Ascorbic Acid and Calcium Silicate Improve Morpho-Physiological Characteristics of Cadmium Stressed Mung Bean Crop

A rise in heavy metal contamination especially in the rhizosphere affecting the growth and yield of crops is a major concern. We aimed to study the influence of using calcium silicate (CS) and ascorbic acid (AsA) supplements on lowering the impact of cadmium-induced toxicity in mung bean. Both the supplements alone or in combination improved growth characteristics of cadmium (Cd) stressed mung bean plants like root-shoot length and fresh-dry weight. Leaf pigments like chlorophyll and carotenoids were also restored. A significant improvement in the relative leaf water content (RLWC) and low electrolyte leakage (EL) at the membrane was recorded. Results were more promising when combinations of CS and AsA treatments were used against the lower concentration of cadmium. Hence, both CS and AsA interact synergistically to alleviate Cd induced metal toxicity in mung bean plants.


Introduction
About 53 elements among the natural components of the earth's crust fall under heavy metals. 1 Although cadmium (Cd) is a non-essential element with no biological significance, it is considered highly toxic being soluble and mobile in a plant-soil system. 2 A longer half-life and non-biodegradability allow Cd to accumulate and persist in the agricultural food chain. Anthropogenic activities are the major cause of Cd contamination. 3 Its chemical similarity with minerals like zinc (Zn), iron (Fe), calcium (Ca), and manganese (Mn), makes its absorption easier at the root system, altering morphological, biochemical, and physiological characteristics of a plant. Among many cascading effects of cadmium, necrosis, chlorosis, and low tolerance index leading to plant death. 4 Cadmium hampers the photosynthetic activity including chloroplast organization, pigments, membrane integrity, stomatal conductance, and water balance. 5 Cadmium is a redox-inactive metal that promotes the formation of ROS through indirect mechanisms inhibiting antioxidative enzymes or stimulating NADPH oxidase, a ROS-producing enzyme. 6 The release of cytotoxic elements like superoxide (O2·ˉ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OHˉ) oxidize membrane lipids to buildup malondialdehyde (MDA) leading to protein denaturation with altered enzyme functions, osmolyte production, and C/N metabolism. 7 Ascorbic acid (AsA), a water-soluble organic compound, is an essential antioxidant in a plant system participating in metabolic activities. It is present in all plant tissues, usually more in photosynthetic cells, meristem, and fruits. 8 AsA, a non-enzymatic antioxidant acts as a redox buffer and ROS scavenger under stress conditions. Its application enhances photosynthesis, preserves chlorophyll contents, and maintains the integrity of cell membranes during stress. 10 Calcium has been reported as an essential plant macronutrient for the growth, cell wall stabilization, cell membrane integrity, ion transportation, photosynthesis, water relations, and enzyme activation. 9 Calcium silicate (CS) used in agriculture for liming also regulates physical, chemical, and biological properties of the soil, and has proved beneficial for plant growth by alleviating stress. 11 CS restricts the availability of Cd to a plant hence promoting the growth profile of the paddy crop. 12 Generation of ROS during stress involves an exchange of signal between ROS, Ca 2+ , and Ca 2+ -binding proteins, such as calmodulin and silicon. 13 Si, the 2 nd most abundant element on the earth is also considered essential for plant growth and development. 14 Silicon improves water relations of the crop with enhanced photosynthetic activity during stress. We hypothesized that AsA and CS would play a key role in crop improvement, therefore, investigated various growth parameters focusing on the underlying mechanisms of Cd stressed plants.

Material and Methods
Seeds of mung bean (Vigna radiata (L.) R. Wilczek var. MH-421) were procured from CCS Haryana Agricultural University, Hisar, Haryana. Healthy seeds were surface sterilized with 0.01% mercuric chloride followed by thorough washing with distilled water. Seeds were inoculated with the appropriate strain of Rhizobium sp. by soaking overnight in a thick slurry of Rhizobium culture mixed with activated charcoal and acacia gum. Trials were carried out in dome-shaped out-houses in the month of March with an average temperature of 30.0 ± 2°C and 34% humidity. Seedlings were raised in perforated polythene-lined earthen pots filled with approximately 5 kg of washed river sand. Calcium silicate (0.6 mM) was added to the soil before the sowing of seeds. 15 After germination, the seedlings were irrigated with distilled water only for the first 14 days, followed by Cd (0.3 and 0.5 mM CdSO 4 .7H 2 O) and Ascorbic acid (0.8 mM) treatments along with the nutrient medium. Trial consisted of 9 sets as Control, Cd(0.3mM), Cd0.3+CS0.6mM, Cd0.3+AsA0.8mM, Cd0.3+CS0.6+AsA0.8mM, Cd(0.5mM), Cd0.5+CS0.6mM, Cd0.5+AsA0.8mM, Cd0.5+CS0.6+AsA0.8mM. Treatments were repeated for fortnightly thrice. Control plants were grown with nutrient medium only. Observations were made at the reproductive stage of the crop, 45 DAS using fresh leaves samples on the same day. 16,17 Standardized procedures were followed to measure parameters like chlorophyll and carotenoid content. Chlorophyll was extracted with 80% acetone repeatedly to ensure complete extraction and the absorbance was read at 480nm, 663nm, and 645nm against 80% acetone. Observations were made with Thermo-Scientific Evolution-201 UV-Visible Spectrophotometer. Electrolyte leakage (EL) was measured according to Lutts et al. 18 Relative leaf water contents (RLWC) were determined according to Chen et al. 19 Root-shoot length (cm) and fresh weight (g) were calculated at the sampling stage.

