A Physiological Approach to Study the Interaction of Cadmium and Zinc in Groundnut (Arachis hypogaea L.) Seedlings

Groundnut (Arachis hypogaea L.) accounts for approximately 50% of the total oilseed production in India (Mulgir et al., 2014). The 70% of area and 75% of groundnut production has been concentrated in Gujrat, Andhra Pradesh, Tamil Nadu and Orissa (Madhusudhana, 2013). Bianucci et al., (2012) reported considerable metal tolerance in groundnut. Cadmium is a group IIB transition element and also a non-essential heavy metal. Globally, cadmium content in soil is about 0.01-2 mg Kg^-1, with an average of 0.35 mg Kg^-1 of soil (Kebata-Pendias and Pendias, 2001). Cadmium toxicity may interfere in many processes, such as carbohydrate metabolism (Sanita di Toppi and Gabrielli, 1999), nitrate absorption and reduction (Hernandez et al., 1996), the uptake and distribution of macro and micro-nutrients (Van Assche and Clijsters, 1990) and photosynthesis (Pietrini et al., 2003). Zinc is an essential micronutrient and a member of group IIB transition element. It is an important component of many vital enzymes like Cu/Zn-SOD and structural stabilizer for many proteins, membrane and DNA-binding proteins (Vallee and Falchuk, 1993). However, like any other element, Zn at high concentration is toxic to the plant and inhibits several metabolic processes. Wallace (1982) noted that more than one metal in metalliferous environments in potentially toxic concentrations may have synergistic, additive or antagonistic effects on plants. Cadmium and zinc have similar valance state and electronic configuration, cadmium is non-essential, while zinc is an essential heavy metal for plant growth (Narwal et al., 1993). Sometimes this two metals are showing antagonistic interactions (Balen et al., 2011), while in some other cases synergistic interactions have been noted (Nan et al., 2002). A number of research works have been conducted in groundnut on the effect of cadmium stress (Gao et al., 2012; Nagaraju et al., 2015) and the effect of application of zinc (Saha et al., 2015; Rahevar et al., 2015; Nadaf and Chidanandappa, 2015). But the information onthe interaction of cadmium and zinc in groundnut is still lacking. In the present study, the effect of Cd-Zn interaction on growth and different physiological and biochemical parameters in groundnut seedlings was investigated.

Plant culture
 
The experiment was conducted with TG-51 genotype of groundnut (Arachis hypogaea L.) and seeds were collected from AICRP on groundnut, Kalyani in the year 2017. The entire experiment was done in research laboratory of Department of Plant Physiology, Bidhan Chandra Krishi Viswavidyalaya. Cadmium was supplemented in the form of CdCl₂.H₂O, while zinc was applied in the form of ZnCl₂. On the basis of results obtained from a preliminary study on growth of embryonic axis and cotyledon biomass using different concentrations of cadmium and zinc, three concentrations of cadmium (Cd 100, Cd 300 and Cd 500 μM) and two concentrations of zinc (Zn 50 and Zn 150 μM) were chosen for further studies. All the possible combinations of cadmium and zinc concentrations (total six) were used to study their interaction effect on groundnut.
 
The seeds were surface sterilized with 0.1% HgCl₂ (w/v) solution for two minutes then washed with distilled water. In each petri dish twenty seeds were arranged on Whatman No.1 filter paper, moistened with solutions of different cadmium and zinc concentrations and allowed to germinate at 28±1^oC temperature and around 80±1% relative humidity. After 72 hours of germination, the observations of fresh and dry weight of cotyledon and embryonic axis were recorded.
 
The selected concentrations and their every possible combinations were used for further study. After 72 hours, the germinated seeds were transferred to plastic beakers containing neutral sand. Five seedlings were raised in each beaker. Full strength of Hoagland solutions were prepared as per Epstein (1972) containing the metals and their combinations and were applied as the nutrient supplement after transplanting. The nutrient solutions were repeated on every 4^th day. A control set was also grown without containing any of the two metals in the Hoagland solution. The seedlings were raised under laboratory condition of diffused light. All the growth and physiological parameters were recorded on 21-day old seedlings.
 
