Foliar Potassium Sulfate Application Improved Photosynthetic Characteristics, Water Relations and Seedling Growth of Drought-Stressed Maize

Changing climates and frequent spells of drought have increased the risk of crop failure, especially in arid and semi-arid regions, thus multiplying the vulnerability of food-insecure populations. The exogenous application of potassium (K) can potentially ameliorate the adverse effects of drought in maize by maintaining cell osmotic potential and turgidity, provided its optimum doses are applied. The present experiment comprised two maize cultivars, viz. Islamabad Gold (drought tolerant) and Azam (drought susceptible), grown under well-watered (80% water-holding capacity (WHC)), mild drought (60% WHC) and severe drought (40% WHC) conditions. Different doses of K, viz. 0%, 1% and 2%, were also tested to screen out the most superior concentration. Drought stress markedly reduced root and shoot lengths (25% and 16%, respectively) along with their dry weights (20% and 10%, respectively). Moreover, a substantial reduction in leaf relative water content (RWC) (24%), stomatal conductance, transpiration and photosynthesis rates, chlorophyll pigments a, b and total chlorophyll contents (31%) were recorded, compared with well-watered conditions. However, foliar application of K2SO4 at 2% concentration outperformed other doses by improving growth attributes, RWC (10%), total chlorophyll (9%) and proline (12%) under severe drought conditions. Our findings confirmed the effectiveness of foliage-applied K2SO4 in ameliorating drought effects in rainfed maize; however, more doses and sources of K could be tested for developing it as a potent source to cope with water stress.

adverse effects of DS and improving water relations, photosynthetic characteristics and the growth of maize genotypes grown under well-watered and DS conditions.

Materials and Methods
This experiment was conducted at the greenhouse of the Department of Horticulture, PMAS-Arid Agriculture University, Rawalpindi, Pakistan, to study the effect of potassium sulfate (K 2 SO 4 ) on two maize cultivars, Islamabad gold (drought tolerant) and Azam (drought susceptible), grown under DS. These cultivars were collected from the National Agricultural Research Council, Islamabad, Pakistan, and are high yielding and locally preferred for cultivation. The design of the experiment was a completely randomized design (CRD) with three replicates for each treatment. Earthen pots 20 cm in diameter and 45 cm in height were filled with an 8 kg mixture of soil and farmyard manure (FYM). Soil and FYM were mixed in a 2:1 ratio, and 6 seeds per pot were manually sown to avoid germination constraints. A soil sample from pot soil indicated that the soil was alkaline in nature with a 7.20 pH, an electrical conductivity of 0.75 dS m −1 , 0.48% organic matter and 0.03% N, along with available phosphorous and K contents of 4.30 and 78 mg kg −1 , respectively. Both maize cultivars were grown in pots under well-watered conditions. Thinning was done manually at the 3-leaf stage, and only 2 plants per pot were kept to maintain the optimum plant population and promote the establishment of seedlings. Drought was imposed after one week of thinning. For imposing drought, pots were divided into three sets, and drought was maintained as follows: (i) well-watered (WW) (80% water holding capacity-WHC); (ii) mild drought (MD) (60% WHC); and (iii) severe drought (SD) (40% WHC). To maintain WHC, pots were weighed after every 3-4 days of drought imposition, and a difference in weight was achieved by adding water to attain the required weight. After one week of imposing drought, a foliar spray of water and K 2 SO 4 was applied twice in 3-day intervals. For this purpose, plants were treated as follows: (i) water spray (control) using distilled water; (ii) 1% K 2 SO 4 (1 g K 2 SO 4 was dissolved in 1L of distilled water); (iii) 2% K 2 SO 4 (2 g K 2 SO 4 was dissolved in 1L of distilled water), whereas Tween 20 at 0.2 mL L −1 was used as a surfactant. Before spraying, the soil surfaces of treated pots were covered with polythene sheets to avoid contamination. NPK, in the form of urea, DAP and SOP, was applied in quantities of 0.6, 0.27 and 0.06 g per pot, respectively. Seedlings were harvested 60 days after sowing for the measurement of morphological traits.

