Acta agriculturae Slovenica, 119/3, 1–18, Ljubljana 2023 doi:10.14720/aas.2023.119.3.12555 Original research article / izvirni znanstveni članek Investigating the growth characteristics, oxidative stress, and metal ab- sorption of chickpea (Cicer arietinum L.) under cadmium stress and in silico features of HMAs proteins Maryam KOLAHI 1 , Elham Mohajel KAZEMI 2 , Milad YAZDI 3 , Mina KAZEMIAN 2, 4 , Andre GOLDSON- BARNABY 5 Received March 01 2023; accepted September 23, 2023. Delo je prispelo 1. marca 2023, sprejeto 23. septembera 2023 1 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2 Department of Plant, Cell and Molecular Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran 3 Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4 Corresponding author, e-mail: Mina.kazemian69@gmail.com 5 Department of Chemistry, University of the West Indies, Mona, Jamaica Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) under cad- mium stress and in silico features of HMAs proteins Abstract: Heavy metal contamination can have a strong effect on the morphological and physiological characteristics of plants. In the present study, Cicer arietinum L. (chickpea) was exposed to different concentrations of cadmium (control, 2, 4, 8 μg Cd g -1 perlite) and the effect on plant growth and antioxidant enzymes were evaluated. The observed morphological changes in chickpea plant included stunted growth, reduced root system development and plant color change. A significant increase in enzyme activity of peroxidase, superoxide dismutase, catalase, and ascorbate peroxidase was observed at 4 μg Cd g -1 perlite, with a subsequent decrease when concentration was increased to 8 μg Cd g -1 perlite in the leaves of the plants. The highest cadmium levels were determined at a concentration of 8 μg Cd g- 1 perlite. With the addition of 2 μg Cd g -1 perlite, manganese uptake in the aboveground part of the plant increased signifi- cantly, but then decrease at higher cadmium concentrations. In addition, zinc and copper levels decrease in the presence of cadmium. These results indicate that chickpea has a relatively high adsorption capacity for cadmium in aboveground tissues and special precautions should be taken when growing chick- pea. In silico analysis led to the identification of 13 heavy metal ATPases (HMAs) in chickpea. These proteins contain 130 to 1032 amino acids with 3 to 18 exons. They are involved in the transfer of cadmium and zinc and help in heavy metal detoxifi- cation of plants. Bioinformatics studies have been conducted to better understand the mechanism by which the plant is able to combat heavy metal stress. Key words: cadmium, chickpea, HMAs, oxidative stress Preučevanje rastnih značilnosti, oksidativnega stresa in pre- vzema kovin pri čičerki (Cicer arietinum L.) v razmerah kad- mijevega stresa in in silico lastnosti HMAs proteinov Izvleček: Onesnaženje s težkimi kovinami ima lahko mo- čan učinek na morfološke in fiziološke lastnosti rastlin. V razi- skavi je bila čičerka (Cicer arietinum L.) izpostavljena različnim koncentracijam kadmija (kontrola, 2, 4, 8 μg Cd g -1 perlita). Ovrednoteni so bili učinki na rast rastlin in na antioksidacijske encime. Opažene morfološke spremembe čičerke so bile zavr- ta rast, zmanjšan razvoj koreninskega sistema in spremembe v barvi rastlin. Značilna porast aktivnosti encimov peroksidaze, superoksid dismutaze, katalaze in askorbat peroksidaze je bila opažena pri 4 μg Cd g -1 perlita s posledičnim upadom, ko se je koncentracija povečala na 8 μg Cd g -1 perlita v listih tretiranih rastlin. Največja vsebnost kadmija je bila določena pri obravna- vanju z 8 μg Cd g -1 perlita. Pri dodatku 2 μg Cd g -1 perlita se je privzem mangana v nadzemnih delih rastlin značilno povečal a se je pri večjih koncentracijah kadmija zmanjšal. Dodatno so se v prisotnosti kadmija vsebnosti cinka in bakra zmanjševale. Ti izsledki kažejo, da ima čičerka relativno veliko sposobnost pri- vzema kadmija v nadzemna tkiva in moramo na to biti pozorni, če jo gojimo v s kadmijem onesnaženem okolju. In silico ana- lize so vodile k prepoznavanju 13 ATPaz (HMAs), povezanih s težkimi kovinami. Ti proteini vsebujejo 130 do 1032 amino kislin s 3 do 18 eksoni. Vključeni so v prenos kadmija in cinka in pomogajo v rastlinah pri detoksikaciji težkih kovin. Za bolje razumevanje mehanizmov s katerimi rastline premagujejo stres težkih kovin so bile izvedene tudi bioinformacijske raziskave. Ključne besede: kadmij, čičerka, HMAs, oksidativni stres Acta agriculturae Slovenica, 119/3 – 2023 2 M. KOLAHI et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) is a major legume crop that is consumed globally especially on the Africa and Asia continents (Kaur et al., 2022). Chickpea has a very high nutritional content and is one of the cheapest sources of protein and an important source of minerals (manganese, molybdenum, phosphorus and potassium) and vitamins (Mohanty et al., 2022), so measures need to be taken to avoid its contamination with heavy metals such as cadmium. Cadmium (Cd) is one of the most important con- taminants due to its high toxicity and high water solubil- ity and is readily absorbed by the root system of many plants (Zulfiqar et al., 2022). High levels of Cd can have detrimental effects on plant physiological and biochemi- cal processes, leading to reduced growth, impaired nutri- ent uptake, and disruption of cellular functions. More- over, Cd toxicity inhibits plant growth by affecting cell division, cell elongation, and differentiation processes (Tuver et al., 2022). It disrupts hormone balance, lead- ing to stunted root and shoot growth, reduced biomass production, and impaired reproductive development. Cd toxicity can interfere with the uptake and transport of essential nutrients such as iron, calcium, magnesium, and zinc (Zhou et al., 2022). It can bind to transporters, enzymes, and carrier proteins, thereby disrupting nutri- ent homeostasis and causing nutrient deficiencies. Fur- thermore, Cd toxicity negatively impacts photosynthe- sis, reducing the efficiency of light absorption, electron transport, and carbon assimilation (Zulfiqar et al., 2022). Plants have evolved several mechanisms to mitigate the toxic effects of cadmium (Cd) and minimize its ac- cumulation in their tissues. One crucial strategy is the sequestration of cadmium into vacuoles, which serves as a storage site for toxic metals (Jogawat et al., 2021). On the other hand, Cd toxicity leads to the generation of reactive oxygen species (ROS) in plant cells, causing oxi- dative stress (Zhang et al., 