Alleviation of Cadmium Toxicity in Zea Mays L. through Up-Regulation of Antioxidant Defense System and Organic Osmolytes under Supplemental Calcium


 Calcium (Ca) is a macronutrient and work as a modulator to mitigate oxidative stress induced by heavy metals. Present work was conducted to elucidate the role of Ca in modulating growth, physio-biochemical traits, and cellular antioxidant defense system in Zea mays L. seedlings under Cd stress. The experiment was designed in a complete randomized design with two levels of Cd (0 and 150 µM) and six levels of Ca (0, 0.5, 1, 2.5, 5 and 10 mM). Maize seedlings exposed to Cd at150 µM concentration showed a notable decrease in growth, biomass, anthocyanins, chlorophylls, and antioxidant enzymes activities. Higher level of Cd (150 µm) also caused an upsurge in oxidative damage observed as higher electrolyte leakage (increased membrane permeability), H2O2 production and MDA accumulation. Supplementation of Ca notably improved growth traits, photosynthetic pigments, cellular antioxidants (APX, POD and ascorbic acid), anthocyanins and level of osmolytes. The significant improvement in the osmolytes (proteins and amino acids), and enzymatic antioxidative defense system enhanced the membrane stability and mitigated the damaging effects of Cd. The present results concluded that exogenously applied Ca can potentially improve growth by regulating antioxidants and enable maize plants to withstand the Cd toxicity.


Introduction
While growing in natural environments, plants are exposed to various environmental stresses that limit yield and productivity (Huybrechts et al. 2019). Heavy metal pollution is spreading in cultivated lands and is causing severe environmental hazards to crop plants, human health and ecosystems (Liu et al. 2014).
Cd is regarded as the most toxic heavy metal, typically when present in agricultural lands due to its higher mobility and toxicity (Goix et al. 2014). Plants can readily absorb Cd through roots directly from the soil along with essential nutrients (Nogueirol et al. 2016). Like other heavy metals, Cd causes structural changes in plants and adversely affects the morphological, physiological, and biochemical mechanisms which eventually lead to loss of agricultural productivity (Kováčik et al. 2014). Cd is typically nonessential for agricultural crops as no known role is ascribed for Cd in growth and development of crop plants (Huybrechts et al. 2019). Therefore, Cd even in minor concentrations disturb the photosynthesis, change the ultrastructure of the chloroplast, increase lipid peroxidation and enhance production of ROS that leads to oxidative damage (Gallego et  Maize is a valuable cereal crop and provides food for humans as well as fodder for the livestock. It contributes to 36 % (782 Mt) in global grain production ). Maize seeds are enriched with energy as 100g seeds contain 365 kilocalories of energy (Nuss and Tanumihardjo 2010). Among worldwide production, 70-80% of maize is used as food and was ranked third in Pakistan for consumption after wheat and rice. Pakistan was ranked 18th in the production with 6130 thousand tons maize produced annually that was cultivated at 1334 thousand hectares . The requirement for the maize production has signi cantly increased recently due to excessive usage in the wet milling industry as well as food for poultry (Boomsma and Vyn 2008

Plant sampling and measurements
Plants material was sampled at the seedling stage to determine plant growth attributes, physiobiochemical traits, ROS, and enzymes of antioxidants defense system. Harvested seedlings were washed with distilled water and growth attributes were recoded. Leaf samples of maize seedlings were frozen at -80°C for physio-biochemical traits and antioxidants. Sampled seedlings were dried in oven at 70 °C to achieve a constant dry weight for determination of root (RDW) and shoot (SDW) dry weight.

Growth attributes
The shoot length (SL) of plants from each treatment was measured from sand level to the topmost leaf of the plant. The roots of seedlings were carefully removed from the sand for recording root length (RL). Root (RFW) and shoot fresh weight (SFW) of seedlings were measured immediately after excision. The leaf area (LA) was estimated by measuring length × width × 0.68.