Estimation of Relative Water Content (%)
For measuring leaf relative water content (RWC), samples were randomly collected from each treatment and packed into polythene bags. The leaves' fresh weight (FW) was measured by using a digital weighing balance. Then, the leaves were soaked in distilled water for 24 h to make them turgid, and then the turgid weight (TW) was measured using a digital weighing balance. Thereafter, the leaves were oven-dried at 75 • C for 72 h, and subsequently, the dry weight (DW) was measured. The RWC was calculated by using the below formula: RWC = (FW − DW)/(TW − DW) × 100 FW = Fresh weight of leaves DW = Dry weight of leaves TW = Turgid weight of leaves.

Estimation of Gas Exchange Parameters
Gas exchange parameters, viz. stomatal conductance (Gs), photosynthesis rate (Pn) and transpiration rate (Tr), were measured using an infrared gas analyzer (IRGA Leaf Chamber Analyzer, Type LCA-4, manufacturer, city and state abbreviation, USA) two weeks after the foliar spray of K 2 SO 4 . For this purpose, fully developed expanded leaves were selected from each experimental pot and inserted in an IRGA Leaf Chamber Analyzer (Type LCA-4, manufacturer, city and state abbreviation, USA) [36]. Measurements were Atmosphere 2021, 12, 663 4 of 13 taken from 9:00 a.m. to 11:00 a.m. on a clear, sunny day by keeping the CO 2 concentration at 400-µmol mol −1 [36].

Estimation of Chlorophyll Content
Chlorophyll (a, b and total) contents were measured by using the method proposed by Ref. [37]. About 1 g of fresh leaves were taken and divided into segments that were kept in 80% acetone solution for a whole night. Then, samples were centrifuged at 14,000 rpm for 5 min, and supernatant was observed at 645 nm and 663 nm wavelengths using a UV spectrophotometer (Unicam 8620). Chlorophyll a, b and total contents were measured using the following formula where V = volume of the leaf extract (mL), W = weight of fresh leaf tissue (g).

Estimation of Proline (mg g −1 )
The proline content was measured according to the procedure developed by [38]. Homogenization of 0.5 g of fresh leaves was done with 10 mL of 3% sulfosalicylic acid (C7H6O6S) solution. Filtration of the homogenized mixture was done with 2 ml of acid ninhydrin and 2 ml of glacial acid in a test tube. The mixture was incubated for 60 min at 100 • C. Then, the mixture was cooled in an ice bath and 4 mL of toluene was mixed into it while stirring simultaneously for 60 s. A spectrophotometer with 520-nanometer wavelength radiations was used to measure chromophore absorption. Proline contents were calculated on the basis of fresh weight from the optimized curve using the following formula: µmol proline g −1 fresh weight = (µg proline mL −1 × mL of toluene/115.5)/(g of sample).

Morphological Traits
Plants were uprooted two weeks after foliar application of K 2 SO 4 from well-watered and drought-stressed treatments, and the shoot length (SL) of plants was measured using a meter scale. After measuring the SL, the roots were separated, and their root length (RL) was measured using the same scale and then averaged to determine the RL per plant. After the measurement of the RL, the shoot and root from each treatment were placed into envelopes and dried in a hot air oven at 75 • C for 72 h. After drying the samples, the shoot dry weight (SDW) and root dry weight (RDW) was measured using a digital weighing balance.

Statistical Analysis
Data for different attributes were analyzed using Statistix 8.1 software to determine the significance among the treatments. Tukey's test at a 5% probability level was executed to find out the significant differences among treatment means [39]. Bar graphs using standard error were prepared in Microsoft Excel 2007.

