Assessment of zinc toxicity and tolerance in chickpea (Cicer arietinum L.) cultivars using physiological, biochemical and metabolomic responses

Assessment of zinc toxicity and tolerance in chickpea (Cicer arietinum L.) cultivars using physiological, biochemical and metabolomic responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of zinc toxicity and tolerance in chickpea (Cicer arietinum L.) cultivars using physiological, biochemical and metabolomic responses shakir ullah, Xingfan Li, Uzma Salam, Ahmed A. Elateeq, Ilbong Ri, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5188964/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Aims : Zinc (Zn) is an essential microelement that plants need for appropriate growth and development. However, high concentrations may hamper the physio-chemical and metabolic processes and weaken plant growth. This study aims to broadly explore the relative tolerance of chickpea ( Cicer arietinum L.) cultivars, and examine their physiological, biochemical, and metabolomics responses under various Zn levels. Methods : Three chickpea cultivars: ICCV89310 (IC8), NC234 (NC2), and ICCV89323-B (IC8-B) were exposed to different Zn levels (Ck, 50, 100, and 150 µM) for one week in a hydroponic medium. Growth and physiological indices, oxidative stress markers, antioxidant enzymes activity, and osmolytes content were detected. Primary metabolites profile and accumulation of Zn were assessed using GC-MS and ICP-OES, respectively. Results : IC8 and NC2 cultivars exhibited more tolerance than IC8-B because of their high biomass and plant height, root-to-shoot ratio, shoot water, and chlorophyll contents under high Zn stress. Besides, Zn contents were higher in the root of IC8-B, while IC8 and NC2 showed high accumulation in the shoot. Under Zn stress, there was an increase in the concentrations of hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), and electrolyte leakage (EL). Additionally, Zn supplementation positively regulated the activities of superoxide dismutase (SOD), peroxidase (POD), and osmolytes (proline, soluble sugars, and total protein), but catalase (CAT) and glutathione reductases (GR) were differential in response to Zn stress. Simultaneously, metabolomics profiling revealed forty-six responsive metabolites in IC8, NC2, and IC8-B, mainly consisting of organic acids, amino acids, amines, alcohols, and sugars. Conclusion : Cultivars IC8 and NC2 displayed superior tolerance to Zn stress compared to IC8-B, showcasing robust growth characteristics and biochemical responses. The relative tolerance potential of IC8 and NC2 may be attributed to different adaptive strategies, such as a well-developed profile of responsive metabolites, such as histidine, asparagine, tryptophan, allantoin and antioxidants. Hence, cultivar IC8-B maybe utilized as a control cultivar under Zn stress to evaluate other chickpeas' tolerance capacity. Besides, IC8 and NC2 can be suggested as promising candidates for Zn-contaminated soil. Cicer arietinum zinc toxicity antioxidants oxidative stress metabolites. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Worldwide, heavy metals (HM) seriously threaten agriculture security and has become an environmental problem (Aithani and Kushawaha 2024 ). Zinc (Zn) is a critical contaminant of the terrestrial and aquatic environment released from anthropogenic and natural sources (Khudsar et al. 2004 ). Although, Zn is an essential trace element for all living organisms, however, at elevated levels (> 50 mg/L), it inhibits the normal ionic homeostasis system, provokes reactive oxygen species (ROS) production, and increases the level of membrane lipid peroxidation (Andrade et al. 2009 ). Additionally, Zn toxicity causes chlorosis in young leaves, hindering photosynthesis and adversely affecting the nutritional status and Calvin cycle (Fariduddin et al. 2022 ). Hence, the capacity of plants to effectively regulate the uptake, transportation, and scattering of Zn within their cells, organs, and intracellular compartments is essential for preserving their vital functions (Zlobin 2021 ). To counteract the toxic effects of HM, plants developed several adoptive strategies, including changing the cells' biochemical, physiological, and molecular status (Chen et al. 2024 ). Besides, plants overcome the toxicity of HM by compartmentalization, vacuolization, excluding, and accumulation or hyperaccumulation (Giannakoula et al. 2021 ). Furthermore, plants have a well-developed system of enzymatic and non-enzymatic antioxidants to detoxify HM stress (Al Mahmud et al. 2017 ). Initially, plant cell walls play an important role as they contain specific polysaccharides that chelate or bind with HM metal ions and protect their entry into the cell (Yu et al. 2024 ). It has been shown that glutathione and phytochelatins enhanced the resistance of non-hyperaccumulators plants to cadmium (Cd) and Zn stress (Jin et al. 2022 ). After getting into the cell, a high amount of Zn is also chelated by organic acids, like citrate and malate (Kupper et al. 2004 ). In addition, plant cells secure themselves against Zn toxicity by making proteins, such as metallothioneins, which bind them in the organelles by sequestration or exporting (Palmiter 2004 ). Besides, plants protect themselves from Zn toxicity by regulating Zn transporters, like ZRT/IRT-like protein (ZIP), metal tolerance protein (MTP), and Zn-induced facilitator (ZIF) (Kaur 2021). Likewise, a specific group of proteins called "plant defense proteins type I" (PDF1s) has been discovered and characterized because of the role they play in making plants resistant to Zn (Mirouze et al. 2006 ). Similarly, plants exposed to Zn excess can develop anti-oxidative defense system, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) to scavenge ROS (Fariduddin et al. 2022 ). Also, stressed plants defend their cells from oxidative damage by producing high levels of osmolytes like soluble sugar and proline (Kumar et al. 2022 ). It has been demonstrated that plant metabolites contribute to metal stress resistance. Amino acids, such as histidine, methionine, and asparagine, can chelate HM ions, including Zn, in plant cells and xylem fluid, eventually enhancing plant tolerance (Ghuge et al. 2023 ). Proline and total amino acids increase in the leaves when there is an excess of Zn in the soil, suggesting their responsive role in maintaining osmotic pressure (Spormann et al. 2023 ). Similarly, phenolic compounds chelate Zn into the vacuole by sequestering or excluding it from the membrane space via transporters, which is essential to their homeostasis (Vega et al. 2022 ). For instance, metabolomics profiling was performed to understand the role of metabolites as stress responses in chickpea cultivars under Zn stress. Chickpea ( Cicer arietinum L.) is a valuable legume crop, rated third after beans and peas globally. The seeds of it are a rich source of protein, carbohydrates, and fats (Ullah et al. 2020 ). Besides, it has significant health benefits and helps to reduce diabetic, cardiovascular, and cancer risks (Merga and Haji 2019 ). There are limited reports on the toxicity of Zn in chickpeas in a hydroponic environment. Hence, the current study aims to broadly explore the relative tolerance of chickpea cultivars, and examine their physiological, biochemical, and metabolomics responses under various Zn levels. Materials and methods Plant material and cultivation The seeds of chickpea ( C. arietinum L.) cultivars, [ICCV89310 (IC8), NC234 (NC2), and ICCV89323-B (IC8-B)] were obtained from the Crop Genetic Resources Institute, Xinjiang Academy of Agricultural Sciences, Xinjiang, China. Before germination, seeds were sanitized with 75% ethanol and 6% NaClO solution and washed several times with distilled water. After soaking for 24 h, the seeds were sown in plastic trays containing vermiculite and incubated in a germinator for 12 days. The growing chamber was climate-controlled, with a day/night temperature of 22–25 o C, 70% relative humidity, and a 14/10 h light/dark cycle. Uniform-sized seedlings were then allowed to grow for 8 days in Hoagland nutrient solution (6 plants/pot). After an initial growth of 20 days, Zn was applied to the nutrient solution as ZnSO 4 in different concentrations (0, 50, 100, and 150 µM). Seedlings were exposed for one week under Zn stress in Hoagland nutrient solution. The concentrations of Zn were selected based on earlier reports (Andrade et al. 2009 ; Cherif et al. 2010 ). The nutrient solution was