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

Introduction

About 53 elements among the natural components of the earth’s crust fall under heavy metals. ¹Although 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. ²A 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. ³Its 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. ⁴Cadmium hampers the photosynthetic activity including chloroplast organization, pigments, membrane integrity, stomatal conductance, and water balance. ⁵Cadmium 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. ⁶The release of cytotoxic elements like superoxide (O₂·ˉ), hydrogen peroxide (H₂O₂), 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.

⁷Ascorbic 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. ⁸AsA, 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. ¹⁰Calcium 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. ⁹Calcium 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. ¹¹CS restricts the availability of Cd to a plant hence promoting the growth profile of the paddy crop. ¹²Generation of ROS during stress involves an exchange of signal between ROS, Ca²⁺, and Ca²⁺-binding proteins, such as calmodulin and silicon.¹³Si, the 2^nd most abundant element on the earth is also considered essential for plant growth and development. ¹⁴Silicon 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. ¹⁵After germination,the seedlings were irrigated with distilled water only for the first 14 days, followed by Cd (0.3 and 0.5 mM CdSO₄.7H₂O) and Ascorbic acid (0.8 mM) treatments along with the nutrient medium. Trial consisted of 9 sets as Control, Cd_0.3mM; Cd_0.3+CS_0.6mM; Cd_0.3+AsA_0.8mM; Cd_0.3+CS_0.6+AsA_0.8mM; Cd_0.5mM; Cd_0.5+CS_0.6mM; Cd_0.5+AsA_0.8mM; Cd_0.5+CS_0.6+AsA_0.8mM. Treatments were repeated for fortnightly thriceControl 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.

^16,17Standardized 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.

Observations and Results

Chlorophyll Content

Cadmium (0.3 and 0.5mM) treatments reduced leaf chlorophyll content up to 19.08% and 30.84%. The pigment loss was only 5.88% (Cd_0.3+CS_0.6mM); 19.08% (Cd_0.5+CS_0.6mM), 2.18% (Cd_0.5+AsA_0.8mM) in CS and AsA supplemented Cd treatments in comparison to control plants. The percentage value of the contents was more than that of control in combination treatments +7.80% (Cd_0.3+AsA_0.8mM); +48.64% (Cd_0.3+CS_0.6+AsA_0.8mM); and +20.18% (Cd_0.5+CS_0.6+AsA_{0.8 mM}). Observations revealed the effectiveness of the CS and AsA combination in neutralizing the deleterious effect of Cd (Fig.1).

Figure 1: Effect of Cd alone and in combination with CS and AsA on total chlorophyll in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=0.21). Click here to view Figure

Carotenoids

The contents of accessory pigment carotenoids were reduced up to 17.72% and 28.09% with Cd (0.3 and 0.5mM) application, respectively. Loss of accessory pigment noticed with CS or AsA supplements was only 7.94% (Cd_0.3+CS_0.6mM); 21.57% (Cd_0.5+CS_0.6mM), 2.27% (Cd_0.5+AsA_0.8mM) in comparison to control. The combination treatments were found to be promotive such as +9.48% (Cd_0.3+AsA_0.8mM); +52.66% (Cd_0.3+CS_0.6+AsA_0.8mM); and 15.96% (Cd_0.5+CS_0.6+AsA_0.8mM). Thus, the effectiveness of combination treatments of CS and AsA was more when used with low cadmium (0.3mM) concentrations (Fig. 2).

Figure 2: Effect of Cd alone and in combination with CS and AsA on carotenoids in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=0.12). Click here to view Figure

Relative leaf water content (RLWC)

RLWC of the leaves dropped with Cd treatments up to 9.31% (Cd_{0.3 mM}) and 11.74% (Cd_{0.6 mM}). Such losses in water content with the application of CS or AsA were only up to 6.07% (Cd_0.3+CS_{0.6 mM}); 8.09% (Cd_0.5+CS_0.6mM); and 4.86% (Cd_0.3+AsA_0.8mM); 7.29% (Cd_0.5+AsA_0.8mM). The results with combination treatments of CS and AsA with Cd were encouraging, minimizing losses of water content to 1.21% (Cd_0.3+CS_0.6+AsA_0.8mM) and 4.05% (Cd_0.5+CS_0.6+AsA_0.8mM). Thus, RLWC could nearly be restored to control levels using a combination of CS and AsA with low concentrations of Cd (Fig.3).

