Selenium Mediated Alterations in Physiology of Wheat under Different Soil Moisture Levels

: Soil moisture stress is one of the most serious aspects of climate change. Selenium (Se) is regarded as an essential element for animal health and has been demonstrated to protect plants from a number of abiotic challenges; however, our knowledge of Se-regulated mechanisms for enhancing crop yield is limited. We investigated the effects of exogenous Se supplementation on physiological processes that may impact wheat productivity during soil moisture stress. The plants were grown in plastic containers under screen-house conditions. The experiment was laid out in CRD consisting of three soil moisture regimes, i.e., control (soil moisture content of 12.5 ± 0.05%), moderate (soil moisture content of 8.5 ± 0.05%), and severe moisture stress (soil moisture content of 4.5 ± 0.05%). Selenium was supplied using sodium selenite (Na 2 SeO 3 ) through soil application before sowing (10 ppm) and foliar application (20 ppm and 40 ppm) at two different growth stages. The foliar spray of Se was applied at the vegetative stage (70 days after planting) and was repeated 3 weeks later, whereas the control consisted of a water spray. The water status, photosynthetic efﬁciency, and yield were signiﬁcantly decreased due to the soil’s moisture stress. The exogenous Se application of 40 ppm resulted in decreased negative leaf water potential and improved relative water contents, photosynthetic rate, transpiration rate, and stomatal conductance in comparison to the control (without selenium) under water shortage conditions except the plants treated with soil application of selenium under severe moisture stress at 70 DAS. Subsequently, Se-regulated mechanisms improved 100 seed weight, biological yield, and seed yield per plant. We suggest that Se foliar spray (40 ppm) is a practical and affordable strategy to increase wheat output in arid and semi-arid regions of the world that are experiencing severe water shortages.


Introduction
In the current climatic change scenario, most crops suffer from soil/atmospheric water deficiency in the root zone due to scarcity and worsening water quality [1].Soil moisture stress, in particular (increased evapotranspiration and decreased precipitation), may become more evident and damaging to crop productivity in years to come [1][2][3][4].The primary causes of these losses are decreased net photosynthetic rates caused by reduced water availability, oxidative damage to chloroplasts, stomatal closure, and poor grain set [5,6].Plants are unable to extract water from the soil, which is required for proper growth and development when subjected to drought-induced osmotic stress.The response of plants to water stress is determined by various factors, including developmental stage, intensity and length of stress, and cultivar genetics, which results in stunted growth and reduced CO 2 diffusion to chloroplasts due to stomatal closing.The majority of plants adopt numerous methods to combat stress-related impacts through changes in developmental, morphological, physiological, and biochemical processes.Such strategies include reduced growth and water absorption, altered stomatal behavior and reduced photosynthetic efficiency, increased osmolyte buildup, disturbed ion balance, and stress-induced gene expression [7].Physiological and biochemical techniques are critical for understanding the varied responses of plants to water scarcity.
Wheat (Triticum aestivum L.) is widely recognized as one of the world's three most important cereals.It is a rich source of minerals, dietary fiber, proteins, and B-group vitamins, making it an ideal health-building food consumed by 36% of the world's population [8].India's contribution to global wheat production was 13.18%, placing it second only to China in production [9].However, its production is threatened by a variety of causes, the most serious of which are climate change and an ever-increasing population.Irrigation covers around 20% of cropland, providing for approximately 40% of global food production, with rain-fed agriculture accounting for the remaining 60%.As a result, the most significant abiotic restriction to agricultural production is water [10].Researchers discovered that even if global warming is limited to 2 • C over preindustrial levels, up to 30% of worldwide wheat production areas could face water shortages [11].Treatment with abiotic stress-relieving chemicals could be used as a more cost-effective way to improve plant drought tolerance.Selenium has been found to be beneficial to plants in sufficient amounts.Selenium's physiological and antioxidant properties benefit plants exposed to a variety of abiotic stress conditions [12].Se, for example, enhances plant tolerance to drought stress by controlling water status, increasing chlorophyll content in plant leaves, and stimulating the activity of antioxidant enzymes [13][14][15][16].Selenium mitigates salt stress [17], heavy metals stress [18], and high-temperature stress [19] by enhancing glutathione peroxidase (GPX) and antioxidant activity while decreasing lipid peroxidation.Furthermore, Se has been shown to increase the yield of grain crops such as wheat [20], rice [21], and maize [22].However, the physio-biochemical mechanisms behind selenium's beneficial benefits on yield during drought stress remain unknown.Keeping all of these factors in mind, the current study was conducted to investigate the effects of Se on reducing soil moisture stress in wheat.There is limited data on the effects of exogenous Se delivery on wheat output during soil moisture stress.We postulate that Se treatment improves wheat seed yield under soil moisture stress by enhancing plant water relations and gaseous exchange traits.