2019). Antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxi- dase (APX), and catalase (CAT), scavenge and neutral- ize ROS, protecting cellular components from oxidative damage (Faria et al., 2022). Moreover, Plants possess transporters that can efflux Cd ions from the cytoplasm to the extracellular space or restrict their entry into spe- cific tissues. ATP-binding cassette (ABC) transporters and heavy metal ATPases (HMAs) are involved in Cd transport across cell membranes. These transporters play a crucial role in minimizing the accumulation of Cd in sensitive tissues and facilitating its sequestration (Tian et al., 2023). HMAs belong to the P-type ATPase superfamily and are localized in the plasma membrane or tonoplast (vac- uolar membrane) of plant cells. HMAs play a crucial role in the detoxification of cadmium by actively transporting it out of sensitive cellular compartments or sequester- ing it into vacuoles. This process contributes to reducing the concentration of free cadmium in the cytoplasm and minimizing its toxic effects on plant growth and devel- opment. HMAs function as efflux pumps, actively trans- porting Cd ions out of the cytoplasm and extruding them from the cell or into specific compartments, such as the vacuole (Fang et al., 2016). By pumping Cd out of sensi- tive cellular regions, HMAs reduce the concentration of free cadmium in the cytoplasm and minimize its toxic ef- fects on cellular processes (Satoh-Nagasawa et al., 2012). HMAs participate in the regulation of metal ion homeo- stasis in plants. They are involved in maintaining the bal- ance between essential metals (such as zinc and copper) and non-essential heavy metals (such as Cd) (Fang et al., 2016). This regulation ensures that essential metals are properly acquired and utilized while minimizing the up- take and accumulation of toxic metals like Cd. HMAs in- teract with metal chelators, such as phytochelatins (PCs), which are small peptides that bind to heavy metal ions, including Cd. This process contributes to the detoxifica- tion and sequestration of cadmium in less sensitive cel- lular compartments (Tian et al., 2023). The purpose of the study is to get insights how chickpea plants respond to cadmium, a harmful heavy metal that can contaminate soil and negatively affect plant health .Understanding the mechanisms of Cd tox- icity in plants is crucial for developing strategies to miti- gate its adverse effects. The first objective is therefore to examined the impact of Cd on the growth characteristics, activity of oxidative enzymes, Cd, zinc (Zn), copper (Cu) and manganese (Mn) content in chickpea. The next objective of this study was to gain a better understanding of the role that HMAs play in chickpea, particularly under conditions of cadmium stress and to provide insights into how chickpea plants respond to cadmium by Bioinformatics analyses such as number of genes, proteins, gene loci, cellular location, phylogenetic relationship, three-dimensional protein structure, con- served domains, similar template and catalytic site. 2 MATERIALS AND METHOD 2.1 PROPAGATION AND CADMIUM EXPOSURE Chickpea (Cicer arietinum L.) seeds were germinat- ed in sterilized Cucupite and Perlite in a greenhouse on the photoperiod of 8 h light and 16 h darkness. Seedlings with leaves were planted in pots (diameter, 12 cm and height 15 cm) under controlled conditions and watered Acta agriculturae Slovenica, 119/3 – 2023 3 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... with distilled water every 3 days. Cadmium chloride were added in four concentrations (control, 2, 4, and 8 μg Cd g -1 perlite) calculated per g of perlite. Plants were watered with Hoagland nutrient solution (Hoagland and Snyder 1933) without cadmium chloride (field capacity was considered). After 10 days of Cd treatment plants were harvested for further investigations. 2.2 GROWTH PARAMETERS The fresh and dry mass of the roots and above ground parts were determined (mg). Plantlet height, leaf area, root length, shoot length and internode length were measured. Stomatal densities on the lower and upper epi- dermis were evaluated. 2.3 ENZYME ASSAYS Enzyme extracts were prepared from fresh chickpea leaves (1 g) with phosphate potassium buffer (5 ml). Ho- mogenous samples were prepared by pulverizing followed by centrifugation (4 °C, 25 min, 15000 rpm) and storage at -80 °C. Catalase enzyme activity was determined by mixing phosphate buffer (2.5 ml, pH 7.5) and hydrogen peroxide (1%, 0.1 ml) in an ice bath and the addition of enzyme extract (0.1 ml) and the rate of disappearance of H 2 O 2 is followed by observing the rate of decrease in the absorbance at 240nm via spectrophotometer. Peroxidase enzyme activity was determined based on the method by Koroi (1989). The reaction mixture consisted of acetate buffer (0.2 M, 2 ml, pH 5), benzidine (0.02 M, 100 ml), hydrogen peroxide (3 %, 200 µl) and enzyme extract (25 µl). The absorption was determined at 530 nm. Ascorbate peroxidase (EC11.1.11.1) activity was determined spectrophotometrically (Nakano and Asada, 1987). To the enzyme extract (100 µl) was added K 2 HPO 4 (0.5 M, 2.5 ml), ascorbate (0.5 mM, 0.1 ml), EDTA (0.1 mM, 0.1 ml) and H 2 O 2 (1 %, 0.2 ml) and the absorbance read at 290 nm. Specific enzyme activity was reported as units/g fresh mass (Nakano and Asada 1987). Total soluble protein was determined utilizing the Brad- ford assay with bovine serum albumin (BSA) as standard. The absorbance was read at 595 nm (Bradford, 1976). 2.4 CADMIUM AND OTHER ELEMENTS MEAS- UREMENT Plant samples were oven dried (72 h, 60 °C) and the dry mass determined. Dried samples were ashed (550 °C, 8 h). The digested extract (1N HCl, 1 mL; nitric acid, 97 %, 1 ml, 1 h) was made to a final 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 scientific equipment, Australia) which has a detection limit of 0.007 µg ml -1 . Cd (II), Zn (II), Cu (II) and Mn (II) standard solution were prepared using their nitrate salts in nitric acid. Bioconcentration factor (BCF) computed as heavy metal accumulated in each plant tissue to that dissolved in the soil medium (Bose and Bhattacharyya 2008). Root bioconcentration factor: BCF = root/soil Shoot bioconcentration factor: BCF = shoot/soil TF = BCFshoot/BCFroot 2.5 BIOINFORMATICS ANALYSIS The gene database of NCBI was searched utilizing the keyword „HMA“. Gene characteristics included lo- cation, exon count and conserved domain. Protein se- quences were used for localization prediction from the Localizer and protein tertiary structure predicted by Phyre2. Potential tunnels within each protein and cata- lytic