Micronutrient and redox homeostasis contribute to Moringa oleifera ‑regulated drought tolerance in wheat

Global food security is being severely affected by the rapid increase in population and drastic climate change. Drought stress is the most important limiting factor for the sustainable production of several important crops, including wheat. The gradual temperature rise and reduced precipitations are likely to cause the frequent onset of droughts around the world. Therefore, alleviation of drought stress in crop plants has become an essential requirement to meet the increasing food demand. The present study explored the role of foliar application of Moringa leaf extract (MLE) in conferring drought tolerance in wheat during the anthesis stage. A wheat genotype of Indo-Gangetic Plains (HI1544) was exposed to drought stress during the anthesis of the spikes and simultaneously foliar sprayed with MLE for 10 days. The results showed the MLE treatment to improve the concentrations of macro- (K, Ca) and micronutrients (B, Cu, Fe, Mn, Zn, Si) in flag-leaves of wheat under non-stressed conditions. Application of MLE also maintained the flag-leaf nutritional contents under drought stress. The micro-nutrients, including Cu, Fe, Mn, and Zn being the co-factors of the enzymes also stimulated the antioxidant enzyme activities; eventually leading to a significant reduction in the reactive oxygen species and malondialdehyde accumulations under drought stress. Furthermore, micronutrients played a crucial role in osmotic adjustment and sustainable plant growth under drought stress. Overall, the study provided insights into the functional role of micronutrients in improving drought tolerance and also indicated the potential to commercialize MLE as an effective bio-stimulant for sustainable agriculture in drought-prone regions.


Introduction
Drought stress regularly affects major wheat cultivating regions throughout the world, significantly reducing the yield when coincides with the flowering or grain filling stages (Zhang et al. 2018). Most of the wheat genotypes cultivated in the Indo-Gangetic Plains (IGP) are developed for the irrigated cropping systems and therefore, are extremely susceptible to drought (Zhang et al. 2017). High temperature-induced severe increase in soil moisture evaporation is the primary reason for drought stress in this particular region. Continuous increase in the average atmospheric temperature and asymmetric precipitation is predicted to intensify the incidence of drought around the world causing about 9-12% increase in yield loss of wheat by the end of the twenty-first century (Leng and Hall 2019). Furthermore, rapid depletion of groundwater tables in the larger part of the IGP may cause the frequent onset of droughts (Nath et al. 2017). Therefore, the amelioration of drought stress has become an essential requirement to encounter the global food security.
Drought can occur at any stage of the plants' life cycle but anthesis is considered as the most vulnerable period related to the production of wheat (Senapati et al. 2019). Therefore, it is a serious need to increase the drought tolerance during the reproductive development in wheat. Drought has significant negative influences on morpho-physiological (Stallmann et al. 2018) and biochemical responses (Hameed et al. 2011) of wheat plants ultimately impeding the crop productivity. Water stress also disrupts the plant-water relationships in wheat affecting the plant growth (Yadav et al. 2019). The chlorophyll biosynthesis process is also inhibited under drought stress that accelerates flag-leaf senescence in plants . Drought stress in plants is associated with the excess production of reactive oxygen species (ROS) that causes severe oxidative damages (Hameed et al. 2011). Accumulation of superoxide anion (B7O 2 − ), singlet oxygen (O 2 *), hydrogen peroxide (H 2 O 2 ), hydroxide ion (OH − ) induced by the water stress triggers the membrane lipid peroxidation and cell death (Basu et al. 2021b).
Mineral nutrients including the macro-and micronutrients also play a major role in drought tolerance in plants. The role of microelements in the regulation of plant growth has been widely studied (Hansch et al. 2009). However, the role of the mineral nutrients in the abiotic stress tolerance is so far less explored. Reduced soil moisture under drought stress impairs the nutrient uptake and aerial translocation in plants (Silva et al. 2011). Drought-induced stomatal closure also restricts the root-to-shoot transport of the mineral nutrients by reducing the transpiration rate. Major inorganic solutes, including potassium (K), calcium (Ca) play a significant role in the osmoregulation and plant growth under different abiotic stresses. The major elements may directly act as osmotic solutes or indirectly as regulators in the biosynthesis of organic solutes intended for water conservation in plants. On the other hand, micronutrients such as, iron (Fe), copper (Cu), zinc (Zn), manganese (Mn) being the major co-factors of the antioxidant enzymes protect the plants from oxidative damage by effective ROS scavenging (Ahanger et al. 2016). Silicon (Si) also prevents oxidative damage in plants by activating the antioxidant defense system . Additionally, boron (B) plays an important role in the maintenance of structural and functional integrity of cell membrane in plants. Moringa oleifera leaf is a natural bio-stimulant that enhances the growth, physiological performances, and yield attributes of different crop plants . Previous studies have established the foliar spray to be an effective way for the application of bio-stimulants as the chemicals provided are readily available to the plants. Several studies on bio-stimulants have focused on the application of different biomolecules, organic fertilizers, or seaweed extracts in ameliorating different abiotic stresses in plants (Wang et al. 2019;Sharma et al. 2019). A recent study has revealed the Moringa leaf extract (MLE) to improve the biochemical traits, productivity, and grain quality of rice under drought stress (Khan et al. 2021). However, the application of MLE in conferring drought tolerance by enhancing the micronutrient levels during the anthesis stage of wheat has not been extensively studied. Therefore, there is a significant gap in the knowledge about the mechanism of action of the MLE, which is required to be explored. A comprehensive understanding of the role of MLE in improving the macro-and micro-nutrient contents and antioxidant defense mechanism may contribute to enhancing the drought tolerance in wheat. The present study explored the role of MLE in conferring drought tolerance in wheat during the anthesis stage. Further, we have evaluated the association of nonenzymatic and enzymatic antioxidant-mediated enhanced ROS detoxification in the flag leaves with the improved drought tolerance in wheat.

