Physiological Divergence in Green Gram [Vigna radiata (L.) Wilczek] Genotypes for Drought and High Temperature Stress Tolerance During Flowering Phase

Green gram is one of the important pulse crops with high nutritional and economic value. It improves the soil fertility by fixing nitrogen and inhibits soil erosion. Due to climate change, high temperature and drought occur simultaneously which severely affect the plant growth and development (Nahar et al., 2017). Among the various stages of plant growth and development, reproductive stage is highly sensitive to drought and high temperature stress across all species. In green gram, a temperature of >40°C was observed to have detrimental effect on flower and pod formation (Kumari and Varma, 1983; Hatfield et al., 2011).
       
The combined drought and high temperature stress affects the photosynthetic rate of the plants (Farooq et al., 2009) and reduction in photosynthesis is mainly due to decrease in leaf expansion, improper functioning of photosynthetic machinery and leaf senescence (Wahid et al., 2007). The adverse effect of drought on plant growth interferes with the realization of yield components such as pod number per plant and harvest index. This is due to the change in level of photosynthetic pigments and damage in photosynthetic machinery under drought condition (Fu and Huang, 2001). High temperature stress disrupts the integrity of the photosynthetic apparatus and its function (Quinn and Williams, 1985) and ultimately there is considerable reduction in plant growth and yield. Under stress condition the production and accumulation of reactive oxygen species (ROS) in the membrane cell wall, which collapse its structural integrity and cause cellular damage. The production of ROS such as superoxide (O₂^-), hydroxyl radical (OH-) and hydrogen peroxide (H₂O₂) may accelerate the progress of many metabolic changes by altering the molar ratio of the biomolecules and structure of biomembranes, which leads to lipid peroxidation, protein denaturation and  eventually leads to localized cell and tissue death (Miller et al., 2008; Choudhury et al., 2013). The increase in the level of ROS might tightly be regulated by the activities of antioxidant enzyme and antioxidants levels. The antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) are involved in quenching of oxidants produced under oxidative stress (Djanaguiraman et al., 2010). The main objective of this study was to evaluate green gram germplasm collection and identification of elite collection that can withstand stresses at reproductive stage.

Plant material and growing condition
 
To study the influence of combined drought and high temperature stress during reproductive stage, the green gram genotypes were sown in pots (six pots were maintained for each genotype of which three were maintained at 100% field capacity (control) and for another three, drought stress (50% field capacity for 5 days) was imposed combined with high temperature stress (36±2°C) during reproductive phase (35 Days after sowing). The experiment was conducted during March-April, 2019, at National Pulses Research Centre, Vamban, Pudukottai district, Tamil Nadu.
 
Measurement of relative water content
 
Relative water content (RWC) of leaf was measured according to Barrs and Weatherley, 1962.
 
Where
FW- Fresh weight.
TW- Turgid weight.
DW- Dry weight.
 
Measurement of chlorophyll content
 
The green gram leaf samples were homogenized with 80% acetone and the absorbance were measured at 663 and 645 nm to quantify the chlorophyll (chl) a, chl b and total chlorophyll content using UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic) (Yoshida et al., 1971).
 
V - Volume, W - Tissue weight, Abs₆₆₃ - Absorbance at 663 nm and Abs₆₄₅ - Absorbance at 645 nm.
 
Measurement of chlorophyll stability index (CSI)
 
Chlorophyll stability index (CSI) was measured according to (Murthy and Majumdar, 1962). Two batches if equal number of green gram leaf discs were taken in two test tubes. One set of test tubes containing sample was kept as control and the other set was placed in hot water bath for 30 min followed by homogenization with 80% acetone and centrifuged at 3000 rpm for 10 min. Absorbance of the supernatant was measured at 652 nm in UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic).
 
Measurement of Superoxide (O₂^-) radical content
 
Superoxide anion radical was quantified according to Chaitanya and Naithani (1994) by homogenizing the leaf samples in ice-cold sodium phosphate buffer (0.2 M, pH 7.2) containing diethyl dithiocarbamate. The homogenate was centrifuged at 3000 rpm for 1 min. Superoxide anion radical was measured by its capacity to reduce nitroblue tetrazolium. Absorbance of the supernatant was measured at 540 nm with a UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic).
 