pocket were predicted utilizing CAVER Web. The Jones-Taylor Thornton model was selected to obtain the phylogenies tree of HMAs from chickpea and Arabidop- sis using the neighbor-joining (NJ) method, with a boot- strap test performed using 1000 iterations in MEGA5 (Tamura et al., 2007). Multiple sequence alignments were performed utilizing the muscle algorithm of mega 7 soft- ware to detect conserved residues (Kumar et al., 2016). HMAs from Arabidopsis were highlighted in green. Some information has been mentioned below: XP_004509102.1: Probable cadmium/zinc-trans- porting ATPase HMA1, chloroplastic [Cicer arietinum], P_004487939: Cadmium/zinc-transporting ATPase HMA3-like isoform X1 [Cicer arietinum], XP_027189340: Cadmium/zinc-transporting A TPase HMA2-like isoform X2 [Cicer arietinum], XP_012573401: Putative inactive cadmium/zinc-transporting ATPase HMA3 [Cicer arieti- num], XP_004488108: Cadmium/zinc-transporting AT- Pase HMA3-like [Cicer arietinum], XP_012573132: Cop- per-transporting ATPase HMA4-like [Cicer arietinum], XP_012574029: Copper-transporting ATPase HMA4- like isoform X1 [Cicer arietinum], XP_027192934: Copper-transporting ATPase HMA4-like isoform X2 [Cicer arietinum], XP_004500941: Cation-transporting ATPase HMA5-like [Cicer arietinum], XP_004511583: Probable copper-transporting ATPase HMA5 [Cicer ari- etinum], XP_004504792: Copper-transporting ATPase PAA1, chloroplastic [Cicer arietinum], XP_004504659: Acta agriculturae Slovenica, 119/3 – 2023 4 M. KOLAHI et al. Copper-transporting ATPase RAN1 [Cicer arietinum], XP_004501429: Copper-transporting ATPase PAA2, chloroplastic [Cicer arietinum]. Q9SH30 (Protein: Probable copper-transporting ATPase HMA5, Gene: HMA5, Organism: Arabidopsis thaliana (L.)Heynh., P0CW78 (Protein: Cadmium/zinc- transporting ATPase HMA3, Gene: HMA3, Organism: Arabidopsis thaliana, Q9SZW4 (Protein: Cadmium/ zinc-transporting ATPase HMA2, Gene: HMA2, Organ- ism: Arabidopsis thaliana, Q4L970 (Protein: Copper-ex- porting P-type ATPase, Gene: copA, Organism: Staphy- lococcus haemolyticus Schleifer & Kloos, 1975 (strain JCSC1435), O32220 (Protein: Copper-exporting P-type ATPase, Gene: copA, Organism: Bacillus subtilis (Ehren- berg 1835) Cohn 1872 (strain 168), Q9S7J8 (Protein: Copper-transporting ATPase RAN1, Gene: RAN1, Or- ganism: Arabidopsis thaliana), Q9M3H5 (Protein: Prob- able cadmium/zinc-transporting ATPase HMA1, chloro- plastic, Gene: HMA1, Organism: Arabidopsis thaliana). 2.6 STATISTICAL ANALYSES Data analyses were performed using the SPSS 20 software package (SPSS Inc., Chicago, USA). All experi- mental 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 (homogeneity of variances and data normally distributed). The level of significance was set at p < 0.05 for all tests. 3 RESULTS 3.1 GROWTH CHARACTERISTICS IN THE ABOVEGROUND PARTS OF CHICKPEA SEED- LINGS AFFECTED BY CADMIUM Observed morphological changes in chickpea seed- lings exposed to cadmium included changes in plant length, coloration and leaf size. Results indicated that stem color changed to a bright green-yellow. Moreo- ver, changes were observed in leaf color (yellow) due to cadmium exposure. There was a significant reduction in shoot and root length. Shorter and less dense roots were observed in the treated samples (Table 1). The fresh and dry mass of the shoots and roots in chickpea plants were also significantly affected by cadmium with the lowest seedling mass being observed at high cadmium concen- trations. Plants treated with 2 μg Cd g -1 perlite had a de- cline in leaf area which was less than half that of the con- trol. At cadmium levels of 2 μg Cd g -1 perlite, the length of the first internodes increased, whereas at higher con- centrations, there was a decrease, while the length of the Fig. 1: Effect of cadmium on chickpea (Cicer arietinum L.) growth under normal and various concentrations of cadmium. a Seed- lings, b Aboveground parts, c Roots, d Leaf areas (control, 2, 4 and 8 μg Cd g -1 perlite) Acta agriculturae Slovenica, 119/3 – 2023 5 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... peroxidase enzyme activity showed that this enzyme was also affected by cadmium exposure. The highest ascor - bate activity was observed in cadmium treatments with 4 and 8 μg Cd g -1 perlite (Fig. 2d). Oxidative 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. 3.3 MEASUREMENT OF CADMIUM CONTENT AND ELEMENTAL CHANGES IN THE AERIAL PARTS OF CHICKPEA SEEDLINGS AFFECTED BY CADMIUM The cadmium content in aerial parts of chickpea grown in different concentrations of cadmium chloride increased significantly. The highest concentrations were observed at cadmium chloride concentration of 8 μg Cd g -1 perlite. A doubling of cadmium accumulation was observed in the aerial parts of the plant when the cad- mium content of the medium was increased from 2 to 4 μg Cd g -1 perlite (Fig. 3a). Moreover, elemental compo- sition was significantly affected by cadmium levels (Fig. 3). Chickpea cultivated in cadmium-containing media showed a significant difference in the amount of manga- nese present in the aerial part of the plant. With the addition of cadmium, manganese uptake increased significantly by approximately three times, second internodes showed only a significant reduction at high concentrations of cadmium (Fig. 1, Table 1). Fur- thermore, with the addition of cadmium (4 μg Cd g -1 per- lite), stomatal densities on the lower epidermis increased significantly but subsequently declined while higher con- centrations of cadmium (Table1). 3.2 EFFECT OF CADMIUM ON SOD, POD AND CAT ACTIVITIES IN THE AERIAL PARTS OF CHICKPEA SEEDLINGS Cadmium stress resulted in a significant increase in POD enzyme activity. The highest ascorbate activity was observed in cadmium treatments at 4 and 8 μg Cd g -1 perlite. Further increase in cadmium exposure result- ed in a decline in POD activity which was however still significantly higher than that of the control and plantlets treated with 4 μg Cd g -1 perlite. The lowest enzyme activ- ity was observed in the controls (Fig. 2a). SOD enzyme activity significantly increased in chickpea with the high- est enzyme activity being observed in plantlets treated with 4 μg Cd g -1 perlite with the lowest enzyme activity being observed in the control (Fig. 2b). There was a sig- nificant increase in catalase enzyme activity. The high- est catalase activity was also observed in plants treated with 4 μg Cd g -1 perlite with a subsequent decline when cadmium chloride concentration was increased to 8 μg Cd g -1 perlite. The lowest level of enzyme activity was ob- served