Experimental Setup andPlant Material
The current study was carried out on the wheat variety WH 1142 at the screenhouse of the Department of Botany and Plant Physiology, CCS Haryana Agricultural University, Hisar, between Rabi 2019-2020 and 2020-2021.The experiment utilized a completely random design with three replications.Wheat plants were grown under natural conditions in plastic pots (20 × 25cm) filled with 10kg of dune sand.Ten seeds were planted in each pot at a similar depth.The following are some of the physio-chemical features of the experimental sand (Table 1).Following a week of germination, only three seedlings with uniform growth were left in each pot for further study.Hoagland and Arnon [23] nutrient solution was applied to the plants at regular intervals (8-12 days).Soil moisture stress was initiated when seedlings were 20 days old.Seedlings were divided into three groups.One group was grown under normal water conditions as denoted by the control environment (12.5 ± 0.05% of soil saturation percent), the second was grown under moderate moisture stress (8.5 ± 0.05% of soil saturation percent), and the other was subjected to severe moisture stress (4.5 ± 0.05% of soil saturation percent).During the experiment, the pots were watered with nutrient solution or water every day after being weighed.The control and water-stressed plants both got the same amount of nutrient solution, but they were supplied with different amounts of water to reach the respective level of soil moisture.The seedlings of each soil moisture level were divided into 4 subgroups: (i) Control (without selenium application), (ii) Soil application of 10 ppm sodium selenite per kg of soil, (iii) foliar application of 20 ppm selenium, and (iv) foliar application of 40 ppm selenium.Soil application of Se was applied by dissolving sodium selenite in water and saturating the soil before sowing.Following 70 and 90 days after sowing, 10 mL of foliar fertilizer was administered to each pot between 18:00 and 19:00.The experiment was conducted within a manually operated rainout shelter fitted with a movable sheet of clear, flexible plastic.The leaf samples were taken in three replications from all the Se treatments and soil moisture levels (control, moderate, and severe moisture stress) for analysis at both sampling stages.

Physiological Traits
The leaf water potential (−MPa) was determined with the aid of a pressure chamber (Model 3005, Soil Moisture Equipment Corporation, Santa Barbara, CA, USA), while the osmotic potential (s) was calculated with a psychrometric technique utilizing a vapor pressure Osmometer (Wescor INC., Lorganan, UT, USA).The method developed by Barrs and Weatherly was used to measure the relative water content (RWC) of flag leaves [24].Gaseous exchange parameters such as photosynthetic rate (µmol m −2 s −1 ), transpiration (mmol m -2 s -1 ), and stomatal conductance (mmol m -2 s -1 ) of flag leaves were monitored from10:30 AM to 11:30 AM using an open system LCA-4 ADC portable infrared gas analyzer (Analytical Development Company, Hoddeson, England).Cuvette conditions were 1000 µmol m −2 s −1 photosynthetic photon fux density (PPFD), 60% relative humidity, 400 ppm atmospheric CO 2 , and 25 • C leaf temperature [25].Chlorophyll fluorescence was estimated in flag leaves of Se-treated and non-treated plants under control and stressed conditions.The chlorophyll fluorescence was measured at the jointing and heading stages of plants using a portable handy PEA analyzer (Hansatech, UK) after adapting the leaves to the dark for 25 min using special leaf clips [25].