Measurement of Hydrogen peroxide
 
Hydrogen peroxide (H₂O₂) level was quantified as per Patterson et al., (1984). Leaf samples were homogenized in 1 mL of cold acetone. 0.1 mL of 20% titanium reagent (20% w/v TiCl₄ in 12.1M HCl) and 0.2 mL of 17M ammonia (NH₃) solution were added in known volume of supernatant. It was centrifuged at 3000 rpm for 10 min at 4oC and the supernatant was discarded. The pellet was dissolved in 3 mL of 1 M sulphuric acid (H₂SO₄). Absorbance was measured at 410 nm using UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic).
 
Lipid peroxidation
 
Lipid peroxidation was determined by malondialdehyde (MDA) content produced by thiobarbituric acid (TBA) as described by Behera et al., (1999). Leaf samples were homogenized in 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm for 5min at 4°C. The aliquot was mixed with 1.2 mL of 0.5% TBA prepared in 20% TCA and incubated at 95°C for 30 min. After stopping the reaction in ice bath for 5 min, samples were centrifuged at 10,000 rpm for 10 min at 25°C. Absorbance of the supernatant was measured at 532 nm with a UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic).
 
Membrane damage
 
Leaf samples were cut into small pieces and washed with deionized water, then incubated in 10 mL of deionized water at 2°C for 4 h in a shaker. The initial electrical conductivity (E1) was measured using EC/TDS hydrotester. The samples were then boiled at 95°C for 60 min in water bath and cooled to 25°C and again the electrical conductivity (E₂) was measured. The relative electrolyte leakage or membrane injury was estimated using the following formula given by Chauhan and Senboku (1996):  
Antioxidant content
 
Proline content
 
For assessing the proline content, leaf samples were homogenized in 3% sulfosalicylic acid and centrifuged at 11,500 rpm. Supernatant was mixed with acid ninhydrin, glacial acetic acid and phosphoric acid. The mixture was incubated at 100^oC for 1 h and cooled down. The mixture was taken in a separating funnel and equal volume of toluene was added and mixed well and the developed pink coloured upper layer was collected to determine the proline content by measuring its absorbance in UV-VIS spectrophotometer (Eppendorf BioSpectrometer kinetic) at 520 nm (Bates et al., 1973).

Catalase activity
 
Catalase activity was estimated according to Aebi (1984). The catalytic activity of the enzyme was measured spectrophotometrically (Eppendorf BioSpectrometer kinetic) by recording the decline of absorbance at 240 nm due to decomposition of H₂O₂.

Relative water content
 
Drought and high temperature stress significantly reduced the RWC in green gram genotypes during reproductive stage as compared to control. The green gram genotypes TARM 1 (87.23%), VGG 17006 (84.05%) showed higher RWC as compared to green gram genotype VGG 17037 (29.58%) under drought and high temperature stress during flowering phase (Table 1).      



Relative water content (RWC) is one of the parameter to measure the water status in the plants (Deivanai et al., 2010).  The loss of turgidity due to reduction in RWC leads to closure of stomata, this in turn reduced the photosynthetic rate. The drought and high temperature stress significantly reduced the relative water content in the green gram genotypes. Higher RWC represent the water status of the plants under stress condition (Nahar et al., 2015). The RWC was affected by the interaction of severity, duration of the drought event and species (Yang and Miao, 2010). Similar results were observed in green gram under drought and high temperature stress (Nahar et al., 2017).

Chlorophyll content
 
There was significant decrease in chlorophyll content in the green gram plants grown under drought and high temperature stress conditions. The chl a, chl b and total chl content were decreased under drought and high temperature stress when compared with the control. The chl a content was higher in green gram genotypes VGG 17004 (1.62 mg g^-1 FW) and VGG 17003 (1.33 mg g^-1 FW), while VGG 17019 (1.01 mg g^-1 FW) and VGG 17010 (0.98 mg g^-1 FW) has recorded higher chl b content. Among these two genotypes, VGG 17010 has recorded high chl a and b content at D+HT stress conditions. This increase may be due to increase in the content of PS I and PS II subunits, which may protect the chl a/b proteins from proteosomal degradation and thus maintains high chl a and b content even under stress conditions (Shan et al., 2018). Among the genotypes screened, VGG 17004 recorded high chlorophyll content (2.53 mg g^-1 FW) followed by VGG 17003 (2.25 mg g^-1 FW) during reproductive stage under drought and high temperature stress condition (Table 1 and 2).
 

       
The reduction in photosynthetic pigments such as chl a, chl b and total chl content under drought and high temperature stress may be due to inhibition of chlorophyll biosynthesis or due to the burst of reactive oxygen species (ROS) induced oxidative stress due to increase in temperature and drought condition (Prasad et al., 2011).
 