in the control (Fig. 2c). Investigation of ascorbate Table 1: Effect of Cd (Control, 2, 4 and 8 μg Cd g -1 perlite) on morphometric features in chickpea (Cicer arietinum L.) Values with different letters are significantly different at p < 0.05 Parameters Control 2 μg Cd g -1 perlite 4 μg Cd g -1 perlite 8 μg Cd g -1 perlite Plant length (cm) 62.76 ± 1.36 a 58 ± 0.0709 a 42.56 ± 1.78 b 37.93 ± 1.78 b Shoot length (cm) 29.33 ± 0.66 a 25.65 ± 0.779 b 22.55 ± 1.35 bc 21.16 ± 1.092 c Root length (cm) 35 ± 0.57 a 30.86 ± 0.69 b 18.56 ± 0.92 c 16.6 ± 0.83 c Plant fresh mass (g) 4.0367 ± 0.043 a 3.442 ± 0.238 b 3.084 ± 0.169 b 1.715 ± 0.042 c Shoot fresh mass (g) 2.291 ± 0.11 a 1.6317 ± 0.14 b 1.297 ± 0.061 c 0.682 ± 0.014 d Root fresh mass (g) 2.24 ± 0.078 a 1.9 ± 0.1 b 1.3167 ± 0.109 c 0.99 ± 0.003 d Shoot dry mass (g) 1.987 ± 0.01 a 1.4783 ± 0.11 b 1.0447 ± 0.029 c 0.606 ± 0.002 d Root dry mass (g) 2.01 ± 0.04 a 1.696 ± 0.063 b 1.123 ± 0.069 c 0.823 ± 0.062 d Leaf area (mm 2 ) 103.33 ± 1.76 a 48.33 ± 2.18 b 20.66 ± 0.666 c 18.33 ± 1.201 c First internode length (cm) 1.1 ± 0.264 b 1.766 ± 0.0577 a 1.3 ± 0.3 b 0.833 ± 0.838 c Second internode length (cm) 2.433 ± 0.513 a 2.266 ± 0.503 a 1.766 ± 0.808 b 1.633 ± 0.850 b Stomatal densities on the upper epidermis 35 ± 0.545 ab 31 ± 0.564 b 24.33 ± 0.413 c 39.33 ± 0.633 a Stomatal densities on the lower epidermis 29.6667 ± 0.448 c 39 ± 0.653 b 46 ± 0.765 a 37.33 ± 0.985 b Acta agriculturae Slovenica, 119/3 – 2023 6 M. KOLAHI et al. while higher concentrations of cadmium reduced the amount of manganese in chickpea plants (Fig. 3b). In- crease in the levels of cadmium in the culture also caused changes in the amount of zinc present in the aerial parts of pea plants. Increasing the levels of cadmium in the medium resulted in a decline in zinc (Fig. 3c). Increasing cadmium concentration, also decreased the levels of cop- per present in the aerial parts of chickpea seedlings. The lowest amount of copper was observed in high-cadmium seedlings (Fig. 3d). The BCF and TF values is greater than one at 8 μg Cd g -1 perlite (Fig. 3 e,f). 3.4 BIOINFORMATICS In the current bioinformatics study of chickpea un- der cadmium stress, HMA proteins were chosen. In silico analysis of chickpea HMAs showed that of the 13 HMA identified, there were three proteins for each HMA3 and HMA4, two proteins for HMA5 and one protein for HMA 2, 6, 7, 8 (Table 2). The ATPase PAA2, chloro- plastic, copper-transporting ATPase RAN1, and copper- transporting ATPase PAA1, chloroplastic identified in chickpea were identified as HMA6, HMA7, HMA8, in Arabidopsis, respectively. HMA7 and HMA8 all contrib- ute to copper transport. The 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 confidence level of predicting the three-dimensional structure of chickpea HMAs proteins is shown in Table 3. Their cel- lular locations are often in the nucleus and chloroplast. Using phyre2, their three-dimensional structure was de- termined. 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. pneu- mophila Brenner DJ, Steigerwalt AG, McDade JE 1979. The patterns of c3j08A and c3j09A are also related to the p-type ATPase copper transporter CopA. Five (5) tem- plates including copper-transporting proteins ATPase ATP7A, apoWLN5-6, domains 3 and 4 of human ATP7B, apo HMA domain of copper chaperone for superoxide dismutase and C2H2 type zinc finger (region 641-673) of human zinc finger protein 473 belong to humans. In to- tal, the HMA studied in chickpeas were found to contain nine domains which are common in the 13 HMAs. The COG4087 domain is listed as Soluble P-type ATPase and pfam00122 as E1-E2_ATPase are present Fig. 2: The activities of a Peroxidase (POD), b Superoxide dismutase (SOD), c Catalase (CAT) and d Ascorbate peroxidase en- zymes in aboveground parts of chickpea (Cicer arietinum L.). Values with different letters are statistically significantly different at p < 0.05 (One-way ANOV A, post hoc Tukey test) Acta agriculturae Slovenica, 119/3 – 2023 7 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... in ten HMAs. Following the prediction of the three- dimensional structure for chickpea HMAs, the longest tunnels for each protein and catalytic pocket predicted by CAVER Web for ion passing was determined. The longest and shortest tunnels predicted belonged to cad- mium/zinc-transporting ATPase HMA3-like and cation- transporting ATPase HMA5-like, respectively. The puta- tive inactive cadmium/zinc-transporting ATPase HMA3 was the largest HMA with 1032 amino acids and 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 the passage of ions rep- resented in color is illustrated in Fig 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 inter- action with ions was determined. For XP_027192934, three catalytic sites with Asp residues at positions 522, 729, 733 with 40 % similarity over a specific reference of active site type and metal ion-binding site were identi- fied. 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 Fig. 3: Effects of different cadmium treatments on a Accumulation of cadmium, b Manganese, c Zinc, d Copper content in the aerial parts of chickpea seedlings after 10 days of cadmium treatment. Values with different letters are statistically significantly different at p < 0.05. (One-way ANOV A, post hoc Tukey test). Bioconcentration factor (e) and translocation factor (f). BCF values > 1 indicate that the concentration in the organism is greater than that of the medium. Translocation factor (TF) values more than one can be considered potential as Cd accumulators for phytoremediation. Mean plant tissues BCF are averages of five BCF values (n = 5) ± SEM Acta agriculturae Slovenica, 119/3 – 2023 8 M. KOLAHI et al. Table 2: An overview of the features of chickpea HMAs proteins structure, genes loci, conserved Protein Domain Family, cellular location, Phyre2 confidence (residues modelled at > 90 % confidence), templates used for 3D prediction and longest tunnel pre- dicted by the Caver Web for transport ions Protein Length Gene Exon count Conserved domain Location Template pattern Longest tunnel XP_004509102.1 839 101490857 Chromosome: Ca7 13 COG4087 TIGR01512 pfam00122 Chloroplast c3rfuC,c1mhsA,c3j08A,c5 mrwF,c4umwA,c3j09A 70.1 XP_004487939 834 101492022 Chromosome: Ca1 9 COG2608 COG4087 pfam00122 Nucleus c3rfuC, c3j08A, c4umwA, c3j09A 85.5 XP_027189340 569 101492022 chromosome: Ca1 9 cl21460 COG2608 - c4umwA, c3rfuC, c3j08A, c3j09A 29 XP_012573401 1032 101505376 Chromosome: Ca7 11 COG2608 TIGR01512 pfam00122 Nucleus c3rfuC,c2emcA, c3j08A,c4umwA,c3j09A 41.7 XP_004488108 832 101497233 