Study of Oxidative Stress Indices, Morphological Response, Mineral Absorption in Chickpea (Cicer Arietinum L.) Under Cadmium Stress and Bioinformatics of HMA Poteins

Cicer arietinum L. (chickpeas) is a widely consumed legume that is impacted by heavy metal contaminants such as cadmium. Cadmium is a chemical hazard and can severely impact the morphological and physiological features of the plant. C. arietinum L. were exposed to cadmium and its impact on plant growth and antioxidant enzyme activity evaluated. Bioinformatic studies were performed to further understand the mechanism by which the plant combats heavy metal stress. Observed morphological changes included stunted growth, poor root development and yellowing of the plant. The study also revealed that increased cadmium resulted in a decline in mineral transportation to aerial regions of the plant. Antioxidative enzyme activity (peroxidase, superoxide dismutase, catalase, ascorbate peroxidase) increased in the leaves suggesting that these enzymes play an integral role in combatting heavy metal contamination. These research showed chickpea has a relatively high adsorption capacity for cadmium in aerial tissues. Special precautions should therefore be taken in the cultivation of chickpea. Increasing the levels of cadmium in the medium resulted in a decline in zinc, copper and manganese in the aerial parts of chickpea seedlings. There appears to be a competitive mechanism for mineral uptake in plants. HMAs play an important role in the transport of metals in plants and provide resistance to the uptake and transportation of metals. In silico analysis led to the identication of 13 Heavy Metal ATPases (HMAs). These proteins contain 130 to 1032 amino acids with 3 to 18 exons and assist in heavy metal detoxication. in their HMAs chickpea of antioxidant enzyme due to of parsley antioxidant enzymes in Crotalaria juncea exposed to cadmium, showed that there no signicant change in CAT activity in the root. At concentrations of 2 mM cadmium, CAT activity in the leaves 6 fold compared to the control. In the study, it observed that of the 4 isoenzymes, two are dependent on manganese and the other two are copper-zinc-dependent. CAT activity was similar to SOD and glutathione reductase activities. Increased activity of some antioxidant enzymes exposed to metals reveal the crucial role that these enzymes play in heavy metal detoxication. Under normal physiological conditions, various antioxidant cycles lead to the production and scavenging of ROS which is in a state of dynamic equilibrium Kisa studied the response of antioxidant systems to stress induced by heavy metals in the leaves and roots of which showed that cadmium treatment signicantly increased the activity of the APX and SOD enzymes. Antioxidant scavenging systems are involved with ROS detoxication which is a defense mechanism employed by plant tissue to combat oxidative stress Tomato plants exposed to cadmium showed signicantly higher SOD activity. There was however a decline in CAT activity.


Introduction
Chickpea (Cicer arietinum L.) is a major legume crop consumed globally. The legume ranks third when compared to other legumes contributing 16.4 % to the world market. In 2010, 10.9 million tons of chickpea was produced from a land mass of 12.0 million hectares. In Iran, 6.2% of total crop area is designated to chickpea cultivation. Of the total Iranian beans grown in Iran, 64.3% is in relation to chickpeas. Chickpea has high nutritional content and is an economical source of protein (12-31%). Chickpea is also a source of minerals (manganese, molybdenum, phosphorus and potassium) and vitamins. Phytoestrogens offer therapeutic bene ts. Considering its economic and nutritional importance, steps need be taken to avoid contamination with heavy metals such as cadmium. Globally, environmental contamination by heavy metals is a major concern. Contamination of waterways, the soil and air are a serious threat to the It has been hypothesized that P 1B -type heavy-metal ATPases (HMAs) are involved in the transportation of both essential and potentially toxic components across cell membranes. Through the use of phylogenetic analyses P 1B -type heavy-metal ATPases can be subdivided into 2 distinct clusters, namely the Cu cluster involved in transporting Cu and Ag, whereas the Zn cluster proteins transport Zn and other heavy metals (e.g. Co, Cd, and Pb) (Satoh-Nagasawa, Mori et al. 2012, Takahashi, Bashir et al. 2012).