Chlorophyll stability index (CSI)
 
Maximum chlorophyll stability index was observed in green gram genotypes VGG 17003 (89.67%), VGG 15029 (86.05%) and CSI was minimum in green gram genotype VGG 17037 (27.69%) green gram genotypes under drought and high temperature stress during reproductive stage (Table 2). It is one of the important traits that reflects the ability of plants to sustain photosynthesis under stress condition (Sayed, 1999).
 
Oxidant content
 
The superoxide radical content was significantly decreased during reproductive stage in VGG 17019 (0.87 change in OD min^-1 g^-1 FW), VGG 15029 (0.90 change in OD min^-1 g^-1 FW) and increased in VGG 17037 (2.77 change in OD min^-1 g^-1 FW) green gram genotypes under drought and high temperature stress (Table 3). The hydrogen peroxide content was lower in green gram genotypes COGG 1332 (8.40 nM g^-1 FW) and VGG 16069 (9.23 nM g^-1 FW) genotypes and higher in VGG 17045 (23.90 nM g^-1 FW) genotype when compared with other green gram genotypes exposed to drought and higher temperature stress during reproductive stage (Table 3). During reproductive stage malondialdehyde content was significantly (P < 0.001, Table 3) decreased in COGG 1332 (10.25 nM g^-1 FW), VGG 16069 (10.35 nM g^-1 FW) genotypes when compared with other green gram genotypes imposed to drought and higher temperature stress during reproductive stage (Table 3).
 

       
Production of ROS was observed mainly in chloroplast (Reddy et al., 2004) and in mitochondria (Moller, 2001), when O₂ reacts with the components of the electron transport chain. Increase in ROS results in oxidative damage to the crop plants, ROS also cause programmed cell death, oxidation of nucleic acids (Moller et al., 2007). Reduction in anti-oxidant defense causes injuries in leaves due to oxidative damage such as degradation of lipids and proteins (Kumar et al., 2012; Fahad et al., 2017) resulting in inhibition in the functioning of cells; precisely, peroxidation of lipids and oxidation of amino acid and carbonylation of protein results in collapse of the membrane structural integrity thereby affecting the physiological and biochemical processes in plants. Lipid peroxidation was observed to be increased by four times in pea (Pisum sativum) under drought stress.
 
Antioxidant content
 
Under drought and high temperature stress the following green gram genotypes viz., VGG 17003 (14.34 μM g^-1 FW), VGG 17019 (13.44 μM g^-1 FW) have accumulated significantly more proline as compared to the green gram genotypes VGG 16027 (1.45 μM g^-1 FW), CO8 (2.30 μM g^-1 FW) as shown in (Table 4). Higher proline content was observed in green gram genotypes VGG 17003 (14.27 μM g^-1 FW), VGG 16069 (12.83 μM g^-1 FW), the proline content was lower in CO 8 (1.13 μM g^-1 FW) (Table 4) as compared to other genotypes during reproductive stage. The catalase enzyme activity was higher in VGG 17019 (32.63 μM H₂O₂ destroyed min^-1 g^-1 FW), VGG 16069 (31.46 μM H₂O₂ destroyed min^-1 g^-1 FW) genotypes and lower in VGG 17036 (7.43 μM H₂O₂ destroyed min^-1 g^-1 FW) genotypes under drought and high temperature stress during reproductive stage (Table 4). To cope with oxidative stress, plants prevent the ROS production by antioxidant defence involving enzymatic or non-enzymatic antioxidants and enzymatic based antioxidant defense mechanism is most effective in combating the oxidative stress induced damages in crop plants (Farooq et al., 2008).
 

In the present study, drought and high temperature stress has negative impact on green gram physiology and this was evident by reduction in relative water content, chlorophyll concentration and reduction in chlorophyll stability index. Among the genotypes screened for their tolerance at reproductive stage, the following green gram genotypes viz., TARM 1, VGG 15029, VGG 17003, VGG 17004, VGG 17006, VGG 17010 and VGG 17019 were found to withstand the drought and high temperature stress and maintain high total chlorophyll content, relative water content and chlorophyll stability index. These green gram gramplasm can be used in pulse breeding program to evolve resilient green gram varieties.

We thank Department of Science and Technology (DST) - Science and Engineering Research Board (SERB), New Delhi for financial support (ECR/2016/001006). We would like to thank National Pulses Research Centre (NPRC), Vamban, Pudukottai for providing the green gram seed material.

The authors declare that they have no conflict of interest.