Estimation of biochemical and physiological parameters
 
Chlorophyll a and b content in the leaf sample was estimated as per the method of Arnon (1949). These following formulae were used to calculate the chlorophyll content.


Where,
V = Volume of the extract (mL).
W = Fresh weight of tissue (g). 
A = Absorbance.

Relative leaf water content (RLWC) was estimated as per Perez et al., (2002). It was expressed as follows- Where,
FW= Fresh weight of tissue (g),
TW= Turgid weight of tissue (g),
DW= Dry weight of tissue (g).

The soluble protein content in the leaf of the seedlings was estimated following the method of Lowry et al., (1951). BSA standard curve was used to calculate the protein content.

Extraction of nitrate reductase and the estimation of enzyme activities were done as per the method of Jaworski (1971).

Total phenol content in the leaves of seedlings was estimated following the method of McDonald et al., (2001). Total phenol content was expressed in terms of gallic acid equivalent.
 
Statistical design and analysis
 
The experiment was conducted in completely randomized design (CRD) with three replications. All the mean data were statistically analyzed by INDOSTAT version 7.1 software.

The data were recorded (Table 1, Plate I) on fresh and dry weight of cotyledon and embryonic axis of seeds after 72 hours of germination and analysis of variance indicated highly significant (P £ 0.05) variation among all the treatments in respect of all the parameters studied. Perusal of means revealed that the fresh weight of both cotyledon and embryonic axis decreased concurrently along with the increase in concentration of the metals in germinating nutrient medium. The range of decrease in case of fresh weight of cotyledon varied from 5.41% at 100 µM to 61.63% at 1000 µM Cd and from 8.88% at 50 µM to 35.78% at 300 µM Zn over control. The corresponding ranges for fresh weight reduction of embryonic axis were from 3.81% at 100 µM to 74.03% at 1000 µM Cd and from 7.37% at 50 µM to 76.70% at 300 µM Zn over control. The dry weight of cotyledon and embryonic axis also followed the same pattern. The ranges of dry weight reduction varied from 3.48% at 100 µM of cadmium to 47.26% at 1000 µM of cadmium, while the corresponding values for zinc varied from 0.71% at 50 µM of zinc to 47.26% at 300 µM. Dry weight of embryonic axis also reduced under the metal treatments having the range from 7.12% at 100 µM of cadmium to 47.46% at 1000 µM and from 3.05% at 50 µM of zinc to 60.68% at 300 µM of zinc. Such inhibitory effects of cadmium and zinc on growth of embryonic axis might be a consequence of disarray in mitotic cycle as well as chromosomal aberration caused by heavy metals resulting in disturbance of cell division (Rajput et al., 2019).  However, from the overall data on growth of embryonic axis as well as fresh and dry weight of cotyledons under different concentrations of cadmium and zinc, three concentrations of cadmium (viz, 100, 300 and 500 µM) and two concentrations of zinc (viz, 50 and 150 µM) and their combinations were finally selected for cadmium-zinc interaction studies in the present experiment because these concentrations of two metals were found to be not too severe nor too mild for embryonic growth of groundnut.





Perusal of data indicated that all the treatments caused significant (P £ 0.05) reduction in root length of groundnut seedling. The percent reduction in root length increased from 24.34% to 35.10% over control as the concentration of cadmium in the medium increased from 100 to 500 µM (Table 2). The two concentrations of zinc used in the experiment also produced negative effects on root length, the severity been increased under higher concentration. The adverse effects of both these metals on root length might be attributed to reduction in mitotic index affecting cell division and elongation as was also noted earlier by Borboa and Delatorre (1996). Zinc supplements reduced the severity of cadmium stress on root length to some extent in all treatment combinations, although the inhibitory effect could not be completely rectified.