In this study, the impact of cadmium on morphological features, antioxidative enzymes, mineral absorption and the cadmium content of chickpeas was evaluated. Genes involved in oxidative stress and their response to heavy metals were studied utilizing bioinformatics. HMAs are transmembrane metaltransporting proteins that play a key role in metal homeostasis. Very little is known about their function in the Fabaceae family. Utilizing bioinformatics, HMAs in chickpea were investigated. The number of genes, proteins, gene loci, cellular location, phylogenetic relationship, three-dimensional protein structure, conserved domains, similar template and catalytic site were determined.

Materials And Method
Propagation and cadmium exposure Kabuli chickpea (Cicer arietinum L.) seeds were planted in sterilized Cucupite and Perlite in a greenhouse at Tabriz University, Iran utilizing an illumination cycle of 8 h light and 16 h darkness. After germination, plantlets (3) at the leaf stage were planted in pots (diameter, 12 cm and height 15 cm) under sterile conditions and irrigated with distilled water (3 days). A hydroponic nutrient solution (Hoagland solution), was then utilized for irrigating the plantlets treated with cadmium chloride in replicates of three at four different concentrations (control, 2, 4, and 8 mM) for 10 days. Plants were then harvested for further investigations.

Morphometric parameters
Fresh and dry weight of the roots and aerial organs were determined (mg). Plantlet height, leaf area, root length, aerial organ length and internode height were measured. Stomatal densities on the lower and upper epidermis were evaluated.

Elemental analysis of Cd, Zn, Cu and Mn
Plant samples were oven dried (72 h, 60 °C) and the dry weight determined. Dried samples were ashed (550 °C, 8 h). After cooling the samples were acid digested (1N HCl, 1 ml; nitric acid, 97%, 1 ml, 1 h). The digested extract was made to a nal volume of 20 ml and the cadmium, zinc, copper and manganese content of the samples measured (Chellaiah, 2018) utilizing a Flame Atomic Absorption Spectrometer (GBC, SAVANTAA scienti c equipment, Australia) which has a detection limit of 0.007 µg/mL. Cadmium, zinc, copper and manganese nitrate in nitric acid was used as the standard.

Bioinformatics analysis
The NCBI gene database was searched utilizing the keyword "HMA". Gene characteristics included location, exon count and conserved domain. Protein sequences were used to predict localization from the Localizer and protein tertiary structure predicted by Phyre2. Potential tunnels within each protein and catalytic pocket were predicted utilizing CAVER Web. The Jones-Taylor Thornton model was used to obtain the phylogenies tree of HMAs from chickpea and Arabidopsis using the neighbor-joining (NJ) method, with a bootstrap test performed using 1000 iterations in MEGA5 (Tamura, Dudley et al., 2007). Multiple sequence alignments were performed utilizing the muscle algorithm of mega 7 software to detect conserved residues . HMAs from Arabidopsis were highlighted in green.

Statistical analyses
Data analyses were performed using the SPSS20.0 software package (SPSS Inc., Chicago,IL, USA). All experimental data were presented as the mean ± SD.
One-way ANOVA was used to test differences between various means followed by the post hoc Tukey test. The level of signi cance was set at p < 0.05 for all tests.

Results And Discussion
Heavy metal pollution is a signi cant environmental problem. Researchers are actively seeking new cost effective and environmentally friendly technologies to be utilized in soil remediation. Increased knowledge of the mechanisms by which plants are able to mitigate heavy metal stress could assist in creating new tools applicable to phytoremediation. Further research regarding heavy metal detoxi cation and signaling pathways in plants will assist in identifying useful targets for biotechnology resulting in increased plant tness in heavy metal polluted sites (Tchounwou, Yedjou et al. 2012).
