Bio-Physico-Chemical Response of Drought Tolerant Chickpeas to Nickel

Heavy metal pollution is increasing in the environment due to mining, industrialization and other anthropogenic activities. Airborne heavy metals fall upon, react with and are absorbed by plants and soils near the sites of pollutants generation. Heavy metals pose a significant threat to food safety due to their mobility in the soil-plant system (Bell et al., 2007). Human-induced interruption of natural bio-geochemical cycles, emphasized accretion of heavy metals is a problem of vital significance for ecological, nutritional and environmental reasons (Nagajyot et al., 2010; Ali, et al., 2013; Uliana Ya. et al., 2018).  Metal pollutants viz; cadmium, chromium, copper, mercury, nickel, zinc and lead are considered to be potential threats to plant and soil. Among heavy metal pollutants nickel and cadmium need special attention due to their widespread occurrence and potential for toxicities. The concentration level representing nickel toxicity in plants varies greatly from 25 to 246 μgg^-1 dry weight (DW) of plant tissue, depending on the plant species and cultivars (Iyaka et al., 2011). Excess nickel negatively affects germination process and seedling growth traits of plants by hampering the activity of the enzymes such as amylase and protease as well as disrupting the hydrolyzation of food storage in germinating seeds (Yadav et al., 2007; Aydinalp et al., 2009; Sethy et al., 2013). Plant growth parameters are also affected by Ni toxicity. Khan et al., (2010) investigating the toxic effect of nickel and cobalt on chickpea (Cicer arietinum L.) showed that toxicity of Ni on the biomass production was more pronounced than Co and both metals led to poor nodulation, resulting in the reduced yield. Antioxidant enzymes activity increases in plant cells as a response to environmental stresses. Environmental stresses can result in the production of Reactive Oxygen Species (ROS) a complex antioxidant system. The primary components of this system include carotenoids, ascorbate, glutathione and tocopherols, in addition to enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), peroxidases and the enzymes involved in ascorbate-glutathion cycle such as ascorbate peroxidase (APX) and glutathione reductase (Baby et al., 2011). Another study indicates that GS/GOGAT cycle rather than GDH was responsible for ammonia assimilation in the nodules of chickpea cultivars and salt stress interferes with the activity of these enzymes (Garg and Singla, 2006). The objective of this research was aimed to determine the uptake and distribution of nickel in two different varieties representing two genetically diverse groups of chickpeas namely PUSA 1103 a drought tolerant Desi variety vs PUSA 1105 a drought tolerant Kabuli variety and effect of the nickel on various growth influencing physiological and biochemical factors viz; chlorophyll pigmentations and three enzymatic activities namely nitrate reductase (NR), peroxidase (POX) and super-oxide dismutase (SOD). Nitrate reductase activity provides a good estimate of the nitrogen status of plants and is correlated with growth and plant yield (Srivastava, 1980). Nitrate reductase mediates the reduction of nitrate to nitrite, which is regarded as a rate limiting step in plant growth and development (Solmonson and Barber, 1990). Superoxide Dismutase (SOD) catalyzes the dismutation of superoxide radical to hydrogen peroxide an important antioxidant enzyme. Peroxidase is an iron (heme) protein referred as non-specific peroxidase or guniacol-peroxidase and mostly located in the cell wall region of the cell and catalyzes the reduction of hydrogen peroxide with a concurrent oxidatior of a substrate in addition to the oxidation of phenolic compounds onwards the synthesis of lignin.
       
The information obtained from this study should be useful for studying genetic behaviour of Cicer arietinum that are contaminated with nickel and other heavy metals.

The experiment was conceptualized, standardized and conducted at Sardar Vallabhbhai Patel University of Agriculture and Technology, Modipuram, Meerut during the period 2010-14. The air dried soil was treated with chloride salts of nickel of different doses viz; 0, 0.62, 3.12, 12.5, 62.5 and 125 µg g^-1 in 35 x 45 cm earthen pots containing 8 kg of soil. Pots without any addition of metal constituted the control. Each treatment was replicated thrice in a completely ran domized block design. Efforts were made to ensure visual uniformity across the plants. Ten seeds were sown equidistantly at 2-3 cm depth in each pot and irrigated after 3-4 days during rabi season 2012-13. After emergence, seedlings were thinned to 5 plants in each pot. Pots were hand weeded time to time to keep plants free from it. The various morphological parameters studied were top length (cm), root length (cm), leaf area (cm²), nodule number, nodule weight (g), biomass of the plants (g), number of internodes, number of primary branches, number of secondary branches, number of pods/plants, number of seeds /plant, weight of seeds (g), test weight of seed (g), dry weight of plants (g). Biochemical parameters as chlorophyll a, chlorophyll b and protein contents in leaves and seeds, enzymatic activities like nitrate reductase (NR), peroxidase (POX) and super-oxide dismutase (SOD) in leaves along with accumulation of nickel in different parts of the plants and soil residues were analyzed during the year 2013-14.
 