Physiological attributes
Chlorophyll contents Chlorophyll contents were assessed as described by Arnon (1949) and carotenoids following the method of Davis (1976). For the appraisal of chlorophyll contents, 0.1 g of leaf sample was grounded in 5 mL of acetone (80 %). Extract was ltered through a Whatman # 02 lter paper (GE Healthcare, UK) and absorbance was recorded through a spectrophotometer (Hitachi U-2910, Tokyo, Japan) at 645, 663, and 480 nm. The values of photosynthetic pigments were calculated by using the following formula.
Here, V characterizes the volume of acetone and (FW) showed the leaf fresh weight.
Determination of relative membrane permeability The fresh leaf samples were collected and washed thoroughly with 4 changes of water to eradicate any adhered electrolytes on the surface. The leaves were cut into small discs with a borer and placed in the small glass test tube containing deionized water (10 mL), The EC o was measured by the help of Cond/Salinity meter (TPS AQUA-CPA). The test tubes were incubated for 24 h at 4 °C and EC 1 was measured. The tubes were then wrapped with aluminum foil, autoclaved for 10 min. at 100 kPa and (EC 2 ) was recorded. The ratio of % ion leakage was computed as designated by Yang et al. (1996).

Assessment of biochemical traits
Anthocyanin contents Anthocyanin content was appraised according to the method of Giusti and Wrolstad (2001). The 0.1 g of leaf was pulverized in trichloroacetic acid (TCA) by using pestle and mortar. The homogenized material was transferred to test tubes and shifted to water bath at 80 °C for 20 min. Homogenized material was centrifuged at 12,000 xg for 10 min. in the absorbance was noted at 516 and 700 nm using a spectrophotometer (Hitachi U-2910, Tokyo, Japan). Acetone was run as blank and amount of monomeric anthocyanin contents was calculated as follows.

Oxidative stress markers (MDA and H 2 O 2 )
Lipids peroxidation (LPX) was quanti ed by means of malondialdehyde (MDA contents) by following Heath and Packer (1968). LPX content was determined by the reaction of thiobarbituric acid-TCA with trichloroacetic acid-TCA. The 0.25 g leaf sample was grinded in 500 µL of TCA (0.1 %) and then centrifuged at 15,000 xg. An aliquot (1 mL) was taken and mixed with 2 mL of 0.5 % of TBA and 20% TCA. Test tubes containing reactants were incubated at 85 °C for 20 min. and reaction was terminated in an icebox. Absorption was recorded at 532 and 600 nm by spectrophotometer (Hitachi U2910, Tokyo, Japan). All absorption ODs (at 532nm) were subtracted from 600 nm. LPX concentration was calculated by using 155 mM cm -1 as an extinction coe cient.
Amount of H 2 O 2 was quanti ed by measuring the oxidation of ferrous ions medicated by peroxidase and ferric ions react with the xylenol (Bellincampi et al. 2000). Leaf sample 0.5 g was grounded in 5 mL of 10 mM sodium phosphate buffer (SPB). Centrifugation of homogenized material was done at 15,000 xg. A 2 mL of aliquot was reacted with the assay reagent containing 200 mM sorbitol, 200 µM xylenol, 50 mm H 2 SO 4, and 500 µM ammonium ferrous sulphate. The reactant material was incubated at 24 °C for a 1 h and absorption was recorded at 560 nm by using a spectrophotometer (Hitachi U-2910, Tokyo, Japan).

Cellular antioxidants (APX and POD)
The maize seedlings' shoot were grounded in liquid nitrogen and extracted with 1 mM L -1 of 5% polyvinylpyrrolidone, and, sodium phosphate buffer (SPB) having pH 7.8. Extracted material was centrifuged at 15,000 xg. Enzyme crude extract was stored at 4 °C for 36 h till analysis.
Ascorbate peroxidase activity (APX) Activity of APX was quanti ed by oxidation of ascorbate (Chen and Asada 1989). Reaction was started by adding 10 µL of crude enzyme extract to 2 mL of assay reagent ( and volume was maintained up to 3 mL. Enzyme activity was measured at 460 nm after 60 s interval through a spectrophotometer (Hitachi U-2910, Tokyo, Japan). Enzyme speci c activity was expressed on the base of proteins.