Statistical Analysis
Data were analyzed using two factorial CRD (Complete Randomized Design) design.Treatments, environments, and their interactions were compared using critical difference (CD) at 5% significance with OPSTAT software.

Plant Water Relations
Leaf water potential decreased (more negative values) significantly under receding soil moisture levels.This decrease was 17.28 and 29.78% in moderate and severe water stress, respectively, as compared to control at 90 DAS.Plants treated with Se showed more leaf water potential (less negative) as compared to the plants without Se treatment.At 90 DAS, a maximum increase of 15.12%, 9.0%, and 12.40% in leaf water potential was observed in plants treated with foliar application of 40 ppm Se under control, moderate, and severe moisture stress, respectively (Table 2).The osmotic potential of the leaf increased from 70 DAS to 90 DAS, and it was 0.16, 0.19, 0.24 (-Mpa) under control, moderate, and severe moisture stress at 70 DAS and increased by 16.85% and 50.56% at 90 DAS under moderate and severe water stress, respectively (Table 2).Selenium treatment helped the plant to increase the leaf osmotic potential.The maximum increase was observed in plants treated with foliar application of 40 ppm selenium in all environments except severe moisture stress at the vegetative stage (70 DAS).Relative water content decreased as the crop advanced (70DAS to 90 DAS), as well as under soil moisture stress.It was reduced from 85.19% to 74.81 and 63.06% at 70 DAS and 83.13 % to 72.26% and 57.37% at 90 DAS under moderate and severe moisture stress, respectively (Table 2).Among the Se treatments, foliar application of 40 ppm selenium showed maximum RWC except for plants under severe stress at the vegetative stage (70 DAS).