Chromosome: Ca1 9 COG2608 COG4087 pfam00122 Nucleus c3rfuC, c3j08A, c4umwA, c3j09A 107.3 XP_012573132 853 101504726 Chromosome: Ca6 7 COG2217 cd00371 pfam00122 - c3rfuC, c4u9rA, c3j08A, c3j09A 43.8 XP_012574029 958 101515614 Chromosome: Ca7 10 COG2217 COG2608 COG4087 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c3j09A 72 XP_027192934 849 101515614 Chromosome: Ca7 10 cd02094 cd00371 cl00207 Nucleus c3rfuC, c4u9rA, c3j08A, c3j09A 95.6 XP_004500941 130 101507723 Chromosome: Ca5 3 pfam00122 - c3rfuC, c3j08A, c3j09A, c2kijA, c2hc8A 11.3 XP_004511583 998 101498342 Chromosome: Ca8 7 COG2217 COG4087 cd00371 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c3j09A 94.3 XP_004504792 934 101496348 Chromosome: Ca6 17 COG2217 COG4087 cd00371 pfam00122 Chloroplast c3rfuC, c4u9rA, c3j08A, c3j09A - XP_004504659 995 101509532 Chromosome: Ca6 10 COG2217 COG4087 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c2crlA, c3j09A 95.9 XP_004501429 884 101500347 Chromosome: Ca5 18 COG2217 COG4087 cd00371 Nucleus, Chloroplast c3rfuC, c3j08A, c3j09A - Acta agriculturae Slovenica, 119/3 – 2023 9 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... 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 (Fig. 5), com- parison of the protein sequences of chickpea HMA with Arabidopsis revealed great similarity between these pro- teins in chickpea and Arabidopsis. HMA 2 and 4 are very similar to Arabidopsis and are next to HMA 3 chickpeas. HMA 3 chickpea is adjacent to HMA 3 Arabidopsis. HMA 1 2 3 chickpea are all 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 simi- lar to the copper-transporting ATPase PAA2 chickpeas. Copper-transporting ATPase PAA1 pea is very similar to Arabidopsis P-type ATPases. In chickpea, copper-trans- porting ATPase RAN1 resembles copper-transporting ATPase HMA5, which is adjacent to copper-transporting ATPase RAN1 Arabidopsis. Cation-transporting ATPase HMA5-like and copper-transporting ATPase RAN1 are also in the vicinity of copper-transporting ATPase RAN1 Arabidopsis. 4 DISCUSSION Heavy metal pollution is a significant environmen- tal problem. Increasing our knowledge of the mecha- nisms by which plants are able to mitigate heavy metal stress could assist in creating new tools applicable to Fig. 4: An overview of the 3D model of chickpea HMAs generated by Phyre2 software. The structures were predicted using coordinate templates represented in Table 2. Colored regions in 3D structure represent the longest tunnel. a XP_004509102.1, b XP_004487939, c XP_027189340, d XP_012573401, e XP_004488108, f XP_012573132, g XP_012574029, h XP_027192934, i XP_004500941, j XP_004511583, k XP_004504792, l XP_004504659, and m XP_004501429 Acta agriculturae Slovenica, 119/3 – 2023 10 M. KOLAHI et al. Table 3: Index, residue, accession code of the reference entry, sequence identity to the reference entry, type, description, neighbor- hood and pocket score features of chickpea HMAs proteins structure Protein accession number Index Residue Accession code of the reference entry Sequence identity Type Description Neighbor- hood Pocket score XP_004511583 656 Asp Q9SH30 73.8 % active site 4-aspartylphosphate inter- mediate VFDKT VFDKT 100 % 860 Asp Q9SH30 73.8 % metal ion- binding Magnesium VGDGI VGDGI 864 Asp Q9SH30 73.8 % metal ion- binding Magnesium INDSP INDSP XP_004488108 591 Asp P0CW78 50.2 % metal ion- binding Magnesium VGDGI VGDG 33 % XP_012573401 590 Asp Q9SZW4 54.6 % metal ion- binding Magnesium LGDGL VGDGL 28 % XP_012574029 838 Asp Q9SH30 56.4 % metal ion- binding Magnesium VGDGI VGDGI 100 % XP_012573132 522 Asp Q4L970 41.3 % active site 4-aspartylphosphate inter- mediate VFDKT VFDKT 6 % 730 Asp Q4L970 41.3 % metal ion- binding Magnesium VGDGI VGDGI XP_027192934 522 Asp O32220 41.6 % active site 4-aspartylphosphate inter- mediate VFDKT VLDKT 68 % 729 Asp O32220 41.6 % metal ion- binding Magnesium VGDGI VGDGI 733 Asp O32220 41.6 % metal ion- binding Magnesium INDSP INDAP XP_004487939 392 Asp P0CW78 49.8 % active site 4-aspartylphosphate inter- mediate AFDKT AFDKT 13 % 591 Asp P0CW78 49.8 % metal ion- binding Magnesium IGDGI VGDGL XP_004504659 649 Asp Q9S7J8 73.4 % active site 4-aspartylphosphate inter- mediate IFDKT IFDKT 100 % 138 Cys Q9S7J8 73.4 % metal ion- binding Copper AACVN AACVN 869 Asp Q9S7J8 73.4 % metal ion- binding Magnesium VGDGI VGDGI 873 Asp Q9S7J8 73.4 % metal ion- binding Magnesium INDSP INDSP XP_004509102 467 Asp Q9M3H5 68.2 % active site 4-aspartylphosphate inter- mediate AFDKT AFDKT 25 % 701 Asp Q9M3H5 68.2 % metal ion- binding Magnesium INDAP INDAP Acta agriculturae Slovenica, 119/3 – 2023 11 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... phytoremediation. It is important to further research processes involved in heavy metal detoxification and signaling pathways in plants so as to identify useful tar- gets for biotechnological applications thereby increasing plant fitness in heavy metal polluted sites (Dala-Paula et al., 2018). Cadmium exposure reduced leaf area, shoot and root length. The effect of cadmium ion suppression on root expansion extends through its effect on cell growth (Hassan et al., 2008). Cadmium attaches to the cell wall and the middle lamella, increasing the bonding between the wall components, ultimately leading to growth in- hibition and a decline in cell and organ development. Cadmium also alters water proportions in plants caus- ing physiological dryness, which leads to metabolic dys- function and production of ROS. These factors reduce growth and impact on plant length and mass (Zulfiqar et al. 2022). Many studies on the mechanism of cadmium blockage on cell growth have shown degradation of cell membranes by cadmium and changes in the degree of cell exchange and cellular depletion (Bücker-Neto et al., 2017). The observed changes in plants exposed to cadmi- um may be as a result of multiple nutritional deficiencies being experienced by the plant. Nutrients serve an essential role in the formation, expansion, and operation of chloroplasts. Cd-phytotox- icity affects the synthesis and extensibility of cell walls (Gomes et al., 2011). Cell wall thickening in root endo- dermal tissue affords a greater surface area over which cadmium accumulation can occur thereby limiting its transportation to the shoot (Zulfiqar et al., 2022). Chlo- rosis observed in the leaves of bean plants exposed to Fig. 5: Phylogenic tree of HMAs from chickpea and Arabidopsis. A phylogenetic tree was constructed using the neighbor-joining (NJ) method, with a bootstrap test performed using 1000 iterations in MEGA5 with the amino acid sequences of HMAs. HMAs from Arabidopsis are highlighted in green 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 leaves are due to the associated toxic effects of cadmium includ- ing mesophyll curvature, decreased leaf thickness and a reduction in the composition of intercellular spaces of spongy parenchyma (Tuver et al., 2022). 