Plant material and experimental design
The present study was accomplished with a wheat genotype of IGP (Triticum aestivum L. cv. HI1544). Seeds of the IGP wheat genotype were surface sterilized with 0.5% sodium hypochlorite (10 min), followed by thorough washing with de-ionized water. Then the seeds were soaked overnight and equal numbers of seeds were germinated for 3 days under dark conditions in pots (diameter 25 cm, height 19 cm) filled with the equal amount of soil. After germination of the seeds the pots were transferred to the greenhouse conditions (22 ± 2 °C; 16 h light/8 h dark cycle; relative humidity 75-80%). Crop management was optimized with the normal irrigation.

Stress imposition and application of Moringa leaf extract (MLE)
Moringa leaf extract (MLE) was prepared according to Kumar et al. (2021). During the anthesis of the spikes the pots were divided into four groups; plants of the first group grown under the normal conditions (Control), plants of the second group was foliar sprayed with the MLE (MLE), plants of the third group was exposed to the drought stress by withholding water for the next 10 days (Drought), plants of the fourth group were also exposed to drought stress (similar to the third group) but simultaneously foliar sprayed with the MLE for 10 days (Drought+MLE) (Fig. S1A).

Measurement of growth parameters and leaf area index
The height of the wheat plants and the spike lengths were measured at their respective anthesis stages. The relative growth rate for each genotype was calculated according to Kumar et al. (2009).
The leaf area was measured according to Dwivedi et al. (2019) and the leaf area index (LAI) was determined from the ratio of leaf area and ground area.

Estimation of relative water content and total chlorophyll content
Relative water content (RWC) was determined according to Weatherley (1950). The RWC was calculated from the fresh weight (FW), dry weight (DW), and turgid weight (TW).
Total chlorophyll was extracted from the flag-leaf samples with 80% chilled acetone. Absorbance was recorded at 645 and 663 nm on a UV--VIS spectrophotometer and total chlorophyll content was calculated following Arnon (1949).

Determination of electrolyte leakage and membrane stability index
Relative electrical conductivity or electrolyte leakage (EL) was estimated according to Kumar et al. (2009). The membrane stability index (MSI) was calculated following the equation proposed by Basu et al. (2021b).

Quantification of proline and total soluble sugar content
Proline was estimated according to Bates et al. (1973). Total soluble sugar (TSS) from flag leaves was quantified according to Yemm and Willis (1954).