Gas ExchangeAttribute
The rate of photosynthesis decreased significantly under soil moisture stress at both 70 DAS and 90 DAS (Table 3).Plants grown under moderate stress and severe stress conditions showed 17.50% and 35% lower photosynthetic rates as compared to normal irrigated control, irrespective of the selenium treatment at 90 DAS.Plants treated with foliar application of 40 ppm selenium (11.50 and 9.85 µM m −2 s −1 ) showed a higher photosynthetic rate under both control and moderate moisture stress, respectively, at 70 DAS (Table 3).While under severe moisture stress, the maximum rate of photosynthesis was observed in the plants treated with soil application of 10 ppm selenium (8.54 µM m −2 s −1 ) at 70 DAS (Table 3).At 90 DAS, foliar application of 40 ppm selenium showed a maximum increase of 14.18%, 11.55%, and 17.71% in photosynthesis under control, moderate, and severe moisture stress, respectively.The transpiration rate was lower under moderate and severe stress environments as compared to control (1.92 to 1.61 and1.32 mM m −2 s −1 ) and (2.65 to 2.19 and 1.79 mM m −2 s −1 ) at 70 DAS and 90DAS, respectively, irrespective of the selenium treatments.The rate of transpiration was higher at flowering than at the vegetative stage and decreased by 32.61% under severe moisture conditions at 90 DAS.Plants treated with foliar application of 40 ppm selenium showed a maximum rate of transpiration irrespective of stage and soil moisture levels.The stressed plants exhibited a significant decrease of 17.49% and 35.40% at 70 DAS and 15.82% and 32.79% in stomatal conductance at 90 DAS under moderate and severe moisture stress, respectively, over irrigated control.A maximum increase of 2.69%, 19.87%, and 20.0% in stomatal conductance was noticed in the plants treated with foliar application of 40 ppm selenium under control, moderate, and severe moisture stress, respectively, at 90 DAS (Table 3).The results of chlorophyll fluorescence (Fv/Fm) presented in Table 3 indicate a significant decrease in the values of chlorophyll fluorescence under depleting soil moisture levels.This decrease was 10.27%, 21.19% at 70 DAS and 12.74%, 24.24% at 90 DAS under moderate and severe moisture stress, respectively.At 90 DAS, foliar application of 40 ppm Se showed a maximum increase of 17.39%, 11.48%, and 17.65% in quantum yield under control, moderate, and severe moisture stress, respectively.The interaction between environment and Se treatment was found to be significant at 70 DAS only.Instantaneous water use efficiency and intrinsic water use efficiency (WUE) were also calculated as photosynthetic rate/transpiration rate (Pn/E) and photosynthetic rate/stomatal conductance (Pn/gS).Results obtained showed that the application of Se, either soil application or foliar application, enhanced instantaneous as well as intrinsic WUE (Figure 1).
Plants treated with foliar application of 40 ppm selenium showed a maximum rate of transpiration irrespective of stage and soil moisture levels.The stressed plants exhibited a significant decrease of 17.49% and 35.40% at 70 DAS and 15.82% and 32.79% in stomatal conductance at 90 DAS under moderate and severe moisture stress, respectively, over irrigated control.A maximum increase of 2.69%, 19.87%, and 20.0% in stomatal conductance was noticed in the plants treated with foliar application of 40 ppm selenium under control, moderate, and severe moisture stress, respectively, at 90 DAS (Table 3).The results of chlorophyll fluorescence (Fv/Fm) presented in Table 3 indicate a significant decrease in the values of chlorophyll fluorescence under depleting soil moisture levels.This decrease was 10.27%, 21.19% at 70 DAS and 12.74%, 24.24% at 90 DAS under moderate and severe moisture stress, respectively.At 90 DAS, foliar application of 40 ppm Se showed a maximum increase of 17.39%, 11.48%, and 17.65% in quantum yield under control, moderate, and severe moisture stress, respectively.The interaction between environment and Se treatment was found to be significant at 70 DAS only.Instantaneous water use efficiency and intrinsic water use efficiency (WUE) were also calculated as photosynthetic rate/transpiration rate (Pn/E) and photosynthetic rate/stomatal conductance (Pn/gS).Results obtained showed that the application of Se, either soil application or foliar application, enhanced instantaneous as well as intrinsic WUE (Figure 1).

Grain Yield and Biological Yield
The decrease in grain yield and biological yield was 12.42% and 25.49% under moderate soil moisture and 38.38% and 46.0%under severe soil moisture stress as compared to control.The application of selenium significantly increased the biological yield and grain yield per plant (Table 4) by alleviating the deleterious effects of water stress.Foliar application of 20 ppm and 40 ppm selenium showed a higher increase than soil application of Se.Application of 40 ppm Se showed a maximum increase of 7.80%, 13.63%, and 17.07% grain yield under control, moderate, and severe moisture stress, respectively (Table 4).

Grain Yield and Biological Yield
The decrease in grain yield and biological yield was 12.42% and 25.49% under moderate soil moisture and 38.38% and 46.0%under severe soil moisture stress as compared to control.The application of selenium significantly increased the biological yield and grain yield per plant (Table 4) by alleviating the deleterious effects of water stress.Foliar application of 20 ppm and 40 ppm selenium showed a higher increase than soil application of Se.Application of 40 ppm Se showed a maximum increase of 7.80%, 13.63%, and 17.07% grain yield under control, moderate, and severe moisture stress, respectively (Table 4).