Morphometric features in aerial parts of chickpea seedlings affected by cadmium Observed morphological changes in chickpea seedlings exposed to cadmium included changes in plant length, coloration and leaf size. Stem color changed to a bright green-yellow hue. Changes were also observed in leaf size and color (yellow). There was a signi cant reduction in shoot and root length. Shorter and less dense roots were observed in treated samples ( Table 1). The fresh and dry weight of the shoots and roots in chickpea plants were also signi cantly affected with lowest seedling weights being observed at high cadmium concentrations. Plants treated with 2 mM cadmium had a signi cant decline in leaf area (less than half that of the control). At cadmium levels of 2 mM, the length of the rst internodes increased, whereas at higher concentrations, there was a decrease. The length of the second internodes showed only a signi cant reduction at high concentrations of cadmium ( Figure 1, Table 1). Hassan et al. (2006) investigated the effect of cadmium on chickpea and found that plant growth and development as well as carbonic anhydrase enzyme activity declined resulting in changes in plant metabolism and photosynthesis. The impact of cadmium ion suppression on root extension extends through its effect on cell growth. Cadmium attaches to the cell wall and the middle wall, increasing bonding between the wall components, ultimately leading to growth inhibition and a decline in cell and organ development. Cadmium also alters the proportion of water in plants causing physiological dryness, which leads to metabolic dysfunction and the production of reactive oxygen species (ROS). These factors reduce growth and decreases plant length and weight.
Many studies on the mechanism of cadmium blockage on cell growth have shown degradation of cell membranes, changes in the degree of cell exchange and cellular depletion (Bücker-Neto, Paiva et al. 2017). The observed changes in plants exposed to cadmium may be as a result of multiple nutritional de ciencies that are being experienced by the plant. Nutrients serve an essential role in the formation, expansion, and operation of chloroplasts. Cdphytotoxicity affects the synthesis and extensibility of cell walls (Breckle and Kahle, 1992 Cell wall thickening in root endodermal tissue affords a greater surface area over which cadmium accumulation can occur thereby limiting its transportation to the shoot (Gomes et al., 2011). Chlorosis observed in the leaves of bean plants exposed to cadmium may be due to loss of magnesium which is an integral structural feature of the porphyrin ring present in chlorophyll. Physiological changes observed in soybean leaves are due to the associated toxic effects of cadmium including mesophyll curvature, decreased leaf thickness and a reduction in the composition of intercellular spaces of spongy parenchyma. At higher doses of cadmium, the thickness of palisade and spongy tissues is reduced. A decline in the dimensions and composition of the main mid-vein bundle suggests that cadmium alters leaf expansion (Cregeen et al., 2015).
A study of the effect of heavy metals on the cell death of Halophyl astipulecea leaves concluded that high concentrations of metal causes necrosis of the epidermal cells and mesophyll, inhibiting surface growth of the leaves. High levels of heavy metal accumulation in plant cells inhibits the process of breathing and energy reactions, which are associated with cell growth (Ayangbenro and Babalola, 2017). A decline in cell division and growth could also be a contributing factor to the observed morphological changes. Additionally, a decrease in photosynthetic rates has been observed in plants exposed to elevated levels of heavy metals. Higher concentrations of cadmium commonly result in root injury, damage to photosynthetic machinery, inhibition of plant growth, reduced nutrient and water uptake. Cadmium may exert its inhibitory effect in different ways, namely binding speci c groups of proteins and lipids thereby inhibiting normal function and possibly inducing free radical formation due to oxidative stress. The former may occur at transport and channel proteins of cell membranes disturbing the uptake of many other macro-and microelements whereas the latter is due to inactivation of antioxidant enzymes (Long, Zhang et al., 2017).

Effect of cadmium on SOD, POD and CAT activities in the aerial parts of chickpea seedlings
There was a signi cant increase in POD enzyme activity in chickpea seedlings exposed to cadmium. Highest enzyme activity was observed at treatments of 4 mM. Further increase in cadmium exposure resulted in a decline in POD activity which was however still signi cantly higher than that of the control and plantlets treated with 4 mM cadmium. Lowest enzyme activity was observed in the controls (Figure 2A). SOD enzyme activity signi cantly increased with highest enzyme activity being observed in plantlets treated with 4 mM cadmium and lowest enzyme activity in the control ( Figure 2B). It should be noted that the 8 mM treated plants were almost completely yellow at the same day of harvest, thus reducing all the enzymatic activities in the 8 mM plants due to possibly more cell death. There was a signi cant increase in CAT enzyme activity with highest levels being observed in plants treated with 4 mM cadmium.
