Effect of Different Biochemical Parameters and Antioxidant Enzymes Activities on Drought Indices in Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is one of the oldest cultivated food legumes with high nutritional, agronomical and economically important crop in the world. It is an important cool season legume crop with a genome size of 738 Mb (Varshney et al., 2013). India is the largest producer of chickpea having annual production of 10.13 million tonnes from a land area of 9.44 million hectares and a productivity of 1073 kgha^-1  (Asati et al., 2023). Among different chickpea-growing countries including Africa, south America and Indian Subcontinents, India alone contributes more than 70% of the world’s total production. Moreover, it is the most important legume crop and a source of nutrition to millions of people globally owing to its richness in protein, fibre and minerals (Asati et al., 2023). Thus, characterization of chickpea genotypes to select superior line (s) resistant /tolerant against different biotic and abiotic stresses is one of the most important requirements being applied widely at present (Sahu et al., 2020a).
       
Abiotic stresses tolerance is a complex trait, owing to the interactions between diverse stress factors and various molecular Tripathi et al., (2023), biochemical Sahu et al., (2022); Tomar et al., (2024); Tiwari et al., (2023a) and physiological mechanisms (Aarif et al., 2021). Affecting plant growth at different developmental stages. Among diverse abiotic stresses, drought is one of the major abiotic constraints that alters the plant growth and development. Even though chickpea is good in storing water in the soil within the root zone, but it suffers from terminal drought which delays flowering. Drought stress inhibits the uptake of nitrogen and subsequent fixation in the roots of chickpea as well. Drought generally affects chickpea production in areas lacking satisfactory and constant rainfall, it affects overall crop performance starting from germination, with losses of up to 40-50% in crop productivity (Sachdeva et al., 2022).
 
Drought stress is known to alter the chlorophylls and carotenoids concentrations in plant tissues (Tiwari et al., 2023b). It is generally associated with the accumulation of osmoprotectants such as proline. Likewise, the role of reactive oxygen species (ROS) in stress signalling have also been extensively investigated in recent years. The reactive oxygen species in plants are removed by a variety of antioxidant enzymes and/or lipid soluble and water-soluble scavenging molecules (Mishra et al., 2023). The antioxidant enzymes being the most efficient mechanisms against oxidative stress (Sharma et al., 2021). Estimation of different biochemical parameters and antioxidant enzymes activities may be effectively employed for selecting putative drought tolerant chickpea genotype(s). The present study was accomplished to screen drought tolerant genotype (s) based on manifestation of diverse biochemical parameters and antioxidant enzymes activities.

A total 83 chickpea genotypes including 63 desi and 20 kabuli were obtained from All India Coordinated Research Project on chickpea, College of Agriculture, Sehore, Madhya Pradesh, India. The genotypes grown in randomized block design with spacing of 30 cm × 15 cm (row to row × plant to plant), in two replications during Rabi 2021-22 at the experimental field of Rajmata Vijayraje Scindia Krishi Vishwa Vidyalaya, Gwalior, India. The soil type of experimental field was sandy loam having pH 7.1 and containing available N, P and K 202.5 kg/ha, 12.4 kg/ha and 249.7 kg/ha respectively with an average organic matter. First irrigation was given to plants at 30 days after sowing then field was kept unirrigated, for imposing drought condition, 40-day old plants were left unirrigated until podding, which occurred when soil moisture content (SMC) dropped to 20-40%. For the well-watered control, plants received water at regular intervals especially after 60 days to keep the SMC at 50 to 55 per cent. Plants were sampled at 75 days after sowing for measurement of different biochemical parameters from control as well as stressed conditions.
 
Estimation of different biochemical parameters
 
Total chlorophyll
 
Total chlorophyll was calculated as per method given by Arnon et al., (1949). It was estimated 2 times first on 30 days and second on 75 days after sowing, Leaf samples was crushed in 10 ml (80% acetone) finely and was transferred into a falcon tube. Centrifuged for 15 minutes at 10000 rpm and the green supernatant was transferred into a fresh 15 ml falcon tube. Then readings were taken in a spectrophotometer at 643 nm, 663 nm and 470 nm wave lengths.
 
Lipid peroxidation
 
MDA (malondialdehyde) was measured by following the procedure as described by Heath and Packer (1968). Twenty-five mg of leaf sample was taken and crushed in liquid nitrogen. Then 500 microliters of 0.1% trichloro-acetic acid were added and vortexed and centrifuged at 10000 rpm for 10 min. Hundred microliters of supernatant in an Eppendorf tube was taken and 200 microliters of 0.5% TBA was added subsequently. The reaction mixture was heated at 95°C for 30 minutes and quickly kept at -80°C for 2 minutes to stop the reaction. After 2 minutes it was centrifuged at 10000 rpm for 10 min in room temperature and supernatant was taken for reading at 532 nm.
 