Estimation of macro and micro-nutrients
Flag-leaf tissue was digested with 0.1% HNO 3 . Ions were extracted in distilled water by boiling it twice for 30 min each. The filtrate was used to measure specifications with a flame photometer. Endogenous K, and Ca concentrations were determined from their respective standard curves.
The micronutrient contents were determined with the elemental analysis by energy dispersive X-ray fluorescence (EDXRF) spectrophotometer according to Kumar et al. (2021).

Assay of reactive oxygen species and MDA content
Hydrogen peroxide (H 2 O 2 ), superoxide anion ( · O 2 − ) were estimated as described by Basu et al. (2020). Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) test according to Heath and Packer (1968).

Estimation of total ascorbate and glutathione content
Total ascorbate (AsA+DHA) and total glutathione (GSH+GSSG) contents were estimated according to Basu et al. (2021c).

Native-PAGE and activity staining for antioxidant enzymes
Plant extracts containing equal amounts of protein were subjected to discontinuous polyacrylamide gel electrophoresis (PAGE) under non-denaturing and non-reducing conditions (Laemmli 1970). SOD activity was detected following the method of Weisiger and Fridovich (1973). The gels were illuminated until colorless SOD bands appeared against a purple background. CAT activity was determined by staining the gels with 1% potassium ferricyanide and 1% ferric chloride (Scandalios 1968). POX activity was detected using the method of Graham et al. (1964). Gels were incubated in darkness, till the POX activity-containing band was visualized carefully. Enzyme activities were estimated by measuring the relative intensities of bands with the Adobe Photoshop version 7.

Western blotting
Western blotting was performed according to Basu et al. (2021a). Anti-catalase and anti-Cu/Zn superoxide dismutase (1:1500 dilutions) were used as primary antibodies. Goat anti-rabbit IgG-conjugated with Horse radish peroxidase and rabbit anti-chicken IgY-conjugated with Horse radish peroxidase (1:1000 dilutions) were used as the secondary antibodies for visualizing anti-catalase and anti-Cu/Zn SOD, respectively. Bands were observed by using the DAB substrate.

Statistical analyses
All treatments were given three replications (n = 3) with proper randomization to eliminate bias, whatsoever. Each value is presented in the form of mean ± standard error (± SE) and the lowest standard deviations of mean with a reading considering at least three samples per experiment for each genotype and each condition. The data were statistically analyzed for analysis of variance (ANOVA) as a 2 × 2 factorial completely randomized block design using SAS 9.3 software by SAS Institute Inc., USA. The influence of the main and interaction effects of the treatments was carefully evaluated. The standard error of mean and differences between the treatments was compared pair-wise by critical difference (CD) at a 5% level of significance.

Micronutrients sustained plant growth, physiological activities, and improved osmotic adjustment under drought stress
Application of MLE as the foliar spray sustained the plant growth of the wheat genotype HI-1544 during the anthesis stage (Fig. S1B, Fig. 1A). In the present study, MLEtreated plants exhibited considerably enhanced growth than that of the control (18.9% increase). Drought markedly reduced the plant growth than the control (18.4% decrease), whereas, Drought+MLE rejuvenated the plant growth and showed increased growth as compared to that of the control (3.4% increase). The leaf area index (LAI) was also significantly decreased under drought stress (38.7% decrease); whereas, Drought+MLE plants sustained the LAI (13% decrease) (Fig. 1B). The MLE treatment was also found to maintain the spike length following drought stress (13.8% decrease) as compared to that of the drought exposed plants (29.8% decrease) (Fig. 1C). Similarly, the flag-leaf RWC under the MLE treatment was higher than the control (85.6 and 80.9%, respectively) ( Fig. 1D). Conversely, the plants had reduced RWC under drought conditions (39.4%). However, the application of MLE under drought stress (Drought+MLE) sustained the RWC almost comparable to that of the control condition (56.9%). Water stress also resulted in increased EL in the flag leaves (Fig. 1E). The results showed MLE plants had the minimum EL than the control (6.1 and 9.5%, respectively). Imposition of drought stress caused an increase in the EL (80.3%); whereas, the Drought+MLE plants maintained considerably higher EL (32.3%). Likewise, the Drought+MLE plants maintained higher MSI (67.8%), while, drought-stressed plants had the lowest MSI (19.7%; Fig. S2). Flag-leaf chlorophyll content was also significantly reduced under drought stress (Fig. 1F). The MLE plants exhibited higher chlorophyll content (1.16 mg g −1 FW) as compared to that of the control plants (0.97 mg g −1 FW). Water stress caused a significant decrease in chlorophyll content (43.6% decrease), whereas, Drought+MLE exhibited the lowest decrease in flag leaf chlorophyll content (9.9% decrease). Drought stress also caused the accumulation of osmolytes in the wheat genotype HI-1544. Control and MLE plants had almost equivalent proline content ( Fig. 1G; 0.27 ± 0.3 mg g −1 DW). Incidence of drought stress during anthesis caused a significant increase in proline content (1.7-fold increase); whereas, the highest increase in proline content was observed in Drought+MLE plants (3.1-fold increase). A similar trend was observed in TSS content (Fig. 1H). Control and MLE plants had the lowest TSS content, ranging between 0.3 and 0.4 mg g −1 DW. Drought stress caused a marked increase in TSS content (2.5-fold increase); whereas, the maximum increase in TSS content was observed in Drought+MLE plants (4.0fold increase).

