Mitigating Drought by Exogenous Potassium-mediated Improvements in Water Relation, Antioxidant Defense, Morpho-physiological and Biochemical Attributes of Black Gram [Vigna mungo (L.) Hepper] 

Recently, drought stress (DS) has emerged as one of the most critical limitations that reduce plant growth and crop yield causing food insecurity and undernourishment in a rapidly increasing human population. It can reduce production efficiency and crop yield by 20% in warm, dry and arid regions. Furthermore, a persistent DS declines the efficiency by 45% of agricultural lands which has impacted over 38% of the world’s population livelihood (Zhang et al., 2023). The underlying reason is DS inhibits vegetative growth parameters, including root and shoot development, resulting in a decrease in plant growth. The DS-induced oxidative stress may impair the synthesis of photosynthetic pigment and reduce the relative water content (RWC), leading to an increase in oxygen radical production (Waraich et al., 2020). The DS tends to impair germination, cell division, root proliferation, leaf growth, stem elongation, photosynthetic efficiency, nutrient mobilization, cell turgidity and gas exchange attributes, resulting in lower crop yield. Moreover, it adversely affected assimilate translocation which impaired flowering and pod formation, resulting in pollen grain sterility (Ul-Allah et al., 2020), therefore, eco-friendly, DS management strategies are critically needed for sustainable and smart crop production, otherwise, zero hunger and poverty alleviation related Sustainable Development Goals (SDGs) may not be achieved.
       
After nitrogen, potassium (K) is the second-most important macro-nutrient and the most prevalent mineral-nutrient in the kingdom Plantae (Waraich et al., 2020). It plays an important role in protein synthesis, which increases resistance to abiotic stresses. Additionally, it may not be the main component of different plant parts and structures, but exogenous and endogenous K may play a significant role in regulating the physicochemical processes involved in better plant morphological growth, yield and quality and mitigating stress and other mineral deficiency in plants in cotton (Zahoor et al., 2017). The K application assisted in upregulating the plant gas exchange and water relation leading to improved crop performance under DS conditions in canola (Waraich et al., 2020). Moreover, Ihsan et al., (2013) opined that K foliage supplementation remained effective in mitigating moisture-induced stresses in mung bean as revealed by growth, photosynthesis, gas exchange capacity and Zn analysis of shoot. Furthermore, Martineau et al., (2017) have also reported a significant role of K in maize leaf carbon exportation under DS conditions.
       
For ensuring the food and nutritional security of a rapidly increasing population, especially in developing countries of South Asia, legumes hold a strategic position (Iqbal et al., 2019a,b). The leguminous crops contribute over 33% of the global human protein requirement (Iqbal, 2018; Iqbal et al., 2018). Among leguminous crops, black gram [Vigna mungo (L.) Hepper] belongs to the Fabaceae family (Sadiq et al., 2023) and is cultivated in various agroecological zones of rain-fed areas of South Asia. It is one of the most important grain legumes in the world due to its high nutritive profile, including vitamins (A, B and C), protein (24.2%), fat (1.42%) and carbohydrate (59.6%) (Hossain et al., 2024). However, DS seriously reduced black gram yield and research gaps exist about the optimized doses of K for mitigating DS effects in black gram.
       
Therefore, to bridge the research gaps, it has become necessary to explore the exogenous application of numeral nutrition and its importance in boosting plant drought tolerance. The prime objective of this investigation was to explore whether and how the exogenous application of K induces drought tolerance by altering a series of morpho-physiological attributes in black gram.  We further intended to optimize the dose of K application for boosting plant defense DS in field conditions.

Experimental treatments and execution
 
Drought tolerant black gram genotype (cv. Arooj-11) seed was acquired from the Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan. The trial was conducted in the Department of Agronomy, University of Agriculture Faisalabad, Pakistan. Sowing was done in plastic pots (28 cm diameter 25 cm length) with a drainage hole at the bottom, filled with 6 kg of silica sand [after calculating its field capacity (FC)] on 15 March 2020 in a wire-house. The gravimetric method was used to determine the field capacity (FC) of sand, while 25 ml water was added to 100 g of sand to attain the saturation point. To determine the FC, the saturated sand was oven-dried (90°C) until it had obtained a constant weight. Black gram seedlings were grown in clay pots and water supplementation was gradually suspended as plants were exposed to DS by declining field capacity (FC) from 100% to 15%. Mild drought started at 30% field capacity; however considerable drought effects started at the FC of 15-20%. The treatments included a (³) control group entailing control (normal watering), water spray (WA, positive control), control+50 mg L^-1 K₂SO₄, control +100 mg L^-1 K₂SO₄, control +150 mg L^-1 K₂SO₄, while (³³) drought treatments group included drought stress (plant exposed to 15-20% by suspending water supply ), water spray (WA, positive control), drought +50 mg L^-1 K₂SO₄, drought +100 mg L^-1  K₂SO₄ and drought +150 mg L^-1  K₂SO₄. The potassium (K₂SO₄) was sprayed during the reproductive stage (45 DAS). The experiment was set up using a completely randomized design (CRD). Three individual replications were considered for recording the response variables.
 