Total sugar
 
Determination of total sugar was done by anthrone reagent method as described by Dubois et al., (1956). Hundred mg fresh random leaf samples) was taken and crushed in a mortar pestle until the leaf completely disappears and fine liquid solution was made with addition of 5 ml of 80% ethanol. The solution was poured in the 15 ml falcon tube and was centrifuged at 1000 rpm for 10 minutes. Then the supernatant was transferred in a fresh 15 ml falcon tube and more 5 ml (80% ethanol) was added into the old tube which has leaf extract and again centrifuged at 10000 rpm for 10 minutes. Total 10 ml (5 ml+5 ml), dry 10 ml supernatant in glass bottle was incubated at 65°C until it gets dried. After drying 1ml distilled water was added and it was left until it has gotten dissolved. Then 100 microlitre was taken from a glass bottle with the help of a pipette then anthrone reagent was added in a falcon tube and it was heated at 100°C for 30 minutes and cooled until it comes at room temperature and then reading was recorded at 630 nm in the spectrophotometer.
 
Proline
 
Free proline content in leaves was determined according to the method proposed by Bates et al., (1973) based on the formation of red coloured formazine by proline with ninhydrin in acidic medium, which is soluble in organic solvents like toluene.
 
Protein
 
Protein content was estimated by the method given by Lowry et al., (1951) Five hundred mg of plant materials were weighed and macerated in a pestle and mortar with 10 ml of 20 per cent trichloroacetic acid. The homogenate was centrifuged for 15 min at 6000 rpm. The supernatant was discarded. To the pellet, 5 ml of 0.1 N NaOH was added and centrifuged for 5 min. The supernatant was saved and made up to 10 ml with 0.1 N NaOH. This extract was used for the estimation of the protein.
 
Estimation of antioxidant enzymes
 
Leaf samples (250 mg) were ground to a fine powder using liquid nitrogen. Ground powder was homogenized in 1.5 ml of ice-cold extraction buffer containing phosphate buffer (100 mM, pH 7.0), 1% PVP and 1mM EDTA before centrifugation at 1000 rpm and 40°C for 15 min. The supernatant was separated and stored at 40°C till the assay of enzymatic activities using spectrophotometer.
 
Catalase (CAT) activity (EC 1.11.1.6)
 
CAT activity was determined by the method of Luck (1965) with some modifications. For assay 100 μl of diluted enzyme extracted from leaves was taken and added with 800 ìl of 50 mM potassium phosphate buffer (pH 7.0). Reaction was initiated by adding 100 μl of 100 mM hydrogen peroxide. The change in absorbance was recorded at 240 nm at an interval of 15 sec for 2 min. The enzyme activity was expressed as unit mg^-1 protein min^-1. One unit can be defined as 0.1 decrease in absorbance.
 
Ascorbate peroxidase (APX) activity (EC 1.11.1.11)
 
APX activity was measured by the method of Nakano and Asada (1981). To prepare reaction mixture for determining APX activity, diluted enzyme extract (20 μl) was added in 50 mM potassium phosphate buffer (880 μl) containing 0.5 mM ascorbate. The reaction was started by the addition of 1 mM H₂O₂ (100 μl). Diminishing absorbance was recorded at 290 nm at 15 sec intervals for 2 min.
 
Superoxide dismutase (SOD) activity
 
Twenty-five mg leaf sample was taken and crushed in liquid nitrogen. Two hundred and fifty microliters of 0.1% trichloro acetic acid was added in an Eppendorf tube. Then it was vortexed for 10 min and centrifuged for 20 minutes at 10000 rpm followed by taking 160 microliters supernatant in an Eppendorf tube and 160 microliters of phosphate buffer was added. Then 680 microliters of 1 M potassium iodide were added. The reaction mixture was kept in dark for 1 hour and then absorption was taken at 390 nm.
 
Peroxidase (POX)
 
POX activity was measured by estimating the oxidation of guaiacol as proposed by Putter et al., (1974). To prepare reaction mixture for determining POX activity diluted enzyme extract (100 µl) were added in 50 mM potassium phosphate buffer (700 µl). Reaction was initiated by adding 100 µl of 100 mM H₂O₂. The change in absorbance was recorded at 15s interval for 2 min at 470 nm.
 
Statistical analysis, hierarchical cluster and expression analysis
 
Statistical analysis of antioxidant enzymes was performed and the coefficient of correlation among the traits was calculated using software SPSS ver 19.0. Heat map and dendrogram analysis was prepared using “heatmap” function of ‘R’ package (Zhao et al., 2014).