Ascorbic acid
Ascorbic acid was determined as described by Nino and Shah (1986). Plant tissues (100 mg) were pulverized in thiobarbituric acid (TCA) and centrifuged 10,000 xg for 10 min. An aliquot (500 µL) was taken with 500 µL of dthiocarbamate (DTC) in glass tubes. Reactants were left for ½ h at 37 °C. Test tubes containing reactant material was transferred to the ice-bath to terminate the reaction. After that, 2 mL of diluted H 2 SO 4 was mixed slowly and left over for ½ h at 37° C in incubator. Extracted material was centrifuged at 12,000 xg. The shift in absorption was measured at 520 nm with the help of a spectrophotometer (Hitachi U-2910, Tokyo, Japan).

Plant growth traits
Growth traits such as SL, RL, SFW, SDW, RFW, RDW and LA signi cantly decreased at Cd applied at150 µm concentration (P ≤ 0.05). However, different levels of Ca signi cantly alleviated Cd toxicity and enhanced all growth traits. The increase in growth traits was more obvious in response to higher level of Ca applied at 10 mM under Cd stress (Table 1). Under Cd stress, a signi cant (P ≤ 0.05) reduction occurred in the concentration of photosynthetic pigments of maize seedlings. Exogenously supplied Ca signi cantly increased photosynthetic pigments both in Cd stressed and non-stressed seedlings. Calcium applied at 10 mM level was more bene cial in increasing chlorophyll and carotenoids contents of maize seedlings (Table 2).  Mean values for antioxidant activity was higher in Cd stressed (150 µM) as compared to non-stressed plants (0 µM). However, the activity of APX signi cantly enhanced as levels of Ca increased both in nonstressed (0 µM) and stressed plants (150 µM). This increase was the maximum in response to Ca applied at 10 mM concentration. Peroxidase activity showed the same trend as noted for APX under Ca and Cd treatments ( Table 2).

Anthocyanin and relative membrane permeability (RMP)
Under Cd stress, maximum RMP values were observed indicating a high level of electrolyte leakage due to membrane damage. A signi cant (P≤0.05) reduction was observed as the level of Ca increased (Fig 1).
Anthocyanin contents under both treatments of Cd signi cantly increased as levels of Ca increased. Maximum anthocyanin contents were noticed at 10 mM Ca concentration (Fig. 1). Cadmium applied at 150 µm level and without any Ca supplementation had the most toxic effects as the highest electrolyte leakage was observed at this treatment level.

Lipid peroxidation (LPX) and ROS
The accumulation of H 2 O 2 and MDA signi cantly increased in maize seedlings under Cd stress. However, the elevated levels of Ca signi cantly reduced the generation of H 2 O 2 and LPX. The LPX in terms of MDA contents signi cantly decreased as the level of Ca increased in growth medium of the seedlings. The maximum decrease was observed under 10 mM concentration of Ca (Fig. 1).

Osmolytes
Osmolyte (proteins and amino acids) production was signi cantly increased in maize seedling in stressed and non-stressed maize seedlings. Soluble proteins were signi cantly higher in non-stressed maize seedlings as the level of Ca increased (Fig 2). In Cd stressed seedlings (150 µM), the concentration of soluble proteins signi cantly increased and the maximum was observed under 10 mM Ca concentration (Fig 2). Applications of Ca substantially increased the concentration of amino acids in both stressed and non-stressed seedlings and almost parallel results were observed as noted for soluble proteins (Fig 2).

Ascorbic acid contents
Ascorbic acid contents were substantially improved as Ca levels increased in maize seedlings under normal and stress conditions. Maximum values of ascorbic contents were observed under 10 mM concentration of Ca in both stressed and non-stressed condition (Fig 2).

Multivariate analysis Principal component analysis (PCAs)
PCAs results demonstrated high variations on the effects of Cd and Ca treatments among different growth and physio-biochemical traits of maize seedlings (Fig. 3). Chl, carotenoids, and LPX with negative eigenvalues (Fig. 3). Cd stress signi cantly increased level of reactive oxygen species, while supplemented Ca signi cantly increased the antioxidative enzymes activity and growth parameters (Fig. 3).