MLE improved macro-and micro-nutrient contents under drought stress
The MLE treatment increased the macro-and micro-nutrient concentrations in both drought-stressed as well as nonstressed plants (Fig. 2). Application of MLE improved the K content in the non-stressed plants of the wheat genotype (9.8% increase than control; Fig. 2A). Drought stress caused a significant decrease in K content (30.7% decrease than control). However, the MLE application maintained the K concentration in the Drought+MLE plants almost equivalent to that of the control conditions (18.4% decrease than control). Likewise, MLE treatment also increased the Ca content in the non-stressed plants (10.9% increase than control; Fig. 2B). Incidence of drought stress during anthesis caused a severe decrease in the Ca content (43.0% decrease than control), which was revived in the Drought+MLE plants (30.2% decrease than control). The micronutrients showed the similar trend as that of the macronutrient contents. Drought stress led to significant decrease (51.8% decrease than control; Fig. 2C) in B content; whereas, MLE treatment upheld the B concentration under drought stress (34.2% decrease than control). Similarly, the Drought+MLE plants exhibited higher Cu content (37.2% decrease than control; Fig. 2D) contrasting with that of the drought-stressed plants Fig. 1 Effect of micronutrients on MLE-regulated drought tolerance in wheat based on morpho-physiological performances during the anthesis stage. Effect of MLE treatment on A plant height, B leaf area index, C spike length, D relative water content, E electrolyte leakage, F total chlorophyll content, G proline content, and H total soluble sugar content of wheat genotype HI-1544 under drought stress. Data represent means ± standard error (n = 3). Values for different letters indicate significant differences at P = 0.05 (21.0% decrease than control). The Fe concentration was also observed to be significantly decreased under drought stress (57.8% decrease than control; Fig. 2E) while maintained in the Drought+MLE plants (27.0% decrease than control). Drought stress also severely impeded the Mn (52.6% decrease than control; Fig. 2F), Zn (49.4% decrease than control; Fig. 2G), and Si (55.4% decrease than control; Fig. 2H) contents in wheat. However, MLE treatment sustained the Mn (32.1% decrease than control), Zn (26.5% decrease than control), and Si (28.5% decrease than control) concentrations in the Drought+MLE plants.