Statistical Analysis
All the values were in triplicate from a single sample and represented as mean ± SE (standard error). Data were statistically analyzed using randomized block design (RBD) one-way ANOVA in SPSS-16 by taking the probability level of 5%. A least significant difference (LSD) post-hoc test was used to compare the multiple comparisons of the mean.

Root and Shoot Length
The overall length of mung bean plants was reduced with Cd treatments. It was found that the percentage decline in the length of roots was 38.44% in Cd 0.3mM and 49.34% in Cd 0.5mM to that of control. The same reduction in root length in CS or AsA supplemented Cd treatments was comparatively lesser, 14.32% (Cd 0.3 +CS 0.6mM ); 32.16% (Cd 0.5 +CS 0.6mM ) and 12.44% (Cd 0.3 +AsA 0.8mM ); 27.75% (Cd 0.5 +AsA 0.8mM ). Their combination treatments with Cd minimized the losses to 5.84% (Cd 0.3 +CS 0.6 +AsA 0.8mM ) and 19.27% (Cd 0.5 +CS 0.6 +AsA 0.8 mM ) (Fig.5).

Discussion
Heavy metal exposure hampers the growth and productivity of a plant. The present investigation indicated that reduced leaf pigments like chlorophyll and carotenoids in Cd-stressed plants could be restored using supplements like CS and/or AsA. 20,21 An improved content of chlorophyll and carotenoids have been linked to the lowering of Cd-induced toxicity with supplements like CS and AsA. 22,23 Different findings correlated CS elevating pigment levels and 24 AsA enhancing chlorophyll and membrane stability index during stress. 25 As a redox buffer, AsA neutralizes the superoxide radicals and other singlet oxygen species, thus, preventing chlorophyll degradation and increasing its content. 26 The decline in photosynthetic pigments like chlorophyll and carotenoids is accompanied by enhanced leakage of ions in Cd-contaminated chickpea genotypes. As noticed in our present study, enhanced electrolyte leakage due to the action of heavy metal could be suppressed largely by using a combination treatment of CS and AsA in Cd-stressed mung bean. 27 Production and storage of ROS in heavy metal stress destroys membrane lipids distorting the structure of lipid-protein membrane which increase membrane permeability. 28 AsA help in combating stress by decreasing electrolyte leakage and maintaining membrane integrity. Negatively impacted RLWC recovered using CS and AsA supplements in Cd treatments. 29 Long term exposure to Cd causes water imbalance leading to decreased RLWC and transpiration. 30 Cd reduces turgor pressure, relative water content, and water potential of plant cells. 31 Excess Cd levels have been reported to alter the osmotic balance and water content. As reported by Kaya et al., 32 RLWC and chlorophyll levels lowered with Cd toxicity were accompanied by enhanced electrolyte leakage in Capsicum annuum. 33 Introduction of CS enhanced water content owing to deposition of silica in the cell wall that reduces transpiration rate to mitigate plant stress. 34 AsA have also been reported to improve water content in various plant species. 35 Higher Cd levels lead to a drop in fresh-dry weight, root-shoot length, and total pod-seeds in different legumes. The impact of heavy metal stress on the growth profile of mung bean plants like root-shoot length, and fresh and dry weight in the present study was based upon the strength of Cd treatment. 36 Lesser growth and yield of Cd stressed plants was related to the suppression of growth rate of cells because of irreversible inhibition of proton pump responsible for the process. 37 A direct interference of Cd with some hydrolytic enzymes was also suggested to play a pivotal role in restricting food supply to root and shoot. Further, both supplements used in combination proved useful in alleviating metal toxicity. 38 Addition of CS improved root-shoot length, biomass, and related yield attributes in Cdstressed Vigna radiata. 39 Silicates (Si) help in cell enlargement by enhancing the tissue extensibility. 40 AsA is also a co-factor for growth hormones like auxin which is essential for cell expansion and hydroxyproline-rich glycoproteins in cell division. Hence, it can be said that the interaction of CS and AsA with heavy metal Cd improved the morpho-physiological characteristics of mung bean by enhancing RLWC, restoring photosynthetic pigments, and suppressing electrolyte leakage to maintain the integrity of membranes.

Conclusion
CS and AsA improve morpho-physiological characteristics by restoring the content of chlorophyll and carotenoids, RLWC, and suppressing EL to mitigate Cd-induced heavy metal toxicity in mung bean.

Acknowledgment
The financial support of the University Grants Commission, New Delhi in conducting present investigations is gratefully acknowledged.

Funding
University Grants Commission, New Delhi.

Conflict of Interest
There is no conflict of interest.