There was a subsequent decline in CAT activity when cadmium concentration was increased to 8 mM. Lowest enzyme activity was observed in the control ( Figure 2C). Investigation of APX enzyme activity showed that this enzyme was also affected with highest APX activity being observed in cadmium treatments of 4 mM ( Figure 2D). Antioxidative enzyme activity (SOD, APX or CAT) was shown to increase in the leaves of plants exposed to cadmium. Increased SOD activity is associated with an increase in the formation of superoxide, which activates gene expression by signal induction. Studies performed by Pereira et al. (2002) on antioxidant enzymes in Crotalaria juncea exposed to cadmium, showed that there was no signi cant change in CAT activity in the root. At concentrations of 2 mM cadmium, CAT activity in the leaves increased 6 fold compared to the control. In the study, it was observed that of the 4 isoenzymes, two are dependent on manganese and the other two are copper-zinc-dependent. CAT activity was similar to SOD and glutathione reductase activities. Increased activity of some antioxidant enzymes exposed to metals reveal the crucial role that these enzymes play in heavy metal detoxi cation. Under normal physiological conditions, various antioxidant cycles lead to the production and scavenging of ROS which is in a state of dynamic equilibrium (Pereira, Molina et al. 2002). Kisa (2018) studied the response of antioxidant systems to stress induced by heavy metals in the leaves and roots of tomato which showed that cadmium treatment signi cantly increased the activity of the APX and SOD enzymes. Antioxidant scavenging systems are involved with ROS detoxi cation which is a defense mechanism employed by plant tissue to combat oxidative stress (Kisa 2018). Tomato plants exposed to cadmium showed signi cantly higher SOD activity. There was however a decline in CAT activity.
Cadmium content in the aerial parts of chickpea seedlings The cadmium content in aerial parts of chickpea grown at different concentrations of cadmium increased signi cantly. Highest levels were observed at a cadmium concentration of 8 mM. A doubling of cadmium accumulation was observed in the aerial parts of the plant when the cadmium content of the medium was increased from 2 to 4 mM ( Figure 3C At concentrations of 0.04 to 0.32 mM, cadmium is non-polluting in soil. Knowledge about the distribution of cadmium in plant tissues is important to better understand the tolerance mechanism and accumulation of heavy metals in plants. Cadmium in plants is transferable from apoplasty pathways of the stems and leaves (Benavides, Gallego et al. 2005). Cadmium affects membrane potential, protein pump activity and can limit corn growth (Karcz and Kurtyka 2007).
Changes in the mineral composition of aerial regions of chickpea seedlings Mineral composition was signi cantly affected by cadmium (Figure 3). Cadmium accumulation in plant species with varying tolerance to other heavy metals determines the effect of cadmium on the absorption of these other minerals in plants. Chickpeas cultivated in cadmium-containing media showed a signi cant difference in the levels of manganese in the aerial part of the plant. With the addition of cadmium (2 mM), manganese uptake signi cantly increased, while higher concentrations of cadmium reduced the levels of manganese in chickpea plants ( Figure 3B). Increase in the levels of cadmium in the culture also caused changes in the levels of zinc present in the aerial parts of pea plants. Increasing the levels of cadmium in the medium resulted in a decline in zinc ( Figure 3A). Increasing cadmium concentration decreased the levels of copper present in the aerial parts of chickpea seedlings. The lowest amount of copper was observed in high-cadmium seedlings ( Figure 3D). Further studies also showed that zinc and copper along with cadmium have an antagonistic effect and that these minerals act in a competitive manner in relation to the transfer processes. Cadmium has a negative impact on the absorption of Manganese plays a role in many biochemical functions, such as enzyme activation in respiration, redox reactions, intracellular electron transfer systems, the Hill reaction in chloroplasts, amino acid synthesis, and hormone regulation. Manganese concentration was higher in the shoots than the roots of plants treated with cadmium. Transfer of manganese to the shoot may in fact be a tolerance mechanism that reduces the effects of cadmium toxicity on photosynthesis. Research suggests that cadmium and manganese compete for the same membrane carriers (Socha and Guerinot 2014). Dias et al. (2013) showed that at cadmium concentrations of 5 and 10 μm there was a signi cant decline in the mineral content of lettuce leaves. At high concentrations of cadmium, a signi cant decline in manganese in the roots was observed. Cadmium appears to interfere with the transmission of macro and micro elements in the leaf (Dias, Monteiro et al. 2013). According to Guerinot, members of the ZIP and NRAMP or Ca channels and transporters which are responsible for the uptake of essential elements are involved in the transport of cadmium via the same route (Guerinot 2000). Imbalance in nutrient level and growth inhibition is ultimately due to competition between nutrients and toxic metals for binding sites in the cell. Sun and Shen (2007)  Some researchers show that increasing zinc concentration along with cadmium reduces cadmium toxicity. There appears to be a common competitive mechanism for absorption of these elements. In our study the concentration of zinc was kept constant while the concentration of cadmium was increased. By increasing cadmium, a signi cant reduction was observed in the levels of copper and zinc in the aerial sections of chickpea seedlings. Lowest levels of copper and zinc were observed at high concentrations of cadmium. At low concentrations of cadmium, the amount of manganese increased. With an increase in cadmium, the level of manganese decreased. In the current study, high concentrations of cadmium did not signi cantly affect mineral concentration. At high concentrations there is saturation within channels or adsorption receptors preventing mineral absorption.

