Role of Potassium on Growth, Nitrogen Fixation and Biochemical Traits in [Vigna radiata (L.) Wilczek] under Water Stress

Water stress is considered to be the leading cause of crop growth, yield and quality losses (Ahmad et al., 2015). Vigna radiata (L.) Wilczek (mungbean), contains very low levels of oligosaccharides and a good protein (23%), phosphorus, carbohydrate, minerals and provitamin A source, with high digestibility and suitable as fodder and green manure (Ihsan et al., 2013). Mungbean is more sensitive to drought at flowering and grain development stage. Application of potassium improved the ability of plant to tolerate osmotic stress, in drought by minimizing its negative effects and enhancing the uptake and translocation of water to make a balance on plant (Adhikari et al., 2019).

The present study was carried out for three years (2010-11, 2011-12 and 2012-13) during summer season under net house conditions at Botany Department, Kurukshetra University, Kurukshetra, Haryana. Sterilized seeds of SML-668 and MH-318 were inoculated with standard Bradyrhizobium sp. S-24. The crop was raised in earthen pots (30 cm in diameter) lined with polythene bags and filled with 7.0 kg of dune sand. Five seeds were sown in each pot at uniform depth and distance and only two plants were retained in each pot after thinning. Sowing was carried out at field capacity of the soil. The experiment was laid out in factorial complete randomized design (CRD) with three replications.
 
Chemical analysis of soil
The experimental soil samples were taken from the depth of 0-15 cm for chemical analysis. The soil samples were air dried, ground and well mixed and analyzed for chemical properties.


 
Level of potassium
 
After germination 7 days after sowing (DAS), potassium was supplied in the soil in the form of muriate of potash in the following concentration in addition to the existing level 1.27 mM (52 ppm) in the soil medium.
a) 0
b) 1.54 mM (60 ppm)
c) 2.31 mM (90 ppm)
d) 3.08 mM (120 ppm)
 
Level of stress
 
The plants were subjected to water stress by withholding the irrigation at flowering stage (i.e. 35-40 DAS):


 
The pots were weighted daily and depletion in soil moisture content (SMC %) was maintained gravimetrically. Each pot was supplied with an equal quantity of nitrogen-free nutrient solution (Wilson and Reisenauer, 1963) at a regular interval of 7-10 days.
       
At flowering stage, three replicates of each treatment along with control were sampled to record the various parameters mentioned below.
 
Growth parameters
 
Fresh weight was determined with the help of an electronic balance. Plant parts dried kept in an oven at 60oC till a constant weight then, the dry weight was measured. The nodules present were first detached and then counted carefully. The plant height was measured using a meter scale and expressed in centimeter. All the leaves were detached from the stem and their area per plant (sq. cm) was determined by using portable leaf area meter (Systronics 211, Ahmadabad, India).
 
Nitrogen fixation and Nitrate reduction
 
All the detached nodules from the roots of the sampled plants were thoroughly mixed and their leghemoglobin content was estimated by the method of Hartree (1955). Nitrogenase activity of nodules was measured by acetylene reduction assay (ARA) of Hardy et al., (1968). The nitrate reductase activity (NRA) was determined by the method of Jaworski (1971). Nitrite Reductase Activity (NiRA) was estimated by the intact tissue assay of Ferrari and Varner (1971).
 
Biochemical estimations
 
Free proline was estimated spectrophotometrically according to Bates et al., (1973). Total soluble carbohydrates (TSC) were estimated by the method of Yemm and Willis (1954). Total soluble protein (TSP) content was measured by Folin-Ciocalteau reagent, Lowry et al., (1951). Free amino acids (FAA) were estimated by the method of Yemm and Cocking (1955).
 
Mineral analysis
 
Leaf total potassium (K) content of leaves was measured using Flame photometer (Model Elico) and expressed as mg g^-1 DW.
 
Statistical analysis
 
The data collected was analyzed statistically by the online Statistical Analysis (OPSTAT, CCS Haryana Agriculture University, Hisar). The significance of data obtained was judged from the critical difference at 5 % level of significance.

Growth and development
 
Water stress caused a significant decrease in root fresh and dry weight in the genotype MH-318 over control at the sampling stage (Table 1). The decrease in fresh and dry weight of roots was due to the low availability of moisture around the roots, decrease in relative turgidity, decreased cell expansion/cell division and restricted proliferation of root biomass. Increase in fresh weight was observed in genotype SML-668 at 3.08 mM potassium concentration (Fig 1). Increase in photosynthesis, leaf area, accumulation of sugar and translocation of photo assimilates under the influence of potassium are the reason of increase in root biomass (Jordan-Meille et al., 2018). Under water stress condition, the decreases in nodule biomass occur due to reduced availability of carbohydrate to the nodules. Nodules showed decreased branching, breakdown of endodermis, greater compactness and decreased vacuolation of cells in the central tissue as compared to the control under water stress (Swaraj et al., 1995). Potassium increase total leaf area and hence, photosynthesis, this further increases progressive translocation of photosynthates towards roots system for use by nodules and hence improves root nodules (Suryapani et al., 2014).
 