The chickpea has narrow genetic variation which extremely affects chickpea enhancement (Sachdeva et al., 2022). Low moisture stress influences the early vegetative growth stages of chickpea plant which ultimately affects yield. Therefore, biochemical and anti-enzymatic activities performances of selected chickpea genotypes were considered for identification of putative drought linked-selection indices with improved drought tolerance under drought stress.
 
Analysis of biochemical parameters for drought in chickpea
 
Total sugar content ranged between19.6 mg/g (RVSSG) to 32.5 mg/g (RVSSG36) under control condition and 30.3 mg/g (RVSSK86) to 38.4 mg/g (JG16) under stressed condition. While proline content arrayed between 1.3 µg/g (RVSSG44) to 2.2 µg/g (JG16) under control and 2.6 µg/g (SAGL162380) to 6.4 µg/g (SAGL190008) under stressed circumstances. Present findings agree with earlier findings that drought stress causes a substantial increase in accumulation of osmolytes i.e., sugar and proline activities (Tiwari et al., 2023a).
       
MDA was evidenced 1.6 nmol/g (for 6 genotypes including JAKI 9218) to 1.9 nmol/g (for 23 genotypes including JG74) under control and 2.2 nmol/g (RVSSG 44) to 3.9 nmol/g (SAGL171017) under stressed conditions. Crops have become more prone to oxidative damage due to unpredicted climate changes by excessive production of toxic ROS such as H₂O₂, superoxide and hydroxyl radicals. The previous investigations on chickpea emphasized that under low moisture stress condition, oxidant status of sensitive chickpea genotypes increased owing to increased oxidative stress (Kaloki et al., 2019; Jameel et al., 2021). In order to assess the severity of plasma membrane damage and the capacity of plants to withstand drought stress, malondialdehyde (MDA), a chemical formed by membrane lipids in response to reactive oxygen species (ROS), may be utilized as a drought indicator (Zhang et al., 2021). There was a positive association between MDA content and lipid peroxidation and it can deteriorate the integrity of cell wall (Rani et al., 2020). The outcomes of present investigation are in accordance with an earlier report, in which tolerant chickpea genotypes accumulated low MDA concentration compared to sensitive genotypes (Chaudhary et al., 2020).
       
Total protein content varied between 20.4 mg/g (SAGL19005) to 24.4 mg/g (RVSSG78) under control and 17.2 mg/g (SAGL152348) to 20.4 mg/g (SAGL152218) under stressed situations. Remarkable reduction was evident in protein content under water stress conditions in the current study. It was stated earlier that low water status in plant caused a noteworthy reduction in protein production, which might be due to involvement of diverse factors. Protein molecules play a crucial role in appropriate functions of cell. Since proteins directly influence the development of novel phenotypes by altering physiological features in response to environmental changes, their role is essential in the stress responses of plants. This reduction in protein content ultimately leads to reduce in plant growth and crop yield in sensitive chickpea genotypes.
       
Chlorophyll ranged between 1.84 µg/ml (BGD112) to 2.11 µg/ml (SAGL162380) under controlled condition whilst 1.03 µg/ml (BGD112) to 2.04 µg/ml (SAGL162380) under stressed conditions. Photosynthesis plays a vital role in deciding growth and development in plants. Pioneer investigations exhibited that abiotic stress caused a decline in photosynthesis rate which can be computed by assessing photosynthetic pigments. Under stress circumstances, chlorophyll a and chlorophyll b were lesser reduced in tolerant genotypes. Identical findings were also addressed in an earlier investigation in which chickpea heat tolerant genotypes depicted high chlorophyll than sensitive genotypes (Kaloki et al., 2019). Our findings are also in agreement with the results of Keerthi et al., (2023) where chlorophyll content decreased in the chickpea cultivars under water stress conditions. The contents of photosynthetic pigments are directly related to water stress tolerance. Reduction in chlorophyll contents may be because of disturbance in biosynthesis or their breakdown under water stress. Correlation coefficient among different biochemical parameters exhibited significant corelation at 0.01 and 0.05 probability level. Total sugar under control condition was positively correlated with total sugar under stress circumstances (r=0.446) at 1% significant level. Similarly, positively significant correlation was also evident among proline content under control with proline under stressed conditions (r=0.441), MDA under stress condition with proline control (r=0.365) and MDA control (r=0.336) at 1% level of significance (Table 1).
 