Perusal of data revealed that treatment with cadmium or zinc alone led to a decrease in chlorophyll a and chlorophyll b content of the leaf and the effect became more drastic along with an increase in the concentration of metal in medium (Table 3, Plate II). The result corroborated the early finding of Sivakumar et al., (2001). However, the reduced biosynthesis and enhanced chlorophyll degradation along with disorganized chloroplasts (Rascio et al., 2008) might be attributed for the observed inhibitory effects of cadmium on leaf chlorophyll, whereas possible obstruction of Fe transport to chloroplast under excess Zn might be the underlying factor for decreased pigment content (Symeonidis and Karataglis, 1992). However, the Zn supplementation (150 µM) of cadmium-treated seedlings not only mitigated the negative effect of Cd 100 µM and 300 µM, but also increased the chlorophyll content above control level. The result was consistent with the finding of Aravind and Prasad (2005).




 
The data on relative leaf water content (RLWC) indicated non-significant differences among the treatments. In all the cases cadmium and zinc alone led to a reduction in RLWC and the highest reduction was noted down in case of Cd 500 µM treatment (Table 4). Supplementation of highest concentration of zinc was able to reduce the Cd-induced negative effects to some extent.


 
The leaf protein content of the seedlings significantly (P £ 0.05) decreased under cadmium treatment with the effect being more adverse along with an increase in cadmium concentration (Table 4). The observed decrease in protein content under cadmium stress might be a consequence of reduction in protein synthesis (Blaestrasse et al., 2003). In contrast, the protein content increased in the Zn-treated seedlings and the corresponding values were 26.09 and 1.31% over control at 50 and 150 µM, respectively. The observation was consistent with the earlier suggestions regarding the essentiality of zinc in many metabolic processes including its role as protein stabilizer (Vallee and Falchuk, 1993). However, the combination of Zn 150 µM with different concentrations of cadmium somewhat lowered the adverse effect of cadmium stress alone, thus, indicating antagonistic effects of this metal combination.

The activity of nitrate reductase (NR) in the leaves of groundnut seedlings decreased significantly (P £ 0.05) under all concentrations of cadmium and zinc as well as their interactions (Table 4). In case of cadmium, the percent inhibition of the enzyme activity over unstressed control was found to be 53.70, 56.83 and 61.02% for Cd 100, 300 and 500 µM, respectively, thus, indicating increases in toxic effect of the metal concomitant with an increase in concentration in the medium. However, there was a little difference between two concentrations of zinc used in the experiment in respect of negative effect on the enzymatic activity. The different treatment combinations of cadmium and zinc could not remarkably change the level of metal toxicity except Cd 100 µM + Zn 150 µM which significantly lowered the negative effect of sole treatment of Cd 100 µM on NR.

The mean data on leaf phenol content revealed highly significant (P £ 0.05) variations among the treatments. Treatment with Cd 100 and 300 µM induced 9.11 and 4.52% increase in phenol content (Table 4), respectively, over control. This observation was corroborative of the early observation by Garcia et al., (2012) and might be interpreted as a consequence of Cd-induced accumulation of cinnamic acid derivatives (Kovacik et al., 2009). The leaf phenol content also increased under zinc treatment at 50 and 150 µM and the corresponding values were 6.81 and 17.97% over control, respectively. Moreover, the supplements of zinc with cadmium also induced positive effect in respect of phenol accumulation in leaf but the level varied under different combinations. The influence of zinc on phenol accumulation in leaf was reported earlier by Chamani et al., (2016). Plant phenolic compounds are potent non-enzymatic antioxidant and thus, increase in their contents in the leaf might lead to scavenging of reactive oxygen species (ROS) that are responsible to create oxidative stress.

The authors acknowledge the assistance extended by AICRP on groundnut, Kalyani centre for supplying plant materials and Department of Plant Physiology, Faculty of Agriculture, BCKV for providing with all the facilities and supports.