Chlorophyll content was estimated following the method of Vernon (1960), Nitrate reductase activity as per Nichloas and Nason (1957), peroxidase activity as per Nakano and Asada (1987). The activity of super-oxide dismutase was studied by following the method of Alhinolsa et al., (1981) and seed proteins as per Bradford (1976). Plant and soil samples were digested with nitric acid and perchloric acid in Hot Block Digestion system and the nickel contents were determined with the help of Atomic Absorption System (Thermo Jarrell Ash Corporation, Franklin, MA, USA). Statistical Analysis was based on analysis of variance, SEM and C.D. 1% and 5%.

Morphological and biochemical traits
 
Top length, root length and biomass of the plants in both the varieties increased significantly in response to applications of 0.62, 3.12 and 12.5 µg g^-1 nickel dose levels. Higher application of nickel resulted in a reduction of more than 20 % in top length, root length and biomass over the controls (Fig 1). In PUSA 1103 (Fig 1a) and PUSA 1105 (Fig 1b),  top length showed an increase of 95.72 % and 8.775 over the controls at nickel dose of 12.5 µg g-¹ and 3.12 µg g^-1 respectively. Application of higher nickel concentrations showed reduction in the plant growth and resulted in delayed flowering in both the varieties. PUSA 1103 was found to be more tolerant in comparison to PUSA 1105. Nickel doses at 12.5 µg g^-1 in PUSA 1103 and 3.12 µg g^-1 in PUSA 1105 significantly increased both nodule number and weight. Number and size of nodulation was more pronounced in PUSA 1105 than PUSA 1103. The number of primary branches was unaffected by metal stress but number of secondary branches was higher at 3.12 µg g^-1 with an increase of 11.75% in PUSA 1103 and 25.02% in PUSA 1105 at 0.62 µg g^-1 nickel levels, over the controls, respectively. It was observed that doses higher than this reduced the growth though magnitude of reduction varied in both the varieties. It is also supported by the findings that nickel at lower concentrations around 10 ppm enhances the root and shoot length in alfalfa by 37% and 36%, respectively (Peralta et al., 2000). Addition of nickel above 12.5 µg g^-1 level reduced the plant growth, biomass and yield of the plants and similar findings have also been observed by Krupa et al., (1993). They concluded that nickel reduces the plant growth and disrupts the metabolic as well as physiological processes like photosynthesis. Rana and Masood (2002) reported that chickpea rhizobium system is more sensitive to heavy metals toxicity.
 

 





 
Yield
 
Maximum seed number was observed at 12.5 µg g^-1 nickel level in Desi variety PUSA 1103 with an increase of 44.8% over the control, whereas maximum seeds output in Kabuli variety PUSA 1105 was recorded at 0.62 µg g^-1 nickel dose with an increase of 73.49%  over the control. Higher nickel doses resulted in a decreased seed yield and in some of the plants seeds were not formed (Fig 2).
 

 
Protein
 
Protein in seeds of the stressed plants showed different results in both the varieties. In case of PUSA 1103 protein content decreased in comparison to the control plants, but in case of PUSA 1105 seed protein enhancement was observed up to 5% at nickel dose of 3.12 µg g^-1 (Table 2). Heavy metals are said to prevent chlorophyll synthesis either by direct inhibition of an enzymatic step or by induction of a major nutrient. Heavy metals cause a decrease of chlorophyll and protein content in plants (Fusum and Porgali, 2006). Metals play a prominent role in the synthesis of protein, nucleic acid and photosynthesis pigments (Oves et al., 2016).
 

 
Chlorophyll ‘a’ and ‘b’ contents
 
Doses of nickel at 0.62 µg g^-1 and 12.5 µg g^-1 resulted in an increase in chlorophyll ‘a’ and ‘b’ contents, respectively in the drought tolerant variety PUSA 1103. In case of Kabuli variety PUSA 1105 maximum increase of  26.61 % and 113.4 % over the controls were observed for chlorophyll ‘a’ and ‘b’ at 3.12 µg g^-1 and 62.5 µg g^-1 nickel doses, respectively (Table 2). Carotenoids protect chlorophyll from photo oxidative destruction (Middleton and Teramura, 1993) and therefore, a reduction in carotenoids could have serious consequences on chlorophyll pigments. Metal stress has been reported to affect photosynthesis, Chlorophyll, fluorescence and stomatal resistance (Medelssahn et al., 2001; Monni et al., 2001). Reduction in chlorophyll contents by excess nickel in the pigeon pea and spinach were also reported by Dube et al., (2002). The effect of metals are connected with inhibition of certain metabolic processes including biosynthesis of chlorophyll and protein (J,Ma et al., 2016; Xue et al., 2014).
       