changed every four days. Measurement of growth and physiological indices After a week of Zn application, plants were collected and divided into roots and shoots. The lengths of root and shoot, as well as fresh weight (FW), were determined. For dry weight (DW), plant tissues were oven-dried at 120°C for 20 min and remained for three days at 60°C to measure their constant biomass. Dried samples of root and shoot were crushed into powder, weighed (0.3 g), and digested with an acid mixture (HNO 3 + HClO 4 ) to determine the content of Zn using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima-8300 DV; Perkin-Elmer, Inc., Waltham, MA, USA). The translocation factor (TF) of Zn from root to shoot and shoot water content (SWC) was calculated using the following formulas: TF = [Zn] shoot /[Zn] root . SWC= (FW-DW)/FW x 100. The growth tolerance index (TI) of root and shoot was measured separately based on DW, according to Chen et al. ( 2011 ). TIr and TIs represent the tolerance indices of root and shoot, respectively. TIr = root biomass (Zn) / root mean biomass (control) x 100, TIs = shoot biomass (Zn) / shoot mean biomass (control) x 100. Determination of chlorophyll contents The chlorophyll contents were quantified following the method explained by Fang et al. ( 2017 ). In brief, 100 mg of fresh leaves were ground, and their extract was combined with 4 mL of a 2:1 acetone and ethanol mixture. The chlorophyll a and b absorbance levels were 665 and 649 nm, respectively. Determination of oxidative stress markers (MDA, H 2 O 2 , and EL) Malondialdehyde (MDA) content in the leaves was estimated as depicted by Hasanuzzaman and Fujita. (2013), using TBA (thiobarbituric acid) as the reactive agent. The absorbance of a colorful supernatant was measured at 532 nm and 600 nm. Hydrogen peroxide (H 2 O 2 ) was determined according to Yu et al. ( 2003 ), utilizing 0.1% TiCl 4 in 20% H 2 SO 4 (v/v) at 410 nm. Electrolytic leakage (EL) was identified according to Callegari et al. ( 2022 ) using the following formula: EL (%) = (EC1/EC2) × 100 Enzyme assays Superoxide dismutase (SOD) activity was assayed using the reduction method of nitroblue tetrazolium solution (NBT) (Zhang et al. 2012 ), whereas peroxidase (POD) activity was detected using the guaiacol method of Afzal et al. ( 2019 ). The absorbances were monitored at 560 and 470 nm, respectively. Catalase (CAT) and glutathione reductases (GR) were assayed corresponding to the protocols of Li et al. ( 2022 ), and the absorbance was read at 240 and 340 nm, respectively. Osmolytes detection Proline (Pro) content was measured using the acid ninhydrin method (Bates et al. 1973 ), and the absorbance was read at 520 nm. Soluble sugars (SS) were determined following the method Irigoyen et al. ( 1992 ) described, and the absorbance was detected at 625 nm. Total protein (Prot) content was assayed using the Bradford reagent (Ali et al. 1999 ). Bovine serum albumin was standard, and the absorbance was measured at 595 nm. Analysis of primary metabolites Primary metabolites were extracted from shoot tissues using the method of Yang et al. ( 2021 ) with minor changes. In total, 90 mg samples were homogenized with 540 µL cold methanol and 60 µL internal standard (L-2-chloro-phenylalanine, 0.3 mg/mL). Following a 30 min ultrasonication period, 600 µL of water and 300 µL of chloroform were added to the samples. After vortexing (2 min) and sonicating (30 min), the samples were centrifuged (10 min) at 12000 rpm and 4°C. Next, the supernatant of 800 µL was moved to a glass sample container and dried using a vacuum (15000 rpm for 8 h at 40°C). The dried residue was redissolved by adding 400 µL of methoxyamine (15 mg/mL in pyridine). Then, samples were vortexed (2 min) and incubated (90 min) at 37°C. The solution was treated with 400 µL of N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) (15 mg/mL in 1% trimethylchlorosilane) and 60 µL of n-hexane successfully. Subsequently, the collected solution was injected into a GC-MS (Agile 7890A-5975C, Agilent Technologies, Inc., Santa Clara, U.S.A.). The separation process was conducted using a non-polar DB-5 capillary column (30 m × 250 µm ID, J&W Scientific, Folsom, U.S.A.) in conjunction with a constant flow rate of 1 mL/min and helium as