Determination of photosynthetic pigments, photosynthesis and transpiration-related parameters
 
Five plants were randomly selected from each treatment for data recording at 64 DAS during the flowering stage. The chlorophyll content was determined using the method outlined by Arnon (1949) and Davies (1976). The gaseous exchange parameters, photosynthetic rate (Pn), inter-cellular CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (Tr) were recorded by using an infrared gas analyzer (CI-340 portable, Hoddesdon, England), with the water vapor pressure in the chamber kept between 6.0 and 8.9 m bar, the molar flow of air per unit leaf area kept at 403.3 mmol m² s^-1, the ambient temperature of 22.4°C-27.9°C, ambient CO₂ concentration (352 mol mol^-1), leaf temperature of 28.4-32.4 °C and atmospheric pressure 99.9 KPa.
 
Estimation of water status and oxidative stress indicators
 
The plant water relations were recorded at 63 DAS during the flowering stage as described by Scholander et al., (1964). Leaf water potential (ψs) and leaf osmotic potential (ψw) were measured with a water potential apparatus and an osmometer, respectively. The pressure potential (ψp) was determined as suggested by Hopkins (1999).
 

 

       
Following the method outlined by Ahmad et al., (2021), the relative water content was determined.
       
Cell membrane injury (CMI) measurements were made by recording the electrical conductivity of leachates from black gram leaves that had been soaked in distilled water at 100°C for an overnight period (Deshmukh et al., 1991). Equal-sized pieces of leaves (100 mg) were placed in tubes of distilled water (10 ml) in two sets. The electric conductivities C1 and C2 of the two sets were measured after one set was left overnight at room temperature and the other set was placed in a boiling water bath for 15 minutes. The CMI was calculated as follows: 
 

 

 

 

 
The malondialdehyde (MDA) content was estimated by following Heath and Packer (1968).
 
Measurement of metabolite accumulation and antioxidant enzyme activity
 
The metabolite content was determined by following the procedure outlined by Lowry et al., (1951) that estimated total soluble protein. A spectrophotometer was used to measure the absorbance at 620 nm and total phenolic content was calculated (Chaovanalikit and Wrolstad, 2004). The phenolic content of an acetone extract was assessed using the Folin Ciocalteu method and an absorbance measurement at 700 nm.
       
Plant sample extraction was made using the protocol suggested by Chauhan et al., (2022) and catalase (CAT) activity was measured (Kabir et al., 2023). Enzyme extracts (100 µL) were combined with 100 L of freshly made hydrogen peroxide (5.9 mM; 35% pure) to begin the procedure. A microplate reader (ELX800, Bio-Tek Instruments, Inc., Winooski, VT, USA) monitored the rate of absorbance fall for three minutes and recorded the H₂O₂ disappearance rate at 240 nm and superoxide dismutase (SOD) activity was assessed (Ju et al., 2021). Each test tube contained KH₂PO₄ (500 µL) buffer (5 pH, 50 mM), 22 µM methionine (200 µL), 0.1 µM Triton X (200 µL), 20 µM NBT (100 µL) and 0.6 µM riboflavin (100 µL) as a substrate. In the end, 800 µL of distilled water was mixed with 100 µL of enzyme extract. At 560 nm, the absorbance of a microplate reader (ELX800, Bio-Tek Instruments, Inc., Winooski, VT, USA) was measured.
 
Statistical analysis
 
The recorded data were arranged and subjected to analysis of variance (ANOVA) with the help of Statistix (version 10.0, USA) statistical package. Thereafter, the least significant difference (LSD) test was employed at the probability level of 5% to determine the significance among treatment means (Gomez and Gomez, 1984).