Figure 3: Effect of Cd alone and in combination with CS and AsA on RLWC in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=1.28). Click here to view Figure

Electrolyte leakage

Electrolyte leakage from membranes increased substantially with the application of Cd and was found to be up 19.02% (Cd_{0.3 mM}) and 27.60% (Cd_{0.5 mM}). Both supplement (CS and AsA) applications in cadmium treatment were able to check the rise in EL and it was up 15.80% (Cd_0.3+CS_{0.6 mM}), 21.27% (Cd_0.5+CS_{0.6 mM}); and 16.36% (Cd_0.3+AsA_{0.8 mM}), and 17.57% (Cd_0.5+AsA_0.8mM). This EL in combination treatments was minimum and recorded as up 4.35% (Cd_0.3+CS_0.6+AsA_0.8mM) and 15.18% (Cd_0.5+CS_0.6+AsA_0.8mM) to that of control (Fig.4)

Figure 4: Effect of Cd alone and in combination with CS and AsA on electrolyte leakage in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=6.19). Click here to view Figure

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).

The length of shoots also declined up to 40.56% (Cd_0.3mM) and 45.59% (Cd_0.5mM) in comparison to the control. The addition of CS or AsA to Cd lowered the drop in height to 36.77% (Cd_0.3 + CS_0.6mM); 39.71% (Cd_0.5+CS_0.6mM) and 28.15% (Cd_0.3+AsA_0.8mM); 36.97% (Cd_0.5+AsA_0.8mM). The combination of CS and AsA to Cd was able to minimize the drop in height to 4.64% in (Cd_0.3+CS_0.6+AsA_0.8mM) and 27.96% in (Cd_0.5+CS_0.6+AsA_{0.8 mM}) (Fig.6).

Figure 5: Effect of Cd alone and in combination with CS and AsA on root length in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=2.38). Click here to view Figure

Figure 6: Effect of Cd alone and in combination with CS and AsA on shoot length in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=2.49). Click here to view Figure

Fresh and Dry Weight of Plants

In Cd alone treatments (0.3 and 0.5 mM), the fresh weight of the plants declined up to 54.57% and 59.86% respectively. It was comparatively lesser in CS or AsA supplemented Cd treatments 31.13% (Cd_0.3+CS_0.6mM); 53.48% (Cd_0.5+CS_0.6mM) and 20.23% (Cd_0.3+AsA_0.8mM); 51.28% (Cd_0.5+AsA_0.8mM). Combination treatments of CS and AsA with Cd checked the fresh weight losses to 13.87% in (Cd_0.3+CS_0.6+AsA_0.8mM) and 35.51% (Cd_0.5+CS_0.6+AsA_0.8mM) to that of control (Fig. 7).

The dry weight of plants was reduced by up to 16.06% and 19.36% in Cd treatments (0.3 and 0.5 mM) in comparison to the control. CS and AsA supplemented Cd treatments had lesser dry weight loss, 13.27% (Cd_0.3+CS_0.6mM); 15.18% (Cd_0.5+CS_0.6mM) and 11.77% (Cd_0.3+AsA_0.8mM); 17.69% (Cd_0.5+AsA_0.8mM). CS and AsA combination treatment with Cd was effective in minimizing dry weight loss to 4.56% (Cd_0.3+CS_0.6+AsA_0.8mM) and 11.69% (Cd_0.5+CS_0.6+AsA_0.8mM) to that of control (Fig. 8).

Figure 7: Effect of Cd alone and in combination with CS and AsA on fresh weight in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=0.47). Click here to view Figure

Figure 8: Effect of Cd alone and in combination with CS and AsA on dry weight in mungbean plants. Each value represents the mean ± SE of three replicates. (LSD0.05=0.10). Click here to view Figure

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. ^20,21An improved content of chlorophyll and carotenoids have been linked to the lowering of Cd-induced toxicity with supplements like CS and AsA. ^22,23Different findings correlated CS elevating pigment levels and ²⁴AsA enhancing chlorophyll and membrane stability index during stress.²⁵As a redox buffer, AsA neutralizes the superoxide radicals and other singlet oxygen species, thus, preventing chlorophyll degradation and increasing its content. ²⁶The 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. ²⁷Production and storage of ROS in heavy metal stress destroys membrane lipids distorting the structure of lipid-protein membrane which increase membrane permeability. ²⁸AsA help in combating stress by decreasing electrolyte leakage and maintaining membrane integrity. Negatively impacted RLWC recovered using CS and AsA supplements in Cd treatments. ²⁹Long term exposure to Cd causes water imbalance leading to decreased RLWC and transpiration. ³⁰Cd reduces turgor pressure, relative water content, and water potential of plant cells. ³¹Excess 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. ³³Introduction of CS enhanced water content owing to deposition of silica in the cell wall that reduces transpiration rate to mitigate plant stress. ³⁴AsA have also been reported to improve water content in various plant species.

³⁵Higher 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.³⁶Lesser 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.³⁷A 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. ³⁸Addition of CS improved root-shoot length, biomass, and related yield attributes in Cd-stressed Vigna radiata. ³⁹Silicates (Si) help in cell enlargement by enhancing the tissue extensibility.⁴⁰AsA 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.

Conflict of Interest

There is no conflict of interest.

Funding Source

University Grants Commission, New Delhi.

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