Discussion
Soil moisture stress lowered relative water content, leaf water potential (Ψw), and leaf osmotic potential (Ψs) considerably (Figure 1).This could be the outcome of stomatal closure and decreased transpiration rate (Table 2), which is the plant's primary and fastest reaction to moisture stress.Plant water potential may have decreased due to lower absorption (due to less water availability in soil) and water translocation as a result of gradient loss between soil and plant root, which functions as the guiding factor for water transport along the transpiration pull [26].Osmotic adjustment is a natural plant response that assists them in maintaining water balance by synthesizing various osmolytes/solutes [27].These solutes safeguard cellular structures and functions while also maintaining water balance and delaying dehydrative damage by preserving cell turgor and other physiological systems under water-stressed environments [28].Selenium treatment increased leaf water potential (Ψw) and leaf osmotic potential (Ψs) under soil moisture stress but did not achieve the value of the control environment (Figure 1).The higher the negative osmotic potential, the more difficult it was to maintain turgor, and this could be one of the reasons for completing various physiological activities even at low water potential.Similar results using Se application under water deficit have been reported by Nawaz et al. [29].Among the treatments, foliar application of 40 ppm selenium exhibited relatively lower Ψw and Ψs under moisture stress at the flowering stage (90 DAS) in wheat, which might be due to the interactive effect of foliar application of 40 ppm selenium sprayed at 65 and 85 DAS.Cell elongation in higher plants under drought stress is inhibited by reduced turgor pressure.The reduction in water uptake is due to loss in turgor which causes a reduction in tissue water content [30].The value of relative water content decreased from the vegetative to the flowering stage, as mentioned in Figure 1, which might be because the plants were progressing towards senescence or reduced water supply to the tissues resulting from decreased water absorption from the soil.Treatment with selenium increased the RWC of leaves in wheat under moisture stress.This enhanced RWC helped the cultivars of wheat to perform various physiological and biochemical processes efficiently under soil moisture stress.However, foliar application of 40 ppm selenium maintained the highest RWC under moderate and severe moisture stress at flowering that accounted for the top economic yield in plants treated with the above-mentioned treatments.Similar results of selenium on RWC were also obtained by Aissa et al. [31] in sorghum and Sattar et al. [32] in wheat.The photosynthetic rate was higher at the flowering stage as compared to the vegetative stage.A consecutive decrease in photosynthetic rate was observed from control to moderate and severe moisture stress (Table 2).Under moisture stress, a decrease in the rate of photosynthesis is contributed by stomatal and non-stomatal factors.Non-stomatal factors associated with decreased chlorophyll stability index and ion uptake are the component of different enzymes involved in metabolic reactions.Stomatal factors such as Ψw, RWC, and stomatal conductance led to conclude that stomatal closure is one of the major causes of reducing photosynthetic rate (Figure 1 and Table 2).Similar results were obtained by Bashir et al. [33] in pea plants and Ahmad et al. [34] in wheat crops.As a result of selenium application, plants improve the water status, which increases the photosynthetic rate by increasing stomatal conductance (Table 2).This relationship between the stomatal opening and the relative increase in photosynthetic activity has also been reported by Hajiboland et al. [35].Among the treatments, foliar application of 40 ppm exhibited a relatively higher photosynthetic rate irrespective of soil moisture level at the flowering stage.The transpiration rate decreased under moderate and severe moisture stress conditions (Table 2), and this decrease seems to be a common phenomenon due to decreased water potential, RWC, and stomatal conductance (Figure 1 and Table 2).A decrease in the rate of transpiration may also be associated with the size of the stomatal pore, which regulates the transpiration rate, as reported by Beltrano et al. [36] and Sharma et al. [37].Selenium application maintains a higher transpiration rate irrespective of the soil moisture level.In the case of transpiration also, the response of foliar application of selenium was found to be better at the vegetative stage (70 DAS) in the plants grown under control and moderate stress, while under severe moisture stress, soil application of selenium showed the maximum transpiration rate.The reasons for this might be (1) "selenium is a non-essential element for normal growth of plants and under moisture stress only, plant uptake it from soil"; (2)"the transporters (K and S) through which plants uptake selenium are activated when plants need them, i.e., at the time of stress"; (3) fine