Bioinformatics
Heavy metal ATPases (HMAs), belong to the large P-type ATPase family. They play an important role in the transport of metals in plants and provide resistance to the uptake and transportation of metals. For the current bioinformatics study HMA proteins were selected. In silico analysis of chickpea HMAs identi ed 13 HMA. There were three proteins each for HMA3 and HMA4, two for HMA5 and one for HMA 2, 6, 7, 8 ( Table 2). ATPase PAA2, chloroplastic, copper-transporting ATPase RAN1, and copper-transporting ATPase PAA1, chloroplastic identi ed in chickpea were identi ed as HMA6, HMA7, HMA8, in Arabidopsis, respectively. HMA7 and HMA8 all contribute to copper transport. HMA 1, HMA 3, g HMA 2, HMA 4, HMA 5, PAA1, RAN1 and PAA2 genes are located on chromosomes 7, 1 and 7, 1, 6 and 7, 5 and 8, 6, 6, 5 respectively (Table 2). These proteins contain 130 to 1032 amino acids with 3 to 18 exons. The con dence level of predicting the three-dimensional structure of chickpea HMAs proteins is shown in Table 3. Their cellular locations are often in the nucleus and chloroplast. Using phyre2, their three-dimensional structure was determined. The protein templates and organisms used to predict the three-dimensional structure of these proteins are listed in Appendix 1. Among these templates, c3rfuC was used to predict all 13 proteins in a study related to copper-transporting PIB-type ATPase from the gram-negative bacterium Legionella pneumophila subsp. Pneumophila. The patterns of c3j08A and c3j09A are also related to the ptype ATPase copper transporter CopA. Five (5)  The COG4087 domain which is listed as Soluble P-type ATPase and pfam00122 as E1-E2_ATPase are present in ten HMAs. The three-dimensional structure for chickpea HMAs, the longest tunnels for each protein and catalytic pocket utilizing CAVER Web for ion passing was determined. The longest and shortest tunnels belonged to cadmium/zinc-transporting ATPase HMA3-like and cation-transporting ATPase HMA5-like, respectively. The putative inactive cadmium/zinc-transporting ATPase HMA3 was the largest HMA with 1032 amino acids which had a short tunnel having a length of 41.7. No tunnel was predicted for copper-transporting ATPase PAA2, chloroplastic and copper-transporting ATPase PAA1, chloroplastic with 934 and 884 amino acids.  The three-dimensional structure with the longest predicted tunnel allowing for passage of ions is illustrated in colour in Figure 4. Based on the software used to analyze 8 of the 13 HMA chickpeas, the catalytic site was determined. From the proposed envelope for the HMAs the catalytic position for interaction with ions was determined. For XP_027192934, three catalytic sites with Asp residues at positions 522, 729, 733 with 40% similarity over a speci c reference of active site type and metal ion-binding site were identi ed. These catalytic sites can be evaluated and compared based on their Pocket score. The neighboring residues of the catalytic position are also presented in the Table 3. In most cases, the amino acid Asp residue is introduced. For XP_012574029 and XP_004504659 the predicted Pocket score was 100% with XP_004504659 having an active site and three metal ion-binding sites (Table 3).
