to provide better protection against the drought-induced organic peroxides, reactive aldehydes, and lipid hydroperoxides [65]. In general, plants treated with ZnSO 4 have higher levels of antioxidant activity than plants supplied with ZnO do, which is correlated with substantially lower buildup of H 2 O 2 and MDA in 'ZnSO 4 + DS' plants compared to that of 'ZnO + DS' plants ( Figures 4A and 5A-D). This might be another reason for ZnSO 4 -treated plants' improved drought acclimatization responses. In support of the current study, higher levels of antioxidant activities upon ZnSO 4 application were also reported in G. hirsutum under heat stress [20], in H. annuus under drought stress [21], and in S. melongena under salinity stress [23].
Under drought stress, plants accumulate a range of osmoprotectants, predominantly proline, to carry out osmotic adjustment [66]. Our observation indicated that despite the high accumulation of proline, proper RWCs were not maintained in 'DS' plants ( Figure 6A,D). Likewise, when we were directly comparing them with 'DS' plants, the proline content was negatively correlated with the RWCs in 'ZnSO 4 + DS' and 'ZnO + DS' plants, suggesting that Zn might contribute to replenishing water loss without significantly accumulating proline ( Figure 6A). Thus, our study supported that the level of proline accumulation was correlated with the severity of cellular dehydration in cotton [67,68]. The extensive accumulation of free amino acids has usually been observed in response to different abiotic stresses, including drought ( Figure 6B) [69]. An inadequate carbon supply due to reduced photosynthesis under drought stress provokes the accumulation of free amino acids (Figures 2A and 6B), which can be used as alternative substrates for mitochondrial respiration [70]. However, increased photosynthetic efficiency may revert the cellular metabolism to more favorable growth conditions and reduce the accumulation of free amino acids [71], as was evident in 'ZnSO 4 + DS' and 'ZnO + DS' plants in the current study (Figures 2A and 6B). Alternately, Zn-supplemented plants might have used more free amino acids to increase their biomass even under drought conditions ( Figure 1A-F), and as a result, there was lower accumulation of free amino acids in the plants ( Figure 6B). Under water-deficit conditions, plants accumulate different low-molecular-weight substances, including WSPs, to maintain the osmotic balance [72]. Moreover, the role of Zn in improving protein synthesis is well recognized [17], as we expectedly recorded in 'ZnSO 4 + DS' and 'ZnO + DS' plants ( Figure 6C). The increased accumulation of WSPs may aid plants in keeping the leaf turgor pressure and stomatal conductance favorable for efficient CO 2 absorption and boosting the capacity of the plant's roots to effectively harness water [73]. A generalized mechanism of Zn-induced drought tolerance in cotton is presented in Figure 7. Function of zinc (Zn) in regulating cotton physiological and biochemical mechanisms to overcome drought-caused negative effects. Zn supplementation (either in the form of ZnSO4 or ZnO) to drought-stressed cotton plants significantly decreased their growth penalty, partly by protecting photosynthetic pigments and improving gas exchange features, which finally enhanced the overall photosynthetic efficiency and growth performance. Zn addition also activated the antioxidant defense mechanism by increasing the activities of enzymatic antioxidants (e.g., CAT, GST, GPOD, and APX), which helped protect Zn-supplemented cotton plants against reactive oxygen species (ROS)-induced oxidative stress and membrane damage. Furthermore, through boosting water-soluble proteins (WSPs) content, Zn aided osmoprotection, which eventually helped preserve leaf water status for osmotic adjustment under water-scarce conditions. Blue arrow represents an upward trend, while red arrow denotes a declining trend. APX, ascorbate peroxidase; Cars, carotenoids; CAT, catalase; Chls, chlorophylls; E, transpiration rate; EL, electrolyte leakage; GST, glutathione S-transferase; GPOD, guaiacol peroxidase; gsw, stomatal conductance to H2O; H2O2, hydrogen peroxide; MDA, malondialdehyde; Pn, net photosynthetic rate; RWCs, relative water contents; Zn, zinc.

Conclusions
The current study revealed the important roles of Zn in the modulation of physiological and biochemical defense mechanisms that assist cotton plants to overcome watershortage-induced negative consequences (Figure 7). When they are contrasted with Znnon-supplemented plants alone, Zn-supplemented cotton plants exhibited a greater root Figure 7. Function of zinc (Zn) in regulating cotton physiological and biochemical mechanisms to overcome drought-caused negative effects. Zn supplementation (either in the form of ZnSO 4 or ZnO) to drought-stressed cotton plants significantly decreased their growth penalty, partly by protecting photosynthetic pigments and improving gas exchange features, which finally enhanced the overall photosynthetic efficiency and growth performance. Zn addition also activated the antioxidant defense mechanism by increasing the activities of enzymatic antioxidants (e.g., CAT, GST, GPOD, and APX), which helped protect Zn-supplemented cotton plants against reactive oxygen species (ROS)-induced oxidative stress and membrane damage. Furthermore, through boosting water-soluble proteins (WSPs) content, Zn aided osmoprotection, which eventually helped preserve leaf water status for osmotic adjustment under water-scarce conditions. Blue arrow represents an upward trend, while red arrow denotes a declining trend. APX, ascorbate peroxidase; Cars, carotenoids; CAT, catalase; Chls, chlorophylls; E, transpiration rate; EL, electrolyte leakage; GST, glutathione S-transferase; GPOD, guaiacol peroxidase; g sw , stomatal conductance to H 2 O; H 2 O 2 , hydrogen peroxide; MDA, malondialdehyde; P n , net photosynthetic rate; RWCs, relative water contents; Zn, zinc.

Conclusions
The current study revealed the important roles of Zn in the modulation of physiological and biochemical defense mechanisms that assist cotton plants to overcome watershortage-induced negative consequences (Figure 7). When they are contrasted with Znnon-supplemented plants alone, Zn-supplemented cotton plants exhibited a greater root biomass, which might contribute to the acquisition of more water and nutrients for maintaining a better water status and photosynthetic performance under drought conditions ( Figures 1E and 2A). Zn application also radically reduced ROS accumulation, along with curtailing MDA and EL levels ( Figure 4A-C), suggesting that Zn might play a key role in reversing ROS-induced oxidative damage to support cotton plants' survival through drought stress. These results were further supported by augmented activities of enzymatic antioxidants, including CAT, GST, GPOD, and APX, which have important implications in ROS elimination ( Figure 5A-D). Increased levels of WSPs upon Zn supplementation also assisted the water-stressed cotton plants to improve the water status and maintain the optimum osmotic balance ( Figure 6C,D). This study also pointed out that ZnSO 4 supplementation improved cotton's drought tolerance compared with that caused by ZnO supplementation. One important reason for this observation may be the neutral pH of the soil used in this study, which might prevent ZnO from dissolving entirely, making it inaccessible to plants. It is also plausible that the dissociation of ZnSO 4 provided sulfur, which could potentiate the formation of small defense molecules such as glutathione for conferring an extra protection layer against drought stress. Altogether, our results suggest that Zn acted as an imperative regulator of several morphological and biochemical processes, which increased the resilience of cotton plants toward drought stress. Moreover, our observations provide important clues at the physiological and biochemical levels that could be taken into account for further evaluation of Zn-regulated drought tolerance networks at the molecular level in cotton plants. Finally, additional research, including field tests and cost-benefit analyses, should be conducted to confirm that Zn application is a successful strategy for minimizing the negative effects of drought and lowering the cotton yield losses in soils with a limited water supply.