Micronutrients decreased oxidative stress under drought stress
Drought stress resulted in significant ROS accumulation in the flag leaves of the wheat genotype studied (Fig. 3).
Enhanced ROS accumulation was determined by measuring endogenous H 2 O 2 content and · O 2 − production rate. In the present study, MLE plants showed considerably lower H 2 O 2 content in flag leaves than the control plants ( Fig. 3A; 11.1 and 15.4 µg g −1 FW). Following drought stress during anthesis, the H 2 O 2 content in flag leaves was significantly increased (P = 0.5) in the wheat genotype studied (2.1-fold increase), whereas, Drought+MLE plants had a minimal increase in H 2 O 2 (1.7-fold increase). A similar trend was observed in · O 2 − production rate (Fig. 3B). The smallest · O 2 − production rate was noted in the control and MLE plants that ranged between 24.5 and 28.2 nmol min −1 mg −1 protein. Drought stress caused a marked increase in the · O 2 − production rate (1.8-fold increase), contrasting with the Drought+MLE plants (1.4-fold increase). Oxidative damage in the drought-stressed wheat genotype was measured from the lipid peroxidation, estimated in terms of MDA content. The MLE and control plants had the lowest MDA content ranging between 2.4 and 2.6 nmol g −1 FW (Fig. 3C). After perceiving drought stress during anthesis, the wheat plants exhibited a significant increase in the MDA content (12.0 nmol g −1 FW), whereas, the Drought+MLE plants maintained considerably lower MDA content (7.0 nmol g −1 FW). Drought-induced ROS overproduction caused a remarkable increase in relative cell death in the wheat genotype studied (Fig. 3D). The lowest cell death was observed in the control and MLE plants (0.95-1.0). Drought stress resulted in a marked increase in cell death (1.6-fold increase) as compared to the control. However, Drought+MLE plants maintained considerably lower cell death (1.2-fold increase).

Micronutrients stimulated antioxidant defense system under drought stress
Redox homeostasis mediated by several key non-enzymatic and enzymatic antioxidants was measured in the flag leaf of the wheat genotype studied under different experimental conditions (Fig. 4). Non-enzymatic antioxidants were estimated in terms of total ascorbate and glutathione content. Following the present study, total ascorbate content under the control and MLE ranged between 4.0 and 4.9 µmol g −1 FW (Fig. 4A). Drought stress caused a significant increase (P = 0.05) in total ascorbate content (39.9% increase), whereas, the maximum increase in total ascorbate content was noticed in the Drought+MLE plants (51.8% increase). Likewise, total glutathione content was also significantly increased under drought stress ( Fig. 4B; 54.7% increase). However, the Drought+MLE plants had the maximum increase in total glutathione content (62.5% increase).
The study of antioxidant enzymes included SOD, CAT, POX, and APX. Under control and MLE conditions, the constitutive level of SOD activity in the wheat genotype ranged between 3.4 and 7.5 Unit min −1 mg −1 protein (Fig. 4C). However, SOD activity was significantly increased under drought stress conditions (78.5% increase). The maximum increase in SOD activity was observed in the Drought+MLE plants (89.4% increase). Similarly, CAT activity was also lower in the control and MLE plants ranging between 6.1 and 8.7 Unit min −1 mg −1 protein (Fig. 4D). Drought stress during anthesis enhanced CAT activity by 37.6% in the wheat plants; whereas, the highest increase in the activity was observed in Drought+MLE plants (60.5% increase). POX activity of the wheat genotype under different experimental conditions also exhibited an increasing trend by water stress (Fig. 4E). The lowest POX activity was observed in the plants grown under the control and MLE conditions, ranging between 4.7 and 6.8 Unit min −1 mg −1 protein. Drought stress caused a 59.9% increase in the POX activity. However, the maximum increase in POX activity was noted in Drought+MLE plants (70.7% increase). APX activity was Values for different letters indicate significant differences at P = 0.05 also significantly increased by drought stress (Fig. 4F). The minimum APX activity was exhibited by the plants grown under control and MLE conditions that ranged between 2.2 and 5.1 Unit min −1 mg −1 protein. Following drought stress, APX activity was increased by 78.9%, whereas, the highest increase in APX activity was observed in Drought+MLE plants (84.8% increase).
The in-gel activities of antioxidant enzymes were well coordinated with the data obtained in the kinetic measurements ( Fig. 5A-D). Native-PAGE revealed a single isozyme band with POX activity in flag-leaves of wheat under different experimental conditions (Fig. 5A). However, the band intensity was considerably higher in plants under drought stress as compared to the control plants (2.7-fold higher than Fig. 4 Effect of micronutrients on redox homeostasis in drought-stressed wheat genotype the anthesis stage. Effect of MLE treatment on A total ascorbate, and B total glutathione contents, C SOD, D CAT, E POX, and F APX activities of wheat genotype HI-1544 under drought stress. Data represent means ± standard error (n = 3). Values for different letters indicate significant differences at P = 0.05