In the phylogenic tree of the HMAs, comparison of the protein sequences of chickpea HMA with Arabidopsis revealed great similarity between these proteins in chickpea and Arabidopsis. HMA 2 and 4 are very similar in Arabidopsis and are next to HMA 3 chickpea. HMA 3 Chickpea is adjacent to HMA 3 Arabidopsis. HMA 1 2 3 chickpea are involved in cadmium and zinc transfer and are in close proximity to each other in the tree. The P-type ATPases of Arabidopsis are very similar to the copper-transporting ATPase PAA2 chickpeas. Copper-transporting ATPase PAA1 pea is very similar to Arabidopsis P-type ATPases. In chickpea, copper-transporting ATPase RAN1 resembles copper-transporting ATPase HMA5, which is adjacent to copper-transporting ATPase RAN1 Arabidopsis. Cationtransporting ATPase HMA5-like and copper-transporting ATPase RAN1 are also in the vicinity of copper-transporting ATPase RAN1 Arabidopsis.
HMAs are classi ed based on substrate binding with one group bound to copper and silver and the other to cadmium, lead and cobalt. HMAs 9 and 8 have been studied in rice and Arabidopsis, respectively. AtHMA1-4 in A. thaliana and OsHMA1-3 in Oryza sativa are in the rst group and AtHMA5-8 and OsHMA4-9 in the second group. The expression of each of these genes is sensitive to heavy metals as indicated by mutagenesis. Typical P1B-ATPase cadmium with the rest belonging to lead, silver and copper. There are 10 HMA genes related to silver and copper in poplar that are signi cantly higher than those in rice and Arabidopsis.2 OsHMA plays an important role in transmitting cadmium from the root to the stem especially in rice grains (Li, Xu et al. 2015).
OsHMA3 transports cadmium to root cell vacuoles. Manipulating and altering the expression of these genes is a useful tool for reducing cadmium concentration in the seeds. AtHMA1 is within the chloroplast and zinc anti-toxic while AtHMA 3 is present in the vacuolar membrane with zinc and cadmium playing a role. The motifs of poplar HMA are very similar to Arabidopsis and rice proteins and it seems that family members of these genes may be functionally divergent due to differences in gene organization and existing motifs. AtHMA 1 and 2 are in the plasma membrane and in zinc and cadmium uxes. OsHMA 1 is involved in zinc transfer. No HMA type has been reported in rice. The number of HMA genes in the soybean genome is higher than that in Arabidopsis and rice, probably due to duplication of the soybean genome. Phylogenetic study of these genes divided them into six groups, based on their divergent gene structure, conserved segments or protein motif patterns. Examination of the cellular location of these proteins indicates that only GmHMA1 is involved in the secretion pathway while 1, 16, 17, 20, 20 peptides are mitochondrial targets, whereas 1, 2, 2, and 2 GmHMA2 are chloroplast peptides ( Conclusion Chickpea seedlings exposed to cadmium exhibited changes in their morphological features. These included changes in plant length, coloration and leaf size. There was a signi cant reduction in shoot and root length. Antioxidative enzyme activities were also affected by cadmium stress. A signi cant increase in POD, SOD, CAT APX enzyme activity was observed at 4 mM cadmium with a subsequent decline when concentrations were increased to 8 mM. Plants treated with 8 mM cadmium were discolored (almost completely yellow) and had a reduction in enzymatic activities possibly due to cell death. Cadmium content in aerial parts of chickpea plants increased signi cantly. Highest levels were observed at a cadmium concentration of 8 mM. Cadmium also affects the uptake of other minerals by the plant with signi cant differences observed in the levels of manganese. With the addition of cadmium (2 mM), manganese uptake increased signi cantly but subsequently declined with higher concentrations of cadmium. Zinc and copper levels however declined in the presence cadmium.
These results suggest that chickpea has a relatively high adsorption capacity for cadmium in aerial tissues. Special precautions should therefore be taken in the cultivation of chickpea. There appears to be a competitive mechanism for mineral uptake in plants. In silico analysis led to the identi cation of 13 HMAs containing 130 to 1032 amino acids with 3 to 18 exons. Comparison of the protein sequences of chickpea HMA with Arabidopsis indicated similarity between the proteins. It is apparent that chickpeas utilize a variety of mechanisms to combat heavy metal stress. Genetic engineering may be utilized to create heavy metal resistant chickpea species.