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 al- ters leaf expansion (Cregeen et al., 2015). A study of the effect of heavy metals on the cell death of Halophila stipulacea (Forssk.) Asch leaves showed that high concentrations of metal causes necrosis of the epi- dermal cells and mesophyll, inhibiting surface growth of the leaves. High levels of heavy metal accumulation in plant cells inhibits the process of respiration and energy reactions, which are associated with cell growth (Ayang- benro, 2017). A decline in cell division and growth could also be a contributing factor to the observed morpholog- ical changes. Additionally, a decrease in photosynthetic rates has been observed in plants exposed to elevated lev- els of heavy metals. Higher concentrations of cadmium commonly result in root injury, damage to photosynthet- ic machinery, inhibition of plant growth, reduced nutri- ent and water uptake (Tuver et al., 2022). Cadmium may exert its inhibitory effect in different ways, namely bind- ing specific groups of proteins and lipids thereby inhibit- ing normal function and possibly inducing free radical formation due to oxidative stress. The former may occur at transport and channel proteins of cell membranes dis- turbing the uptake of many other macro- and microele- Acta agriculturae Slovenica, 119/3 – 2023 12 M. KOLAHI et al. ments whereas the latter is due to the inactivation of anti- oxidant enzymes by cadmium (Long et al., 2017). The results showed that oxidative enzymes activ- ity (SOD, APX, POD and CAT) increased in the leaves of chickpea exposed to cadmium. Similar observations have been observed in CAT and POD enzymes present in cereals and squash (Ashraf, 2003). Increased activ- ity of these enzymes is a consequence of lipid peroxida- tion. The effect of cadmium on growth and antioxidant enzymes in two varieties of Brassica napus showed that cadmium decreased the growth indices, nitrate reduc- tase activity and leaf water potential while antioxidant enzyme activities increased. The highest level of enzyme activity was in relation to SOD enzymes, which showed more than 80 % increase in activity. The least increase in enzyme activity was observed in the catalase enzyme (Irfan et al., 2014). Increasing the absorption and accu- mulation of heavy metals in plants causes changes in cell metabolism, oxidative stress and cell destruction which is induced by ROS. Cadmium can induce mineral stress that reduces plant dry mass (Zhou et al., 2022). Tabarzad et al. (2017) showed that wheat seedlings grown in the presence of cadmium had changes in the level of SOD and POD activity. The observed decline in enzyme activ- ity suggests a weakening of the oxygen and superoxide water scavenging system. Reduced activity of the other antioxidant enzymes in some tissues, is due to poor performance in oxygenate decomposition in cadmium treated tissues. ROS activity increased significantly un- der cadmium stress due to an increase in wall oxidation. Reduced SOD activity is justifiable as cadmium is known to be an enzyme inhibitor (Tabarzad et al., 2017). Schutzendubel (2001) showed the inhibition of SOD, POD and total inactivation of APX in pine roots after 48 days of cadmium treatment. An increase in the activity of these enzymes under cadmium stress has been observed in other studies (Schutzendubel et al., 2001). Li et al. (2013) examined the effect of cadmium stress on growth and antioxidant enzymes and lipid oxidation in two Kenaf (Hibiscus cannabinus L.) species. In the study, glutathione reductase activity (GR) was greater than that of the control. The general trend was that of an increase in SOD, CAT and POD activities in the roots of cadmi- um-stressed plants followed by a decline. POD activity however remained relatively unchanged at all stress lev- els (Zhou et al., 2022). Ulusu et al. (2017) investigated the antioxidant capacity and cadmium accumulation of stressed parsley. In the study, enzyme activity increased for catalase and ascorbate peroxidase, (75 to 150 μM cad- mium), while decreasing at 300 μM. The results showed that antioxidant enzymes activity was suppressed due to the accumulation of cadmium in parsley leaves and in- creased non-enzymatic antioxidant activity (Ulusu et al., 2017). Pereira et al. (2002) studied the activity of anti- oxidant enzymes in Crotalaria juncea L. which showed that under the influence of cadmium, catalase activ- ity did not show any significant changes in the root. At concentrations of 2 mM cadmium, catalase activity in the leaves increased 6 fold compared to the control. In- creased activity of some antioxidant enzymes exposed to metals reveal the crucial role that these enzymes play in detoxification (Pereira et al. 2002). Various antioxidant cycles under normal physiological metabolism, results in the production and scavenging of reactive oxygen spe- cies which is in a state of dynamic equilibrium (Zhou et al., 2022). Kisa (2018) studied the response of anti- oxidant systems to stress induced by heavy metals in the leaves and roots of tomato which showed that cadmium treatment significantly increased the activity of the APX and SOD enzymes. Antioxidant scavenging systems are connected with ROS detoxification which is a defense mechanism employed by plant tissue to combat oxidative stress (Kisa, 2018). Tomato plants exposed to cadmium showed significantly higher SOD. Catalase activity was however reduced. The cadmium content in aerial parts of chickpea grown in different concentrations of cadmium increased significantly. Research conducted by Tang et al. (2022) revealed that cadmium concentrations in the seeds of beans from different regions and varieties is based on complex genetic factors and the environment. For dif- ferent legume varieties, environmental factors such as climate, soil, agricultural and geological techniques, in comparison to genetic factors, are more important in the accumulation of heavy metals such as Cd. Compared to the genus and plant species, the accumulation of heavy metals seems to be more influenced by the genetic poten- tial of the plant (Tang et al. 2022). The ability to absorb and distribute cadmium to the aerial regions of the plant is related to its attachment to the extracellular matrix, root flow, intracellular