a carrier gas. The initial programming temperature for the gas chromatography (GC) was 50°C, subsequently increasing by 8°C/min. The oven temperature was scaled from 125°C, 15°C/min to 170°C, 4°C/min to 210°C, 10°C/min to 270°C, 5°C/min to 305°C, and remained constant for 5 min. The electron impact (EI) ion source was maintained at 70 eV, and the acquisition rate was 20 spectra/sec for a scanning range of 50 to 500 m/z. Data analysis The results underwent statistical analysis using Duncan's Multiple Range Test (DMRT) at a significance level of p < 0.05. All collected data were analyzed for variance (ANOVA) using the COSTAT computer package version 6.4 (CoHort software in Monterey, California, USA). Pearson's correlation and principal component analysis (PCA) were conducted to examine the correlation between morpho-physiological and biochemical indicators using OriginPro-2024. Hierarchical clustering analysis (HCA), partial least squares-discriminant analysis (PLS-DA), and sparse partial least squares-discriminant analysis (sPLS-DA) were performed using MetaboAnalyst 6.0 to analyze the data. Subsequently, KEGG database ( https://www.metaboanalyst.ca/MetaboAnalyst/Secure/enrichment/OraView.xhtml ) was used to construct metabolic pathway. The data represent the average of at least three replicates per treatment under the same conditions (n = 3). The values of the results were displayed as the mean ± standard error (SE). Results Effects of Zn on growth and physiological parameters All chickpea cultivars (IC8, NC2, and IC8-B) responded differently to the applied Zn levels in the growth medium. The decrease in root and shoot length was concentration-dependent in all cultivars compared to control plants. For instance, the length of root and shoot was significantly ( p < 0.05) higher in IC8 (7.34 and 14.3 cm) than in NC2 (4.4 and 11.3 cm) and IC8-B (2.9 and 6.8 cm) at excess of Zn (150 µM). The decrease of root and shoot length in IC8, NC2, and IC8-B was 425.3%, 706.6%, 832.1%, and 719.7%, 729.9%, and 451.3%, respectively, relative to the control. Additionally, the FW of the three chickpea cultivars was examined and decreased significantly ( p < 0.05). At 150 µM of Zn, total FW decreased 560.1%, 534.3%, and 686.5% for IC8, NC2, and IC8-B, respectively, compared to control plants (Table S1). After three days of Zn exposure, the symptoms of toxicity were noticed. The visual observations revealed that IC8-B exhibited more toxicity symptoms of shoot and root compared to cultivars NC2 and IC8 (Fig. 1 A). The phytotoxicity trend at 150 µM of Zn was IC8-B > NC2 > IC8 > control plants. The decrease in DW was more prominent in the root compared to the shoot. Under high concentration of Zn, IC8 recorded the highest DW (0.15, 1.64, and 1.79 g), followed by NC2 (0.10, 1.35, and 1.45 g), and IC8-B (0.04, 1.09, and 1.13 g), receptively, (Fig. 1 B, C, and D). Considering the growth attributes, IC8 was potentially a more tolerant cultivar than NC2, while IC8-B was more sensitive, especially at 150 µM of Zn exposure. Furthermore, several physiological indices were considered to investigate the overall health of cultivars IC8, NC2, and IC8-B seedlings. An elevated concentration of Zn in the growth medium led to a substantial decrease ( p < 0.05) in the root-to-shoot ratio (R/S). Under Zn excess, the R/S ratios of IC8, NC2, and IC8-B were 0.09, 0.07, and 0.03, respectively. No statistical difference in R/S was observed between the IC8 and NC2 cultivars (Fig. 1 E). Moreover, the content of water in shoots (SWC) was lowered ( p < 0.05) with the excessive dose of Zn (150 µM). In contrast, no such change was noticed at 50 and 100 µM relative to control (Fig. 1 F). Substantially, Zn at 150 µM was highly detrimental and disturbed the SWC more severely in NC2 and IC8-B than in cultivar IC8. All cultivars significantly declined the tolerance index (TI) values under Zn stress relative to control. Zn at 150 µM was more acute and reduced the TI of root (TIr), where IC8 showed the highest value (48.90), while IC8-B exhibited the lowest value (12.97) (Fig. 1 G). Similarly, TI of shoot (TIs) were significantly higher ( p < 0.05) in IC8 compared to NC2 and IC8-B at 150 µM of Zn. The values of TIs for cultivars IC8, NC2, and IC8-B ranged from 