 
Analysis of antioxidant enzymes activities
 
POX ranged from 7.8 U/mg (RVSSG36) to 12.8 U/mg (SAGL152405) under controlled condition and 10.2 U/mg to 25.1 U/mg (RVSSG78) under stress condition. Whereas APX arrayed between 1.4 U/mg (SAGL152348) to 3.8 U/mg (SAGL154805) under control and 2.9 U/mg (SAGL162380) to 6.9 U/mg (SAGL 152216) under stress circumstances. CAT varied between 12.2 U/mg (SAGL162380) to 42 U/mg (JG14) under control and 22.3 U/mg (SAGL162380) to 69.2 U/mg (RVG201) under stress situations. Whilst SOD ranged between 31.6 U/mg (RVSSG68) to 100 U/mg (JG14) under controlled and 87.6 U/mg (RVSSG44) 194.4 U/mg (SAGL190028) under stressed conditions respectively. In present investigation, the influence of drought stress was exhibited least in drought tolerant genotypes compared to rest of the genotypes which might be because of accumulation of osmolytes and increased activities of antioxidant enzymes. Chickpea stress tolerant genotypes accumulate more osmolytes for instance, proline, glycine betaine and reduced level of glutathione. To detoxify the effects of ROS generated under abiotic stresses, specialized enzymatic antioxidants, i.e., SOD, CAT, POD and APX get triggered and act as first line of defence (Tang et al., 2019). Increased activity of antioxidants enzymes was evident in tolerant genotypes under drought stressed circumstances compared to normal conditions. It has been earlier investigated that drought tolerance in chickpea is strongly correlated with increased antioxidant enzyme activities (Tiwari et al., 2023a). A remarkable enhancement in POD was investigated under combined (drought plus heat) stress in D-09027 and CH24/07 in comparison to control by (Jameel et al., 2021).
       
Correlation coefficient analysis among different antioxidant enzymes of chickpea genotypes under controlled and drought stresses conditions was also analysed. POX under control was significantly correlated with POX under drought stress (r=0.428) at 1% level of significance (Table 2). Likewise, significant positive correlation was also evident among APX under stress with POX under stress (r=0.373) and APX under control (r=0.445), CAT under stress with POX under stress (r=0.511), APX under control (r=0.302), APX under stress (r=0.344) and CAT under control (0.687) at 1% probability level. Positively significant correlation was also found between SOD under stress with SOD under control (r=0.665), POX under stress (0.449), APX under stress (0.423), CAT under control (0.471) and CAT under stress conditions (0.374) at 1% significant level (Table 2).
 

 
Phylogenetic cluster analysis and expression profiling
 
Based on different antioxidant enzymes activities, two major groups were formed where the first group is further divided into one cluster and one subgroup. Genotype JG315 alone represented the cluster-1 and the subgroup included 40 genotypes which further divided into two clusters where cluster-2 includes 25 genotypes and cluster-3, 15 genotypes. Another major group consists of 42 genotypes which further divide into two subgroups. Genotypes viz., RVG 205, RVSSG30, SAGL15221 and JSC37 are considered into subgroup forming cluster- 4 and other 38 genotypes were comprinted into a subgroup further grouped into two clusters namely cluster-5 including 17 genotypes and cluster-6 having 21 genotypes (Fig 1). Diversity assessment based on heatmaps for representing expression levels of different antioxidant enzymes showing variations among studied genotypes (-2 to 2). The increasing intensity of color from pink to black and then to green represents increasing values accordingly (Fig 1). Similar kind of studies were also performed by Sharma et al., (2021) and Tomar et al., (2022) as they have also estimated different biochemical parameters and represented heat map for showing the level of expression. Current research work found significant difference in different biochemical parameters and anti-enzymatic activities under imposed drought conditions in chickpea.  
 

Chickpea is one of the most important legume crops economically and a prime source of proteins. Among several abiotic stresses drought is most devasting problem due to changing scenario of environment. Estimation of biochemical parameters and antioxidant enzyme activity is one of the important aspects to study drought effect. Total sugar, proline and malondialdehyde, has been increased under drought conditions whilst total chlorophyll and protein decreases under drought stress conditions. Likewise, antioxidant enzymes viz., POX, APX, CAT and SOD level increased under drought stress conditions. Highest sugar was found in chickpea genotype JG16, proline in SAGL190008, MDA in SAGL 171017 and highest protein was recorded in genotype RVSSG 78. Whereas, the highest POX enzyme activity was investigated in genotype RVSSG78, APX activity in genotype SAGL152216, CAT in RVG201 and highest SOD was recorded in genotype SAGL190028. Biochemical parameters and antioxidant enzymes play very crucial role to observe effect of drought stress in chickpea genotypes. Such kind of studies are important to select superior chickpea genotype(s) for further breeding programme.