Plant growth attributes
 
The results revealed that K application had a significant (P≤0.05) effect on root length, shoot length, leaf area, shoot fresh weight, shoot dry weight, root fresh weight and root dry weight, respectively, by 12.9%, 0.85%, 11.8%, 18.9%, 9.06% and 16.9% in comparison to the control treatment. However, the maximum improvement in growth attributes of black gram was noted for K₁₀₀ (100 mg L^-1 potassium) followed by K₁₅₀ (150 mg L^-1 potassium) and K₅₀ (50 mg L^-1 potassium) (Table 1). Root length and leaf area in black gram were significantly affected by the interactions between DS and K applications. Black gram yield components were significantly (P ≤0.05) affected by the DS and K application in different concentrations. Compared to control (no stress) plants, the number of pods per plant, 1000-seed weight and yield decreased by 21.2%, 21.06% and 37.80% under DS, to counteract the negative effects of DS, K was applied; this increased the number of pods per plant, 1000-seed weight and yield when compared to the control (no potassium applied). Exogenous potassium application increased pods per plant, 1000-seed weight and yield under DS conditions by 5.14, 14.6 and 27.6%, respectively, in comparison to control (no potassium applied). However, when compared to control (no K applied), the maximum improvement in seed yield and associated characters was observed for K₁₀₀ which was followed by K₁₅₀ and K₅₀ (Table 1). For yield and associated traits in black gram, the interaction between DS and K application was not significant.



Photosynthetic pigments
 
According to the results, DS significantly (P≤0.05) declined chlorophyll a, b and total chlorophyll in black gram by 23.1, 46.9 and 25.4%, respectively to control treatment (Fig 1). The K₁₀₀ significantly improved the chl a, b and total chlorophyll compared to NA (no application of K with normal watering) and WA (only water spray). It was revealed that when K₁₀₀ was applied under DS, chlorophyll a, b and total chlorophyll were increased by 3.93%, 34.1% and 6.77%, respectively than control (no K applied) (Fig 1). Overall, the maximum enhancement in photosynthetic pigments was noted for K₁₀₀, that was followed by K₁₅₀ which in turn was followed by K₅₀ (Fig 1). The DS and K application interaction was also significant for chlorophyll content in black gram.


 
Photosynthesis-associated parameters
 
The result exhibited that DS significantly influenced the photosynthesis-associated traits (photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO₂ concentration were decreased by 34.2%, 73.02%, 37.1% and 27.3%, respectively compared to control treatment) of black gram (Fig 2). The K application increased photosynthetic rate (12.03%) and transpiration rate (14.7%). However, due to the improvement of photosynthesis rate (Pn) in black gram in response to DS, the transpiration (Tr) was enhanced compared to other K treatments. Additionally, K supplementation significantly improved stomatal conductance by 31.4% and sub-stomatal CO₂ concentration by 9.84% (Fig 3). Out of these K treatments (K₅₀, K₁₀₀ and K₁₅₀), the K₁₀₀ showed better performance for improving photosynthesis system in black gram under DS. 



Water relations
 
The K application remained effective in significantly (P≤0.05) influencing the water relations in black gram under DS conditions. It was noted that the water potential, pressure potential, osmotic potential and leaf-relative water content decreased by 39.3%, 21.5%, 9.64% and 24.2%, respectively, compared to the control treatment (Table 2). On the other hand, the K application increased the water relations, as well as the water potential and pressure potential under DS control. The water potential and leaf relative water content were improved under DS by 9.27% and 9.83%, respectively in comparison to the control treatment. The highest increment was observed for K₁₀₀ when compared to the control-no K application during DS conditions. Nonetheless, in comparison to control (no K-applied) plants, pressure potential increased by 47.03%, 36.1% and 26.4% in response to K₁₅₀, K₁₀₀ and K₅₀, respectively (Table 2). The pressure potential was also significantly influenced by the interaction of DS and K dose application.


 
Metabolites regulation
 
Stress-induced metabolites mediate antioxidant defenses under oxidative stress in plants, while these also help to cope with stress in plants. The DS and K application had a significant (P≤0.05) effect on the osmolyte content of black grams (Table 3). Additionally, the DS increased total soluble protein and total phenolics than the control treatment. Exogenous K application, on the other hand, increased total soluble proteins and total phenolics compared to the control (no potassium applied). Under DS, K application increased total soluble proteins and total phenolics by 7.53 and 30.7%, respectively, when compared to no potassium. Furthermore, the maximum increment in osmolyte content was observed for the K100 treatment. Nonetheless, K application increased total phenolics content by 4.57%,16.9% and 10.04% by K₁₀₀, K₁₅₀ and K₅₀, respectively than the control plants receiving no K-application (Table 3). Moreover, the interaction of DS and K application was not significant for osmolyte content in black gram.