root systems under moisture stress help the plant to uptake more selenium than seemed to be leached out under control and moderate stress, respectively.However, at the flowering stage (90 DAS), the additive effect of foliar application of 40 ppm selenium showed better results for transpiration under the control environment.Such an increase in the rate of transpiration has also been observed with the application of selenium by Hajiboland et al. [35].Soil application of selenium might show a dilution effect which is why its effect on transpiration rate is less than the foliar application of selenium.The results presented in Table 1 show decreases in stomatal conductance under moisture stress.Reduction in stomatal conductance may also be due to decreased water potential and relative water content under soil moisture stress which led to a loss of leaf turgor and ultimately decreased stomatal conductance (Table 2).Similar findings have been reported previously by Ahmad et al. [34] in wheat.Among the selenium treatments, soil application of selenium was found to be more effective under severe moisture stress as selenium is more available to the plants under lower water availability [16].Foliar application maintained maximum stomatal conductance at the flowering stage.The possible reason for this maybe the dilution effect of soil application of selenium [21] or that the interactive effect of foliar applications was greater than soil application of selenium in increasing the stomatal conductance.Results obtained are in accordance with the findings of Hajiboland et al. [35], who reported that the selenium helped in stomatal movement resulting in improved transpiration and maintained relative water content to induce drought tolerance.The intriguing result of such responses was a significant drop in water use efficiency under moisture stress and an improvement in water use efficiency in response to the selenium treatment (Figure 1).Stiller et al. [38] postulated that any improvement in components of water use efficiency (WUE) would be expected to mitigate some of the negative consequences of water stress.
Soil moisture stress reduces the source strength by reducing photosynthesis (Table 2) and decreasing metabolite translocation, both of which contribute to yield.Biomass is one of the most important yield determinants and accounts for a large fraction of the total variation in yield [39]. Stress caused by lack of water at the soil surface hastens crop maturity and restricts leaf area development and leaf expansion, both of which contribute to the plant's survival, putting the final economic output at risk [40].Among the two Se application methods, the positive effects of foliar application of selenium were greater than that of soil application of selenium.This increase in yield and its attributing traits might be the result of increased water status and gaseous exchange traits in response to selenium application (Figure 1 and Table 1).Liang et al. [41] reported that Se leads to a significant increase in yield and yield-related traits first and then decreases when Se application levels increase.In wheat, the beneficial effects of foliar Se treatment on yield were stronger than those of soil Se at low Se concentrations (11.5 ppm and 5 ppm), whereas the negative impacts of foliar Se application on yield were more evident than those of soil at high Se concentrations (23 ppm and 10 ppm).Se-mediated growth and productivity gains in different crops have been linked to different mechanisms.For instance, exogenous Se boosted wheat yield by improving the antioxidative system [41].In contrast, Se treatment did not influence biomass but increased seed output by 43%in the seed production in Brassica rapa L. [42].

Conclusions
From the current study, it was concluded that soil moisture stress decreased plant water relations and gaseous exchange traits that affected yield.However the application of selenium during the vegetative and flowering stages may have delayed the effects of water stress that wheat plants experienced and enhanced their ability to absorb additional water with the help of a fine root system or, alternatively, have a greater ability to control

Figure 1 .
Figure 1.Effect of selenium application on instantaneous (A/E) and intrinsic water use efficiency (A/gS) under soil moisture stress in wheat.

Figure 1 .
Figure 1.Effect of selenium application on instantaneous (A/E) and intrinsic water use efficiency (A/gS) under soil moisture stress in wheat.

Table 1 .
Physio-chemical features of the experimental soil.

Table 2 .
Effect of selenium application on plant water relations under soil moisture stress in wheat.

Table 3 .
Effect of selenium application on photosynthetic rate (A), transpiration rate (E), stomatal conductance (gS), and chlorophyll fluorescence (Fv/Fm) under soil moisture stress in wheat.

Table 4 .
Effect of selenium application on grain yield and biological yield under soil moisture stress in wheat.

Table 4 .
Effect of selenium application on grain yield and biological yield under soil moisture stress in wheat.