Heavy metals are reported to prevent chlorophyll synthesis either by direct inhibition of an enzymatic step or by reduced supply of a major nutrient. Heavy metal causes a decrease of chlorophyll and protein contents in plants (Yurekle and Pogali, 2006). In addition, metal induced nutrient imbalances in the plants may indirectly affect photosynthesis and growth. Metal inhibits iron uptake in plants and subsequently reduces the chlorophyll content of he leaves (Patsikka et al., 2002). Our results were in agreement with previous reports regarding the effect of heavy metals on the growth performance of plants.
 
Enzyme activities
 
In both the varieties highest NR activity was recorded at 0.62 µg g^-1 Ni dose with an increase of 73.50% and 7.58% over the controls in PUSA 1103 and PUSA 1105, respectively. Maximum peroxidase enzymatic activity was recorded at 125 µg g^-1 Nickle dose whereas, minimum activity was obtained at 0.62 µg g^-1 Nickel dose in both the varieties. Superoxide dismutase enzyme activity also showed similar trend as peroxidase. The enzymatic activity increased in the plants with increasing plant concentrations of Nickel (Fig 2). Heavy metals prevent chlorophyll synthesis either by direct inhibition of an enzymatic step or by reduced supply of a major nutrients. Heavy metal causes a decrease of chlorophyll and protein contents in plants (Yurekle and Pogali, 2006). In addition, metal induced nutrient imbalances in the plants may indirectly affect photosynthesis and growth. Metal inhibits iron uptake in plants and subsequently reduces the chlorophyll content of the leaves (Patsikka et al., 2002). Our results were in consonance with previous reports regarding the effect of heavy metals on the growth performance of plants. For example, nickel at 200 ppm adversely affected the pigmentation of leaf margins and significantly suppressed the plant growth and seed protein and nodulation in chickpea (Khan et al., 1996). Metal may be part of active sites of enzymes (cofactor) and participate directly in catalysis. Different heavy metals affect the enzyme in different ways, depending on their affinity for sulphydryl and carboxylic groups (Miller et al., 1993). Sulphydryl containing enzymes like NR are very sensitive to heavy metals, whereas peroxidase is relatively resistant. Antioxidant defense system comprise of a variety of antioxidant molecules and enzymes such as superoxide dismutase and peroxidase. The peroxidase take part in the lignifications, suberization and cross linking of cell wall polymers (Passardi et al., 2005). Lignin serves as a defense material, providing both a physical barrier and a chemical deterrent to foreign attack. The response of peroxidase to elevated levels of heavy metals varies depending on plant species and heavy metals (Gratao et al, 2005). Metals play significant task in plants and are reported for significant constitutions of various enzyme activities (Emamverdian et al., 2015).
 
Metal uptake
 
The addition of nickel to the soil resulted in metal accumulation. In both the varieties (PUSA 1103 and PUSA 1105) shoots of the plants had higher levels of metal than the roots. Maximum amount of nickel accumulated in shoots of  PUSA 1103 at 125 µg g^-1 nickels dose. The control plants of both the varieties accumulated very low amount of nickel in their tissues. As the concentrations of soil applied nickel was increased, an increase in the concentrations of nickel in root as well as shoot was observed. Metal accumulation was found to be higher in PUSA 1103 as compared to PUSA 1105. With the increase of nickel contents in soil, an increase in the content of nickel in root as well as top was observed (Fig 3).
 

       
Plant uptake of heavy metals is partly determined by the concentration and speciation of the metals in soil solution (Bingham et al., 1984, 1986). Organic matter makes strong complexes with heavy metals (Bloom 1981, Krogstad, 1983). Solid organic matter may retain metals in the solid phase of the soil, whereas dissolved organic matter may increase mobility of the metals (Lo et al., 1992). The availability or uptake by plant roots may differ for metals bound in soluble complexes and free metals. As organic materials influence the binding of heavy metals in soil and speciation in soil solution (Casstilho et al., 1993), it may also affect plant uptake (Mc Bridge et al., 1981). Soil pH is another factor influencing the bioavailability of metals both in terms of absorption in the soil and speciation in the soil solution. In conclusion, nickel excess may lead to unbalanced nutrients uptake and decreased upward translocation in plants. Drought tolerant chickpea variety (PUSA 1103) expressed resilience to the nickel dose for the grain yield as compared to PUSA 1105.

The increased concentrations of the soil applied nickel demonstrated an increase in the content of nickel in roots as well as shoots. The shoots accumulated higher nickel contents than the roots of the plants. The Desi chickpea variety PUSA 1103 accumulated higher content of nickel and also yielded higher grain yield. Thus, the Desi chickpea variety PUSA 1103 may be utilized in crop improvement strategies to breed new chickpea genotypes for nickel and drought resistance.

The authors express their sincere thanks to UP Council of Agricultural Research, UP Govt. and DST, Govt. of India for providing financial assistance.

The work described has neither been published before nor under consideration for publication anywhere else and its publication has been approved by all co-authors. The authors declare that they have no conflict of interest.