Potassium-induced antioxidant defense
 
The results of the analysis of variance revealed that exogenous application of K and DS had a significant (P≤0.05) effect on the antioxidant activities in black gram. The DS had a significant impact on the antioxidants, such as catalase, peroxidase and superoxide dismutase in the leaves of black gram compared to the control (Table 3). Under DS, exogenous K application increased the activities of antioxidants such as superoxide dismutase, catalase and peroxidase by 9.45, 8.82 and 7.49%, respectively, in comparison to the control (no potassium application) treatment. On the other hand, when compared to the control, the highest antioxidant activity was found at K₁₀₀ followed by K₁₅₀ and K₅₀. Compared to control plants without potassium supplementation, the K-supplemented plants increased the superoxide dismutase activity by 22.9%, 13.9% and 8.17% by the K₁₀₀, K₁₅₀ and K₅₀, respectively (Table 3). The 100 mg/L treatment showed comparably better performance for boosting antioxidant activity. However, the antioxidant enzymes increased significantly in response to drought stress after K supplementation, which assisted plants to survive under DS.
 
Oxidative stress mitigation
 
Regarding the stress indicators in black gram, an analysis of variance revealed a significant (P≤0.05) effect of K exogenous application on membrane stability by accumulating more malondialdehyde and H₂O₂ and inflicting more damage to cell membranes in DS conditions than in control with no stress (Table 4). Results showed that MDA content increased up to 12 folds while H₂O₂ and cell membrane injury in leaf tissues increased by 42.2 and 44.6%, respectively, under DS as compared to control. When compared to control with no potassium applied, K application reduced the accumulation of malondialdehyde, H₂O₂ and cell membrane injury. Malondialdehyde, H₂O₂ and cell membrane damage under DS were all reduced by 12.7%, 8.70% and 17.6%, respectively, when K was applied than the control treatment. Maximum reduction of MDA content, H₂O₂ and cell membrane injury at K₁₀₀ followed by K₁₅₀ and K₅₀. Malondialdehyde content in black grams significantly increased because of the interaction between DS and K application.


       
In this study, K supplementation in varying concentrations ameliorated significantly (P≤0.05) the negative effects of DS in black gram. Shoot length, leaf area and shoot fresh and dry weight all drastically decreased in response to DS, while root length was pronouncedly increased. With K supplementation, black gram yield and yield components improved in a water-limited environment, likely because K served as a primary osmotic solute for plants under DS. Loss of turgidity is directly related to cell expansion and growth, so water stress has a major impact on plant growth. Likewise, the results showed that the leaf area and the photosynthetic pigments were negatively impacted by DS, leading to a decrease in photosynthetic efficiency. Black gram’s chlorophyll content and leaf area were decreased due to water scarcity, which could have diminished the plants’ light-harvesting efficiency and led to an excess of oxygen radicals produced by the photosynthetic apparatus (Ihsan et al., 2013). The higher yield was primarily associated with greater leaf area, which provided more surface area for light interception and photosynthesis. Plants under DS were unable to uptake sufficient water, resulting in a reduction in pressure potential and, in turn, a decrease in turgor potential. This impeded plant growth by drying out the protoplasm. Under DS, plant water status and gas exchange attributes were negatively affected, as shown by a disruption in the source and sink ratio in this study, which indicated that leaf water content had a strong correlation with plant’s drought tolerance (Khorsand et al., 2021). Further, under DS, stomata begin to close, decreasing stomatal conductance and, consequently, reducing CO2 availability in the chloroplast, which may result in diminished carbon assimilation. The K application increased stomatal opening and plant growth in response to DS by increasing osmotic water uptake and preserving cell turgidity. The DS disrupted the plant’s metabolism and reduced crop productivity, whereas exogenously applied K downregulated the lipid peroxidation (malondialdehyde content), H₂O₂ concentration and cell membrane injury along with upregulating gas exchange attributes, plant water relations and antioxidant defense system (Kumar et al., 2020).
       