Correlation matrix
In control plants, anthocyanin contents (Antho-C) was positively correlated with RFW, RDW, SL, LA, Caro, Chl b and TSP. The RMP, H2O2, and RMP were negatively correlated with RFW, RDW, Chl a, b, RL, LA, A-AC and APX (Fig. 4a). Under Cd stress, a highly positive correlation was assessed between POD, SFW, and ASC.A, APX, Chl a, SL, and RL. However, a strong negative correlation was assessed between H 2 O 2 , RMP, and antioxidant enzymes under Cd-150 µM stress (Fig. 4b).

Response of different traits under stressed and non-stressed conditions
In non-stressed conditions (0 µM), a conspicuous positive response was observed for the growth traits (RL, SL, SFW, SDW, and LA) and chlorophyll (Chl a, Chl b and T. Chl) as Ca levels increased (Fig 6a).
Organic osmolytes (TAA, TSP), anthocyanin contents (AC) and ascorbic acid (A,ASc) showed a sharp positive response with increasing Ca regimes (Fig 6b). H 2 O 2 , MDA and RMP exhibits a strong negative response with an increase in Ca levels, however APX and POD exhibits increasing pattern in curve with elevated Ca gradients (Fig 6c). In Cd stressed conditions (150µM), growth traits (RL, SL, SFW, and SDW, LA) and chlorophyll (Chl a, Chl b and T.Chl) displayed a strong positive response and in response to Ca levels (Fig 6d). Concentration of TAA, TSP, AC and AA were the maximum with positive response (Fig 6e).
A strong positive response was noted in activity of APX and POD along increasing Ca levels. In contrast, a strong negative response was assessed for H 2 O 2 , MDA and RMP with increase in Ca regimes (Fig 6f).

Discussion
Calcium plays an essential role in mitigation of abiotic stresses and protection from drastic impacts Reduction in growth traits under Cd toxicity is directly linked to the reduction of photosynthetic contents. As anticipated, the photosynthetic pigments signi cantly declined under 150 µM Cd treatment level.
However, higher levels of Ca signi cantly improved carotenoids, total Chlorophyll (Chl), Chl a, b pigments in maize seedlings under Cd stress (Table 2) (Mittler 2002;Huang et al. 2017). Enzymes like APX and POD also take part in the detoxi cation of free radicles and lead to sequestering of H 2 O 2 (Sharma and Dietz 2006). APX is mainly localized in chloroplast, apoplast, cytosol, mitochondria, and peroxisome and POD in cell walls, cytosol, and vacuoles, Both APX and POD are mainly implicated to the scavenging of H 2 O 2 (Mittler 2002). Their e ciency enhanced during Cd stresses and that greatly imparts stress tolerance and modulates the physiological process in maize seedlings in this study (Ahmad et al. 2010;Siddiqui et al. 2011).
Plants exposed to metal stress showed alterations in cell membrane permeability (RMP) and consequently cell loses membranes integrity (O'Lexy et al. 2018). Cell membrane integrity is considered as a tool to regulate the ionic movements and use as a selection criteria to quantify damage magnitude.
In current results, the relative RMP markedly increased under Cd stress. However, the RMP signi cantly was markedly reduced by the Ca treatments that alleviated the damaging consequences of Cd. In plants increased under Cd stress, however, the addition of Ca considerably reduced the production of ROS in maize seedlings (Fig. 1).
The anthocyanin contents remarkably increased in present study that was more pronounced in highest levels of Ca (Fig. 1) (Türkan et al. 2005). In present study, ascorbic acid in maize seedlings was signi cantly enhanced by the addition of Ca (Fig. 2).

Conclusions
In conclusion, Cd induced oxidative stress caused negative in uences on growth and physio-biochemical traits of plants. The addition of Ca signi cantly enhanced the growth and physio-biochemical traits.
Exogenously applied Ca ameliorated the oxidative stress by increasing the APX and POD activities, and ascorbic acid contents to withstand Cd toxicity and increased tolerance. Calcium treatments signi cantly

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Ethics approval and consent to participate: Not applicable.
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