Results
Various levels of DS and foliar K 2 SO 4 significantly affected the RL of maize. Under drought regimes, increased RL was measured in WW conditions, while drought levels decreased the RL, and the lowest RL was recorded under severe drought (SD), where 40% WHC was maintained ( Figure 1A). Similarly, maize cultivars varied significantly in terms of RL, and a higher RL was recorded in the Azam genotype, compared with Islamabad Gold ( Figure 1A). Drought effects were mitigated through the foliar application of potassium sulfate, which improved the RL, and a 2% solution of potassium sulfate produced the highest RL compared to other treatments ( Figure 1A). The interactive effect of both factors was significant regarding the RL (Table 1), and the highest RL (35cm) was recorded in Islamabad Gold under well-watered conditions with the foliar application of 2% potassium sulfate solution. The minimum RL (22 cm) was recorded by Azam under DS, where 40% WHC was maintained through spraying water only ( Figure 1A). From the results of this study, it is shown that various drought conditions and K applications had significant effects on the RDW. Under drought regimes, the maximum RDW was measured under WW conditions, while the minimum RDW was measured under SD ( Figure 1B). Similarly, maize cultivars were also found to be significant regarding RDW. The maximum RDW was obtained in Islamabad Gold, compared to Azam. Foliar application of potassium sulfate improved the RDW, and the highest RDW was recorded with the 2% potassium sulfate solution, compared with the control and the 1% solution ( Figure 1B). Significant interactive effects were found, and the highest RDW was measured in Islamabad Gold with the 2% potassium sulfate solution under WW conditions, while the least RDW (8g) was measured for Azam with simple water spraying under SD ( Figure 1B). Results also revealed that drought stress levels and potassium sulfate doses significantly influenced the SL of maize (Table 1; Figure 1C). The highest SL was measured under WW conditions while the least SL was recorded under SD ( Figure 1C).
Atmosphere 2021, 12, x FOR PEER REVIEW 5 of 13 the highest RL compared to other treatments ( Figure 1A). The interactive effect of both factors was significant regarding the RL (Table 1), and the highest RL (35cm) was recorded in Islamabad Gold under well-watered conditions with the foliar application of 2% potassium sulfate solution. The minimum RL (22 cm) was recorded by Azam under DS, where 40% WHC was maintained through spraying water only ( Figure 1A). From the results of this study, it is shown that various drought conditions and K applications had significant effects on the RDW. Under drought regimes, the maximum RDW was measured under WW conditions, while the minimum RDW was measured under SD ( Figure 1B). Similarly, maize cultivars were also found to be significant regarding RDW. The maximum RDW was obtained in Islamabad Gold, compared to Azam. Foliar application of potassium sulfate improved the RDW, and the highest RDW was recorded with the 2% potassium sulfate solution, compared with the control and the 1% solution ( Figure 1B). Significant interactive effects were found, and the highest RDW was measured in Islamabad Gold with the 2% potassium sulfate solution under WW conditions, while the least RDW (8g) was measured for Azam with simple water spraying under SD ( Figure 1B). Results also revealed that drought stress levels and potassium sulfate doses significantly influenced the SL of maize (Table 1; Figure 1C). The highest SL was measured under WW conditions while the least SL was recorded under SD ( Figure 1C). Similarly, exogenous K increased the SL and SDW, and the longest and heaviest shoots were produced by Islamabad Gold with the 2% potassium sulfate solution under WW conditions, while the minimum corresponding values were recorded for Azam with water spraying under SD ( Figure 1C,D).    Similarly, exogenous K increased the SL and SDW, and the longest and heaviest shoots were produced by Islamabad Gold with the 2% potassium sulfate solution under WW conditions, while the minimum corresponding values were recorded for Azam with water spraying under SD ( Figure 1C,D).