 

 

       
The average plant height under control condition was observed as 24.1 cm in SML-668 while 21.4 cm in MH-318 (Table 1). Water stress affected plant height due to hormonal imbalance, the expression of enzyme-coding genes involved in the biosynthesis and the production of ethylene increased after potassium deprivation (Shin and Schachtman 2004). Moreover, proteins involved in the biosynthesis of Jasmonic acid increase during potassium starvation and rapidly decrease after K resupply (Armengaud et al., 2004). All these factors lead to changes in cell wall extensibility. Water stress reduces nutrient uptake by roots and transport from roots to shoots because of restricted transpiration rates, impaired active transport and membrane permeability, all these reasons leads to reduced plant height. In potassium treated plants there is increment in plant height in both the cultivars (Fig 3). The increase in plant height with potassium occurs due to improvement in many physiological processes such as activation of enzymes, photosynthesis and maintenance of turgor and translocation of photosynthates (Mengel and Kirkby, 2001). Similar increase was observed by Ahmad et al., (2019) in wheat.
       

 
Significant decrease in leaf area under water stress was observed in both the cultivars (Table 1). Drought-induced reduction in leaf area is ascribed to suppression of leaf expansion through reduction in photosynthesis and relative water content. Leaf injury during water deficit stress is correlated with vulnerability to oxidative stress, accompanied by chlorophyll loss and decreased soluble protein content (Farouk, 2011). Potassium brought a significant expansion of leaf area under control as well as stress conditions at all the stage as it greatly improved the retention of water in the plant tissues under conditions of water stress. The increase in leaf area also occurs due to high maintenance of nutrient concentration in leaf tissues under sustained supply of potassium.
 
Nitrogen fixation and Nitrate reduction
 
Maximum reduction (35.5%) in leghemoglobin content was recorded in the genotype MH-318 under stress conditions without potassium application (Table 2). Nodule dehydration and tissue damage in nodules, low O₂ diffusion, a decrease in sucrose synthase activities, limitations of carbon flux and inhibition of protein synthesis have all been reported concerning the sensitivity of nitrogenase activity to moisture stress. Moreover, the inhibition of nitrogenase activity is often correlated with an increase in resistance of the oxygen diffusion barrier of the nodule cortex. Water stress acts directly on nodule activity by decreasing nitrogenase-linked respiration. A decrease in nitrogenase activity results in low ammonia production and a decrease in water stress enzymes that assimilate ammonia. The decline in nitrogenase activity when subjected to drought was observed by Tint et al., (2011) in soybean and El-Enany et al., (2013) in cowpea. Increasing potassium concentrations showed a considerable increase in leghemoglobin content and nitrogenase activity of nodules under control and stress conditions in both the cultivars. Potassium application increased the nodule size, biomass, nitrogen fixation and N-turnover due to increase in translocation of photosynthates from leaves to the nodules hence, enhanced nitrogenase activity. Nodulation, nitrogenase activity and dry matter increased with incremental potassium supply in broad bean grown at moisture level of only 1/4^th of field capacity (Abd-Alla and Abdel-Wahab, 1995). Nitrate and Nitrite reductase activity decreased under water stress condition as compared to normal conditions. It is possible that reduced import of sucrose and water into nodules via the phloem may also reduce the export of nitrogenous products into the xylem stream. Improved supply of carbohydrates to nodules resulting in an enhanced turnover of tricarboxylic acid cycle in bacteroides, thus providing higher rate of ATP and reducing electrons to nitrogenase. Water stress decreases the nitrate reductase activity either by inhibiting nitrate uptake or protein synthesis. The peak activity of nitrate reductase activity coincides with the maximum chlorophyll content and thereby complementing the carbon-nitrogen balance in plant (Dhumal and Laware, 2003). Drought stress was found to induce a greater reduction in NRA of the drought-sensitive cultivar than of the drought-tolerant at different growth stages (Zhang et al., 2014). Umar (2006) also reported that potassium absorption increased the nitrate reductase activity. Increase in leaf SPAD values by potassium enriched plant is confirmed with the knowledge of nutrients that increases activity of nitrate reductase enzyme. Mohammad and Naseem (2006) declared that the potassium by increasing the nitrate reductase activity, leads to efficient formation of molecules with nitrogen in their structure, which is responsible for synthesis of proteins and enzyme.


 
Biochemical estimations
 
The TSP significantly decline under water stress in both the cultivars (Table 3). Drought stress injury causes damage to protein synthesizing mechanism, which leads to increased activity of proteases enzyme this in turn, promote the breakdown of the proteins and consequently decrease the protein amount. Water stress conditions also activate the pathway of proteins breakdown, because the plant use the proteins for the synthesis of nitrogen compounds as amino acids that might auxiliary to  the plant osmotic adjustment (Sankar et al., 2007). The alteration in protein synthesis is one of the fundamental metabolic processes that influence water stress tolerance (Jiang and Huang, 2002). Moreover, due to decreased photosynthesis the materials for protein synthesis were not provided therefore, protein synthesis is dramatically reduced. Similar results on reduction in proteins were found by Ramos et al., (1999) under water stress in Phaseolus vulgaris. Potassium is required for maintaining the activation of enzymes and protein synthesis in plants because the whole structure of proteins and protein activity needs high concentrations of potassium in the cytosol for optimum plant functions, it also increases synthesis of amino acid into protein (Cherel, 2004).
               