Fig. 5
Effect of micronutrients on the activities of POX (A), CAT (B), and SOD isozymes (C) in wheat genotype HI-1544 during the anthesis stage under drought stress. D Relative intensity of bands in the respective gels. Data represent means ± standard error (n = 3). Western blot analyses using antibodies against E CAT and F Cu/ Zn-SOD control). Drought+MLE plants showed the maximum band intensity (3.1-fold higher than control). CAT activity also showed only one isozyme band in native gels under different experimental conditions (Fig. 5B). Similar to the CAT activity, drought-stressed plants showed higher intensity bands as compared to the control conditions (6.5-fold higher than control). The highest band intensity was observed in Drought+MLE plants (7.6-fold higher than control). Regarding SOD activity, three isozymes bands for MnSOD, FeSOD, and Cu/ZnSOD were observed in native gels under different experimental conditions which followed a similar trend (Fig. 5C). Three isozymes exhibited a modest increase in activities represented by higher intensities of bands in the plants under drought stress compared to the control (2.1, 6.9, and 2.2-fold higher than control in MnSOD, FeSOD, and Cu/ZnSOD, respectively), whereas, the highest band intensity was observed in the Drought+MLE plants (2.5, 7.9 and 2.7-fold higher than control in MnSOD, FeSOD, and Cu/ ZnSOD, respectively).
Western blot analysis also exhibited harmony with the spectrophotometric and ingel analyses of CAT and Cu/ ZnSOD enzyme activities ( Fig. 5E and F). CAT proteins showed differential expressions in western blot (Fig. 5E). Control and MLE plants showed the minimum CAT expression. However, drought stress significantly up-regulated the CAT expression level. However, the maximum level of expression was observed in the Drought+MLE plants. In contrast, control and MLE plants had the minimum increase in the CAT expression. The Cu/Zn SOD expression showed a similar trend as that of the CAT (Fig. 5F). Control and MLE plants showed the minimum Cu/Zn SOD expression contrasting with the drought-stressed condition showing a marked increase in the expression. The maximum Cu/Zn SOD expression was observed in the Drought+MLE plants.