Photosynthetic characteristics including transpiration rate (Tr), stomatal conductance (Gs) and photosynthesis rate (Pn) were significantly affected by the different levels of DS, the foliar doses of potassium sulfate and the maize cultivars (Table 1). However, their interaction effect varied significantly for those parameters. The interaction effect for transpiration rate was found to be significant, while for photosynthesis rate and stomatal conductance, the interaction effects were found to be non-significant. The interaction effect between variety and drought levels on the stomatal conductance was found to be significant ( Table 1). The highest transpiration rate was measured in WW Islamabad Gold with a 2% spray of potassium sulfate, while the lowest transpiration rate was measured in the SD-grown Azam cultivar (Figure 2A). On the other hand, maize varieties differed significantly for stomatal conductance, and higher stomatal conductance was recorded in Islamabad Gold, compared with Azam. Moreover, drought levels also significantly affected stomatal conductance, and the value decreased with the increasing stress levels. However, the foliar application of potassium sulfate increased stomatal conductance, compared to water spraying. Maize cultivars, by virtue of their genetic makeup, differed significantly in photosynthetic rate under different levels of DS and foliar K levels: Islamabad Gold performed better than Azam. Moreover, DS reduced the photosynthetic rate of both cultivars, compared to WW conditions. Approximately a 28% increase in photosynthetic rate was recorded in maize under WW condition compared to SD ( Figure 2C). However, the foliar application of potassium sulfate (2%) improved photosynthetic rate by over 10% compared to the control ( Figure 2C). The proline contents of maize were significantly affected by drought and K application. The maximum proline content was measured in the Islamabad Gold cultivar under SD conditions, while the minimum was observed in Azam under WW conditions ( Figure 2D). Foliar application of K also improved proline contents, compared with water spraying ( Figure 2D). Drought levels, maize cultivars and foliar application had significant effects on RWC, chlorophyll (chl) b and total chl contents, while insignificant effects were recorded for chl a ( Table 2). The interaction effect for RWC and chl b was also found to be significant. Maize plants maintained superior RWC under WW condition, while DS reduced the RWC. However, the foliar application of potassium sulfate (2%) improved RWC, especially for Islamabad Gold under WW conditions ( Figure 3A). The interaction effects for varieties × drought and varieties × K were found to be significant ( Table 2). Increase in chl a content was noticed in Islamabad Gold under WW, whereas it was reduced in Azam under SD ( Figure 3B). Similarly, the foliar application of 2% potassium sulfate increased chl a in Islamabad Gold, while a decrease in chl a was observed in Azam grown with water spraying ( Figure 3B). Likewise, the foliar application of 2% potassium sulfate increased chl b in Islamabad Gold (0.19 mg g −1 ) under WW conditions, while less chl b (0.01 mg g −1 ) was observed in Azam when it was grown under SD conditions, and only water was sprayed ( Figure 3C). Moreover, total chl contents of maize were significantly affected by varieties, drought and K application, while their interaction effect was found to be insignificant. Higher total chl contents were produced in Islamabad Gold, compared with Azam. Similarly, the highest total chl contents were observed under WW conditions, whereas drought stress reduced total chl contents. Nevertheless, the foliar application of potassium sulfate improved total chl contents, and the highest total chl contents were observed under 2% foliar spray, compared with the control ( Figure 3D). Drought levels, maize cultivars and foliar application had significant effects on RWC, chlorophyll (chl) b and total chl contents, while insignificant effects were recorded for chl a ( Table 2). The interaction effect for RWC and chl b was also found to be significant. Maize plants maintained superior RWC under WW condition, while DS reduced the RWC. However, the foliar application of potassium sulfate (2%) improved RWC, especially for Islamabad Gold under WW conditions ( Figure 3A). The interaction effects for varieties × drought and varieties × K were found to be significant ( Table 2). Increase in chl a content was noticed in Islamabad Gold under WW, whereas it was reduced in Azam under SD ( Figure 3B). Similarly, the foliar application of 2% potassium sulfate increased chl a in Islamabad Gold, while a decrease in chl a was observed in Azam grown with water spraying ( Figure 3B). Likewise, the foliar application of 2% potassium sulfate increased chl b in Islamabad Gold (0.19 mg g −1 ) under WW conditions, while less chl b (0.01 mg g −1 ) was observed in Azam when it was grown under SD conditions, and only water was sprayed ( Figure 3C). Moreover, total chl contents of maize were significantly affected by varieties, drought and K application, while their interaction effect was found to be insignificant. Higher total chl contents were produced in Islamabad Gold, compared with Azam. Similarly, the highest total chl contents were observed under WW conditions, whereas drought stress reduced total chl contents. Nevertheless, the foliar application of potassium sulfate improved total chl contents, and the highest total chl contents were observed under 2% foliar spray, compared with the control ( Figure 3D).