Foliage application caused a K influx within the stomatal guard cells, which in turn caused a buildup of water, swelling of the guard cells and a stomatal opening that allowed CO₂ and transpired water vapors to freely move into, out of and throughout the plant tissues (Zhang et al., 2023). The K efflux from the guard cells was stopped and the pores were securely closed to prevent any water loss that would have hampered plant development and cell turgidity. However, the optimized concentration of K boosted plants’ ability to absorb and use water under DS. The results showed that K treatments enhanced leaf water relations by decreasing cumulative transpiration water loss. The K foliar feeding supported the critical osmotic pull that aided in drawing water from the roots, whereas its deficiency increased plant susceptibility to adverse effects of DS (Zahoor et al., 2017).
       
In this study, it was recorded that the oxidative stress indicators such as the MDA concentration increased as a signal of deterioration in plant cells owing to the biosynthesis and excessive accumulation of reactive oxygen species (ROS) by 42.2%. Likewise, the membrane stability was also adversely affected due to the high amount of MDA, which indicated the extent of lipid peroxidation in black gram. Moreover, over-accumulation of reactive oxygen species (ROS) has also been reported as a vital indicator of oxidative damage and the cytotoxic products produced due to lipid peroxidation (Alkhsabah et al., 2018). Overproduction of reactive oxygen species (ROS) caused serious injuries and caused biological membranes to become leaky, allowing electrolytes to move freely within the cell. To reduce the negative effects of drought, black gram plants increased accumulation of plant metabolites such as total phenolics, total soluble proteins and enzymatic antioxidants such as catalase, peroxidase and superoxide dismutase under DS. The findings were consistent with those of Helaly et al., (2017), who found that malondialdehyde and hydrogen peroxide were toxic under DS and were responded to by a significant increment in the concentration of enzymatic antioxidants with K supplementation.
       
These findings corroborated previous research whereby DS caused oxidative stress whereas K application mitigated its deleterious effects by triggering the biosynthesis of antioxidant enzymes and a wide range of plant metabolites (total phenolics, superoxide dismutase, total soluble sugars, peroxidase and catalase) (Jothimani and Arulbalachandran, 2020). By converting O₂ into H₂O₂, which peroxidase also helped to eliminate, superoxide dismutase assisted in the detoxification of O₂. Additionally, peroxidase enzymes are essential for the detoxification of hydrogen peroxide, the oxidation of phenolics and the regulation of cell elongation (Martineau et al., 2017). It was also found that the antioxidant system regulated the delicate balance of oxygen radicals’ production and detoxification, which inhibited lipid peroxidation and improved plant water status, gas exchange characteristics, crop growth and seed yield. Moreover, plants with an exogenously applied K had a higher capacity to scavenge ROS (Ul-Allah et al., 2020).
       
The K supplementation exhibited pronounced potential to protect chlorophyll content and biological membranes under DS by upregulating water relations. The application of K has been shown to reduce ROS biosynthesis by blocking electron transport from photosynthesis to molecule O₂ (Kumaret_al2019). In an experiment with sunflower (Helianthus annuus L.), an adequate supply of K greatly reduced the MDA concentration, demonstrating the importance of K in reducing oxidative stress (Soleimanzadeh et al., 2010). The outcomes have shown that several K-induced mechanisms, including (i) efficient photosynthetic rate and maintenance of turgor pressure, (ii) prevention of drought-induced accumulation of malondialdehyde content, reactive oxygen species (ROS) and cell membrane injury and (iii) upregulation of antioxidant defense system, contribute to the mitigation of DS in black gram.

The results were per the postulated hypothesis as black gram under drought stress responded significantly to the exogenous potassium application. The recorded findings of this study imply that the K supplementation holds the potential to alleviate the deleterious impacts through improvements in water relation, morpho-physiological and biochemical traits of black gram, which led to DS tolerance. This study further explored the optimum dose (100 mg/L) of K that could be useful for inducing antioxidant defense and plant fitness against DS. This study provides a mechanistic overview of K involving the enhancement of plant fitness, agronomic yield and DS tolerance in black gram. Exogenous supplementation of K induced a series of morpho-phycological and biochemical traits, which helped to improve plant robustness, agronomic yield and DS tolerance in black gram. These findings might be helpful to pulse breeders and farmers in improving plant fitness and drought adaptation. However, the limitation of this study necessitates conducting future studies to explore the underlying K-mediated mechanisms that impart tolerance against drought stress by neutralizing or minimizing the deleterious effects of reactive oxygen species. 

All the authors are thankful to the Researchers Supporting Project number (RSPD2024R561), King Saud University, Riyadh, Saudi Arabia.

This Research was funded by the Researchers Supporting Project number (RSPD2024R561), King Saud University, Riyadh, Saudi Arabia.

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

No animals or humans were subjected to experimental treatments in this trial.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.