detoxification and transfer effi- ciency (Akhtar and Macfie, 2012). Cadmium is absorbed in the root of the plants subsequently accumulating in the aerial parts, which often limits the absorption and distri- bution of other elements (Gomes et al., 2013). Cadmium binds to the functional epidermis through direct bind- ing to ion carriers via production of oxygen species that are associated with membrane affects (Altaf et al., 2022). Ling Liu et al. (2012) showed that legumes can increase the accumulation of cadmium in adjacent plants. Cad- mium increase in plants was a direct result of planting crops in proximity to legumes. The study suggests that the system of cultivation of beans should be redesigned to prevent food contamination with cadmium (Liu et al., 2012). Vijendra et al. (2016) showed that in Moth bean (Vigna aconitifolia L.) cadmium concentrations in- Acta agriculturae Slovenica, 119/3 – 2023 13 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... creased significantly in the leaves and roots. Cadmium reaches the aerial sections via the xylem of the plant (Vi- jendra et al., 2016). 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 accu- mulation of heavy metals in plants. Cadmium in plants is transferable through apoplast pathways of the stems and leaves (Benavides et al., 2005). Cadmium affects mem- brane potential, protein pump activity and can limit corn growth (Karcz & Kurtyka, 2007). The result indicated that increasing cadmium con- centration, also decreased the levels of copper and zinc present in the aerial parts of chickpea seedlings. Further studies also showed that zinc and copper along with cad- mium have an antagonistic effect and that these minerals act in a competitive manner in relation to the transfer processes. Heavy metals, such as copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe), serve as essential mi- cronutrients for an array of metabolic processes. These micronutrients serve as cofactors, participate in cellular redox reaction and affects protein structure (Schutzen- dubel & Polle, 2002). At toxic levels Cu will however interfere with physiological processes. Zn also serves as a micronutrient but can be toxic if present at high con- centrations (Schutzendubel & Polle, 2002). To minimize the potential effects of excess metal contaminants, the plant utilizes various homeostasis mechanisms which include the use of specialized transport proteins which serve as carriers mediating the transfer of heavy met- als across cell membranes (Lee et al., 2007). Cadmium has a negative effect on the absorption of essential nu- trients. It reduces ATPase activity and decreases the ex- change of ion H + /K + in the plasmalema surface (Brzoska & Moniuszko-Jakoniuk, 2001). Page and Feller (2005) showed that the transfer of zinc, manganese, cobalt and cadmium in the leaves and roots of wheat were selective. When other minerals are in close proximity to cadmium, the amount of zinc in the root decreases (Page and Feller, 2005). Santos et al. (2014) showed that in the family of legumes, lead and cadmium adsorption was competitive. In this study, the concentration of zinc was eight times higher than that of cadmium, which indicates that zinc adsorption is preferable to cadmium. In plants treated with zinc and lead, lower concentrations of cadmium were observed in plant tissues in comparison to plants treated with cadmium alone. Zinc and lead along with cadmium compete for the sites of absorption and transfer (dos Santos et al., 2014). Chen et al. (2007) showed that manganese reduces the toxic effects of cadmium in corn. This suggests that manganese can be utilized to manage cadmium contamination (Chen et al., 2007). Zinc acts as a micro-element that is essential for plant growth and is part of the structure of regulatory enzymes and proteins. Zinc is very important in reducing cadmium toxicity and decreases the oxidative stress induced by cadmium. Some studies describe zinc and phosphorus interactions in plants (Marques et al., 2013). The phosphorus content in the aerial parts of plants treated with cadmium is relat- ed to the low zinc content in these sections. The negative correlation between zinc and phosphorus content in the shoots of cadmium treated plants explains the high con- tent of phosphorus in these plants (Sarwar et al., 2010). Analysis of cadmium and manganese content in this study supports the competitive theory of absorption of these two elements. The precise mechanism for promot- ing growth and reducing the toxic effects of cadmium is not well known. The uptake of various cations (K + , Ca 2+ , Mg2 + , Mn 2+ , Zn 2+ , and Fe 2+ ) is severely affected by the presence of cadmium (Linger et al., 2005). Different types of proteins and adsorption carriers for cadmium are known such as NRAMP family (Thom- ine et al. 2000), P-type ATPase (Morel et al., 2009), ABC transporter (Kim, Gustin et al., 2004), CAX family, ZIP family (Pence et al., 2000), LCT transporter and CE fam- ily (Guerinot, 2000). Researchers report that cadmium has an antagonistic and synergistic effect on the micro- elements and macro elements in wheat. Many studies on the effect of cadmium inhibition on cell growth suggests the destruction of cell membranes and changes in min- eral levels (Rietra et al., 2017). Jibril et al. (2017), showed that the content of micronutrients and macro elements in different varieties of lettuce is significantly affected by cadmium levels. The study showed that cadmium (12 mg l -1 ) reduced essential elements by 72, 69, 56, 61 and 52 % (nitrogen, phosphorus, potassium and calcium, respec- tively). Copper content was higher in the root than the shoot of cadmium treated plants. This therefore reduces the effect of cadmium toxicity. Indeed, cadmium increas- es the absorption of copper, but prevents it from transfer- ring to the shoots (Jibril et al., 2017). Gomez et al. (2013) examined the effect of cadmium on nutrient distribution in Pfaffia glomerata (Spreng.) Pedersen. Plants were cul- tured with different minerals and cadmium concentra- tion was simultaneously increased over a 20 day period. The study showed that cadmium strongly affects the dis- tribution of microelements and macroelements in the roots and shoots. Despite the high toxicity of cadmium, the micro and macro nutrients present in plants are able to survive in contaminated environments (Gomes et al., 2013). Present study detected that at low concentrations of cadmium, the amount of manganese increased. With an increase in cadmium concentration, the level of manganese decreased in