Discussion
This study was conducted to optimize the dose of foliage-applied potassium sulfate for maize cultivars under varying levels of DS. The results revealed that all morphological and photosynthetic traits, as well as chl a, chl b and total chl contents, were decreased by drought, while proline contents were increased (Figures 1-3). Nonetheless, the foliar application of potassium sulfate increased all these traits under DS (Figures 1-3), because K+ is one of the essential macronutrients that triggers plant growth and development, even under unfavorable environmental conditions. Moreover, K is an integral part of various plant physiochemical and biological cellular mechanisms associated with osmotic adjustments, photosynthetic efficiency, the movement of photosynthates and the activation of enzymes in plant biomes [40]. A deficiency in K under water stress disturbs enzyme activity and the transportation of metabolites and decreases the production and assimilation of photosynthates [41].
Both drought levels significantly reduced the RL, RDW, SL and SDW compared to WW conditions in the current investigation, but foliar-applied K ameliorated the adverse effects of drought ( Figure 1). Furthermore, better root and shoot characteristics were recorded in the drought-tolerant maize genotype, which might be due to the deep root system that assists maize plants in maintaining cellular hydration by increasing water uptake, leading to better growth and higher productivity [42,43]. Reduced RL, RDW, SL and SDW under drought stress were also reported in previous studies [44,45]. The foliar application of K improved the root and shoot characteristics in our study, which might be due to an increased root surface area that improved nutrient and water uptake by roots, and thus root growth was enhanced [46]. The exogenous application of K not only improved root dry matter but also improved shoot dry matter under drought conditions, which might be attributed to enhanced cell membrane permeability under DS, indicating its effectiveness as a drought mitigation agent [47,48].
In addition to morphological traits, photosynthetic characters Tr, Gs, and Pn were also significantly affected by the imposition of DS and the foliar application of K as potassium sulfate in the current investigation ( Figure 2). Reduced photosynthetic characteristics were observed with lower doses of foliage-applied K, and the same was observed in previous studies, wherein K deficiency reduced chlorophyll concentrations in plant leaves [49]. Furthermore, K deficiency prohibited plant growth, while its balanced application improved photosynthetic rate, root and shoot ratio under moisture stress conditions [50]. This was attributed to the improved transpiration activity of plants caused by exogenously applied K, which maximized water loss through transpiration, enhanced nutrient assimilation in the roots and improved the efficiency of damaged tissues, leading to a reduction in osmotic stress under moisture deficit conditions. Additionally, the foliar K also enhanced RWC through the higher rate of transpiration activity [51][52][53][54].
Regularity in stomatal conductance has also been considered an important mechanism for energy generation during the photosynthesis process and for plant cooling, along with the transportation of moisture and mineral nutrients. In the present study, stomatal conductance was significantly improved by the exogenous application of K, probably owing to the modulation of stomatal opening and closing by K, thereby better regulating the gaseous movement between plants and the environment, leading to improved stomatal conductance. In addition, K application also regulated evapotranspiration via pores under moisture-stressed environments and helped the plants survive under moisture deficit conditions [55]. Previous studies report that K plays an integral part in carbohydrate synthesis and the transportation of metabolites, which enables plants to survive moderate spells of drought stress. The increase in photosynthetic efficiency under drought circumstances with K treatment is due to the improvement in leaf number and leaf size [40,56]. The improvement in leaf number and size latterly accelerates the photosynthesis rate per leaf area unit, which profoundly contributes to an overall improvement in the number of photosynthates available for plant growth [40]. Moreover, K regulates photosynthetic efficiency by increasing sunlight interception [57]. In our study, increased proline contents under SD in drought-tolerant cultivars ameliorated drought effects through improved osmoregulation [58,59]. It was also inferred that proline act as a signaling molecule to influence cell proliferation or cell death, and it triggered specific gene expression for plant recovery from stress [60].
Drought levels, maize cultivars and foliar application had a significant effect on relative water contents; however, drought-tolerant cultivars exhibited higher RWC, as they preserved more water under drought by virtue of osmotic adjustment and stomatal regulation [59]. Moreover, foliar application of K improved RWC (Figure 3), which might be due to the role of K in stomatal regulation and osmotic adjustment [25]. Chlorophyll a, b and total contents were reduced under moisture stress conditions due to the destruction of chlorophyll and reduced photosynthetic activity [61]. The inhibition of chlorophyll content in most stressed plants was probably due to the disorganization of thylakoid membranes and degradation of chlorophyll through the formation of proteolytic enzymes such as chlorophyllase [62]. Moreover, chlorophyll tends to decline under stress due to the biosynthesis of O 2− and H 2 O 2 , which causes lipid peroxidation and ultimately chlorophyll degradation [61]. However, the foliar application of K in higher concentrations enhanced the chlorophyll contents, and similar results have also been reported by [63].

Conclusions
It is inferred that foliar potassium sulfate ameliorated the adverse effects of various levels of drought stress in maize genotypes. Foliage-applied potassium sulfate in a 2% concentration boosted root and shoot lengths (25% and 16%, respectively), along with the root dry weight. Moreover, a foliar spray of 2% K 2 SO 4 improved proline and chlorophyll contents, which in turn enhanced photosynthetic activity and maintained higher relative water content under severe drought conditions. Moreover, we concluded that maize genotypes differ in their potential to cope with drought stress, and the screening of drought-tolerant cultivars of maize combined with foliage-applied potassium sulfate holds significant potential in increasing maize production under changing climates. However, there is a dire need to perform screening studies to identify more genetically superior cultivars of maize, along with testing other concentrations and sources of potassium to alleviate drought effects under changing climate scenarios.