The increase under stress in the FAA under water stress is due to high synthesis of amino acids from protein hydrolyses. FAA is utilized by the plant to reduce the effects of water deficit through organic solute accumulation and this way increased the water retention capacity (Sircelj et al., 2005). Further, increase in the content of free amino acids could either be due to disruption in protein synthesis and its partial hydrolysis or reduced flow of amino acids to physiologically active sink. Result on increase in the free amino acids was found by Asha and Rao (2002) working with Arachis hypogaea. Application of potassium resulted in the decreased FAA content under stress and normal conditions in both the cultivars. Potassium functions in the formation of an effective polyribosome complex that apparently preceded the actual incorporation and synthesis of amino acids into protein. Plant suffering from potassium deficiency showed relatively higher content of free amino acids.
 
Water stress resulted in a conspicuous accumulation of proline in both the cultivars (Table 3). SML-668 showed higher proline accumulation under stress and marginal edge in control plants over cv. MH-318. Proline accumulation is believed to protect plant tissues against stress by acting as nitrogen storage compound, serves as a sink for energy to regulate redox potentials, osmosolutes and as a solute. Proline protects macromolecules and cellular structures against denaturation and as a means of reducing acidity in the cell. A similar trend was obtained by Jayant and Sarangi (2014), where resistant varieties accumulate higher level of proline under water stress. Proline contributes to a protective role as scavenges of reactive oxygen species (ROS), resulted in improved adaptation ability and growth of plants under drought conditions. A significant decrease in proline level was observed with applied potassium under control and stress conditions. The accumulation of proline was less under stress conditions as compared to control with potassium application. Similar findings were observed by Sharma et al., (2008) in Brassica. Such decrease in proline content with potassium application may be ascribed to improved turgor through osmotic adjustment hence, could be used as potential osmotica under water deficit.
 

               
Total soluble carbohydrates (TSC) of leaves increased under stress conditions in both the cultivars (Table 3). The increase in TSC in stressed plants was 25.7% in cv. SML-668 and 20.2% in cv. MH-318 as compared to control plants. The accumulation of TSC is probably because its normal utilization and translocation is inhibited during water stress and accumulated carbohydrates have been accounted as part of the osmotic adjustment. The TSC progressively increased in plants under water deficit and in this way improved the resistance of the plants to water deficit (Li and Li, 2005). The sucrose level progressively increased in the plants under water deficit due the sucrose biosynthesis, which is promoted by the increase in sucrose phosphate synthase enzyme activity, which in turn protects membranes and integrity proteins. Accumulation of dissolved sugar compounds interact with cellular macromolecules as enzymes and stabilize their structure. Significant increase in TSC of leaves in response to applied potassium was observed under normal and stressed plants in both cultivars, this occurs due to enhanced photosynthetic rate through improved turgor and stomatal conductance (Fanaei et al., 2011). 
 
Mineral analysis
 
A significant increase in potassium content of leaves was observed under water deficit conditions over control in both the genotypes (Table 4). An important effect of water deficit is on the acquisition of nutrients by the root and their transport to shoots. Higher potassium accumulation has been reported in drought stressed okra (Kusvuran et al., 2011) and common bean (Zadehbagheri et al., 2012). In stressed plants, large numbers of organic or inorganic ions were accumulated which provide resistance against drought. This can be a reason for accumulation of potassium under drought stress. Treatment with potassium resulted in increased potassium content of leaves in both the cultivars under both the conditions. The percent increase in potassium content with potassium application was higher in stressed plants than control ones. Application of potassium enhanced K uptake, irrespective of soil moisture regimes (Baque et al., 2006). Furthermore the application of potassium fertilizers in combination with organic matter improved soil physical and chemical properties by enhancing biological activity and soil organic carbon accumulation thus helping in the uptake of nutrients.
 

Under water stress potassium improved the nodulation, nitrogen fixation and enzymes of nitrogen assimilation. Proline accumulation significantly increased under water stress and decreased with potassium application. Osmotic adjustment is an important mechanism in mungbean genotypes under water stress conditions. Organic and inorganic compounds such as proline, soluble carbohydrates, total free amino acids and potassium play key roles in this regard. Genotype SML-668 able to do better osmotic adjustment under water stress through accumulation of more proline and total soluble sugars hence, proved more water stress resistant than MH-318.

The authors are grateful to the International Potash Institute, Switzerland for the financial support and Kurukshetra University, Kurukshetra for providing the laboratory facilities.