Discussion
Drought stress severely affects the anthesis stage of wheat leading to dramatic yield loss. Gradual increase in average atmospheric temperature and irregular rainfall is predicted to exacerbate the conditions (Leng and Hall 2019). Therefore, enhancing drought tolerance in wheat, particularly during the reproductive development has become an essential requirement to support the global food security (Senapati et al. 2019). Several studies have revealed the potential role of different bio-stimulants in improving abiotic stress tolerance in plants (Sharma et al. 2019). However, the role of micronutrients in the MLE-mediated drought tolerance in wheat has not been studied so far. The present study explored the potential role of the micronutrients in improving drought tolerance in Indian bread wheat during the anthesis stage grounded on the morpho-physiological and biochemical evaluations. The study also critically analyzed the association of the micronutrient-induced drought tolerance with the redox homeostasis.
Drought stress induces the ROS accumulation in plants leading to the oxidative stress. Following the present study, the wheat plants showed increased H 2 O 2 content and · O 2 − production under drought stress (Fig. 3 A-B). The excessive ROS burst resulted in increased MDA accumulation, loss of membrane integrity, and cell death, consequently impeding the plant growth (Hameed et al. 2011;Dwivedi et al. 2018). The increased ROS accumulation and loss of membrane integrity might be attributed to the drought-induced B and Zn deficiency in plants (Hajiboland and Farhanghi 2011). The Fe deficiency under drought stress also led to the H 2 O 2 accumulation in plants by inhibiting the activities of the haem-containing enzymes POX and CAT (Rotaru 2011). Likewise, ROS accumulation was also induced by the loss of activities of the Cu, Zn, and Mn containing antioxidant enzymes Cu/ZnSOD and MnSOD due to their deficiencies under drought stress (Ahanger et al. 2016).
Drought tolerance in wheat is associated with the conservation of the micronutrient homeostasis with the higher level of micronutrients in the plant cells. Following the study, MLE application improved the concentrations of B, Cu, Fe, Mn, Zn, and Si in the non-stressed wheat plants and also upheld their level under the drought stress ( Fig. 2C-H). The micronutrients (Fe, Mn, Cu, Zn, and Si) induced the activities of the antioxidant enzymes, including SOD, POX, CAT, and APX in the Drought+MLE plants and protected them from the ROS-induced oxidative damages (Khan et al. 2021). Additionally, B, Cu, and Fe maintained the flag leaf chlorophyll content in the Drought+MLE plants. Increased accumulation of micronutrients in the Drought+MLE plants was also responsible for the osmoregulation and rejuvenation of the plant growth during the drought stress adaption (Figs. 1 and 2; Wu et al. 2019). The MLE-induced conservation of B concentrations helped in sustaining the water uptake and cell turgidity by directly influencing the uptake of K and solutes into the cells. On the other hand, B, Zn, and Si increased the water retention of cells, thereby maintaining the cellular membrane integrity and chlorophyll content under drought stress . Micronutrients also stimulated the accumulation of osmoprotectants consequently improving drought tolerance in wheat. The present study showed a significant increase in proline and TSS content in the Drought+MLE plants, which conserved the flag leaf RWC by acting as a potential osmoprotectant and ROS scavenger ( Fig. 1G and H ;Yadav et al. 2019).
Micronutrients also stimulated the antioxidant enzyme activities that played an important role in the ROS detoxification in wheat under drought stress. Correspondingly, Drought+MLE plants had enhanced antioxidant enzyme activities in the flag leaves that facilitated in eliminating oxidative damage through successful scavenging of ROS (Figs. 4 and 5;Wu et al. 2019). Furthermore, the present study illustrated the increased accumulation of non-enzymatic antioxidants, including ascorbate and glutathione contents in the Drought+MLE plants, which played a significant role in the ROS detoxification ( Fig. 4A and B; Basu et al. 2021c). Effective ROS detoxification in the flag leaves significantly decreased the MDA content and cell death in Drought+MLE plants, thereby sustaining the plant growth (Fig. 3C and D;Stallmann et al. 2018). In addition, micronutrient-induced ROS detoxification reduced the damage of photosynthetic pigments and maintained higher chlorophyll content in the MLE-treated plants under drought stress ( Fig. 1F; Basu et al. 2017).
Following the study, MLE treated plants exhibited micronutrient-mediated potential drought tolerance through enhanced ROS detoxification and osmoregulation (Fig. 6). Micronutrients facilitated sustainable plant growth by protecting the cells from the ROS-induced oxidative damage. Micronutrient-mediated exceptional osmotic adjustment in Drought+MLE plants also helped in conserving the plant water status in the flag leaf cells. Considering the present study, MLE might be considered as the potential bio-stimulant for enhancing drought tolerance in the Indo-Gangetic wheat genotype during the anthesis stage.

Conclusions
The present study explored the role of micronutrients conferring the MLE-mediated drought tolerance in wheat during the anthesis stage. Drought stress-induced excessive ROS accumulation caused cellular membrane damage ultimately leading to cell death. Application of MLE conserved the concentrations of the micronutrients in plants under drought conditions leading to the activation of the antioxidant defense system that played a significant role in the ROS detoxification and amelioration of the oxidative stress. The sustainable micronutrient contents rejuvenated the plant growth and physiological activities of wheat under drought stress. The micronutrients also increased the proline and TSS contents that contributed to the osmotic adjustment and plant growth under drought stress. As a whole, the study provided insights into the functional role of the MLE in improving drought tolerance and indicated the potential to commercialize the MLE as an effective bio-stimulant for sustainable agriculture in drought-prone regions of IGP.

Fig. 6
Mechanisms underlying the contribution of micronutrients on MLE-regulated drought tolerance in wheat. Micronutrients in drought-exposed wheat plants led to effective ROS scavenging through enhanced non-enzymatic (ascorbate, glutathione) and enzymatic (SOD, POX, CAT) antioxidants, consequently minimizing oxidative damages. Micronutrients also stimulated endogenous macro-and micro-nutrient contents, thereby conserving the ion homeostasis. Micronutrients also improved the proline and total soluble sugar contents, thereby maintaining the osmotic balance under drought stress. Thus micronutrient mediated conservation of redox homeostasis and osmoregulation promoted plant growth in wheat under drought stress