chickpea. Manganese plays a role in many biochemical functions, such as activating Acta agriculturae Slovenica, 119/3 – 2023 14 M. KOLAHI et al. enzymes involved in respiration, redox reactions, intra- cellular electron transfer systems, and the Hill reaction in chloroplasts, amino acid synthesis, and regulation of hormones (He et al., 2022). Manganese concentration was higher in the shoots than the root of plants treated with cadmium. The 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 & Guerinot, 2014). Dias et al. (2013) showed that at cadmium concentrations of 5 and 10 μm there was a significant decline in the min- eral content of lettuce leaves. At high concentrations of cadmium, a significant decline in manganese in the roots was observed. Cadmium appears to interfere with the transmission of macro and micro elements in the leaf (Dias 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 ele- ments 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) explained that the decrease in concentrations of Mn, Fe, Mg, S, and P in the leaves of Cd-sensitive cultivars under cadmium stress is a con- tributing factor to the decline in photosynthesis and the decrease of cabbage growth (Sun & Shen, 2007). Heavy metal ATPases (HMAs), belong to the large P-type ATPase family located in the plasma membrane or tonoplast. They play an important role in the transport of metals in plants and provide resistance to the uptake and transportation of metals. The identified HMAs may contribute to the mechanisms by which chickpea plants manage, detoxify, or tolerate cadmium exposure. Under- standing the structure, function, and localization of these HMAs could offer new strategies for enhancing cadmium tolerance in chickpea, a crucial crop in many parts of the world. HMAs are classified based on substrate binding with one group bound to copper and silver and the oth- er to cadmium, lead and cobalt (Chkadua et al., 2022). HMAs 9 and 8 have been studied in rice and Arabidopsis, respectively. AtHMA1–4 in A. thaliana and OsHMA1–3 in Oryza sativa L. are in the first group and AtHMA5–8 and OsHMA4–9 in the second group. The expression of each of these genes is sensitive to heavy metals as indi- cated by mutagenesis. Typical P1B-ATPase proteins have been studied in various barley plants, Arabidopsis and poplar as well as in Thlaspi caerulescens J.Presl & C.Presl (Takahashi et al. 2012). In poplar (Populus trichocarpa Torr. & A.Gray ex. Hook), seventeen HMAs are known. PtHMA1 – PtH- MA4 belong to the subgroup of metals on cadmium, lead and cobalt. PtHMA5 – PtHMA8 belonging to the silver and copper groups have been identified. Most of these genes are located on chromosome 1 and 2 of poplar. On both sides of the P 1B -ATPase C and N terminals there is also a metal binding site HMA4 in poplar which pro- duces mature RNA transcripts during alternative splic- ing of mRNA, containing approximately six hundred and twenty-six amino acids with an amino acid aver- age of ninety-eight. PtHMA in poplar are all in plasma membrane except PtHMA1 and PtHMA5.1 which are located in the cytoplasm. Poplar HMAs have 5 to 16 in- trons, PtHMA6, 5 introns, 8 PtHMA has 16 introns and 1 PtHMA has 5 introns with the remaining possessing 10 introns (Li et al., 2015). PtHMA1 – PtHMA4 belong to the subgroup of metals consisting of cobalt and cad- mium with the rest belonging to lead, silver and copper. There are 10 HMA genes related to silver and copper in poplar that are significantly higher than those in rice and Arabidopsis.2 OsHMA plays an important role in trans- mitting cadmium entry from the root to the stem and especially to rice grains (Li et al., 2015). OsHMA3 trans- ports 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 AtH- MA 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 ex- isting motifs (Tian et al. 2023). AtHMA 1 and 2 are in the plasma membrane and in zinc and cadmium fluxes. OsHMA 1 is involved in zinc transfer. No HMA 4 type has been reported in rice. The number of HMA genes in the soybean genome is higher than that in Arabidop- sis and rice, probably due to duplication of the soybean genome. Phylogenetic study of these genes divides them into six groups, based on their divergent gene structure, conserved segments or protein motif patterns. Examina- tion 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 (Fang et al., 2016). Researchers have identified nine typi- cal P 1B -ATPase in barley. HvHMA2, a P (1B)-ATPase is highly conserved among cereal crops with functionality in the transportation of zinc and cadmium. Addition- ally, HMA4 (Heavy Metal ATPase 4) has a key role in the translocation of cadmium in non-hyperaccumulating dicots, such as Arabidopsis thaliana (Mills et al., 2012). Acta agriculturae Slovenica, 119/3 – 2023 15 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... 5 CONCLUSION Chickpea seedlings exposed to cadmium exhibited changes in their morphological features which included changes in plant length, coloration and leaf size. The re- sults indicated that shoot and root length were signifi- cantly reduced. With the addition of cadmium (4 μg Cd g -1 perlite), stomatal densities on the upper epidermis decreased significantly but subsequently increased while higher concentrations of cadmium. Oxidative enzyme activities were also affected by cadmium stress. Oxida- tive enzyme activity (peroxidase, superoxide dismutase, catalase, ascorbate peroxidase) increased in the leaves of plants exposed to cadmium suggesting that these en- zymes play an integral role in combatting heavy metal contamination. Cadmium content in aerial parts of chickpea increased significantly. The study also revealed that by increasing cadmium concentration there was a significant reduction in the amount of copper and zinc transported to the aerial regions of the plant. Moreo- ver, at low concentrations of cadmium, the amount of manganese increased It has been suggested that there is a competitive mechanism for mineral uptake in plants. One may therefore be able to manage cadmium accumu- lation by varying the type of fertilizers utilized in culti- vating plants. In silico analysis led to the identification of 13 Heavy Metal ATPases (HMAs) in chickpea. These proteins contain 130 to 1032 amino acids with 3 to 18 exons. Comparison of the protein sequences of chickpea HMA with Arabidopsis indicated that there was great similarity between these proteins. The presence of a va- riety of genes indicates the various mechanisms utilized by chickpeas to combat heavy metal stress. 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