90–100%, 75–92%, and 57–93%, respectively. Exceptionally, cultivar NC2 accounts for a higher TIs value (74.70) than IC8-B at 150 µM of Zn (Fig. 1 H). Besides, to better understand the phytotoxicity and tolerance of IC8, NC2, and IC8-B, Zn content was studied in the root and shoot tissues of seedlings. Generally, the amount of Zn is enhanced in the root and shoot with increasing Zn levels in the nutrient solution. The content of Zn was higher in the root than in the shoot. The root of cultivar IC8-B seedlings exhibited high Zn content (47.41–3106.80 µg/g DW), followed by NC2 (39.22-2997.20 µg/g DW), while IC8 possessed the lowest Zn content (31.16–2866.70 µg/g DW). Conversely, high Zn content was recorded in the shoots of cultivars IC8 (645.56 µg/g DW) and NC2 (415.36 µg/g DW), while IC8-B showed the lowest Zn content (186.51 µg/g DW) under high Zn treatment (Table 1 ). On the other hand, the values of translocation factor (TF) decreased in all cultivars with the application of Zn in growth medium. However, IC8 and NC2 revealed comparatively higher values of TF than IC8-B at 150 µM of Zn. Besides, the highest TF values were recorded in control plants for IC8 and NC2, followed by IC8-B (0.47, 0.30, and 0.12), respectively. Considerably, the transfer of Zn (TF) was more significant at 50 and 100 µM of stress levels (Table 1 ). Table 1 Zn content analysis and translocation factor (TF) of three chickpea cultivars grown hydroponically at various concentrations of Zn for 25 days Cultivars Treatments (µM) Zn content in root (µg/g DW) Zn content in shoot (µg/g DW) TF IC8 Control 31.16 ± 1.34 f 14.65 ± 1.58 h 0.47 ± 0.030 a 50 926.24 ± 9.78 e 154.37 ± 14.20 f 0.17 ± 0.014 d 100 2367.27 ± 54.74 d 342.84 ± 3.79 c 0.15 ± 0.003 d 150 2866.70 ± 64.01 b 645.56 ± 16.48 a 0.23 ± 0.008 c NC2 Control 39.22 ± 0.26 f 11.81 ± 0.94 h 0.30 ± 0.020 b 50 967.55 ± 16.71 e 145.48 ± 1.80 f 0.15 ± 0.003 d 100 2567.84 ± 128.3 c 336.73 ± 4.64 c 0.13 ± 0.008 de 150 2997.20 ± 76.63 ab 415.36 ± 3.22 b 0.14 ± 0.005 de IC8-B Control 47.41 ± 2.32 f 5.66 ± 0.98 h 0.12 ± 0.029 de 50 984.08 ± 4.77 e 122.61 ± 5.92 g 0.12 ± 0.003 de 100 2665.53 ± 64.74 c 258.95 ± 10.71 d 0.09 ± 0.003 ef 150 3106.80 ± 65.77 a 186.51 ± 7.42 e 0.06 ± 0.005 f The data is shown as mean ± SE (n = 3). Values in columns with the same characters imply a non-significant variation depending on DMRT at p < 0.05. Effects of Zn on chlorophyll contents and oxidative damage Adding Zn to the growth medium considerably impacts the chlorophyll (chl) contents. The toxicity of Zn reduced chl a, chl b, and total chl in a concentration-dependent manner (Fig. 2 A, B, and C). Cultivar IC8, NC2, and IC8-B revealed non-significant differences ( p < 0.05) in the control medium, while total chl and chl b were slightly increased at 100 µM of Zn. High Zn stress was more harmful in IC8-B compared to IC8 and NC2. In contrast, cultivars NC2 and IC8 showed the lowest reduction of chlorophyll contents. Oxidative stress indices (H 2 O 2 , EL, and MDA) significantly enhanced in the shoots of IC8, NC2, and IC8-B under different Zn levels related to control plants (Fig. 2 ). The highest increase in oxidative stress markers was observed at 150 µM of Zn. At the same time, the lowest contents were noticed in control seedlings. Among the studied cultivars, IC8-B documented the highest contents of H 2 O 2 and EL (32.4 µmol/g FW and 58.5%), followed by NC2 (23.02 µmol/g FW and 49.1%) and IC8 (20.97 µmol/g FW and 46.3%), respectively, at 150 µM (Fig. 2 D and E). A similar trend was observed in MDA content under control and Zn-treated plants. The content of MDA in cultivars IC8-B, NC2, and IC8 increased by 44.2%, 37.02%, and 35.4%, respectively, at 150 µM of Zn related to control (Fig. 2 F). Effects of Zn on antioxidants and osmolytes contents Different enzymatic and non-enzymatic activities and their ratios were assessed under different Zn treatments to examine the defense system of chickpea cultivars. Compared to control plants, the activity of SOD and POD were stimulated significantly ( p < 0.05) by Zn supplementation in all cultivars (Fig. 3 ). Cultivars IC8, NC2, and IC8-B treated with 150 µM of Zn showed an intensification in the activity of SOD and POD by 9.1% and 8.6%, 7.6% and 7.3%, and 8.1% and 6.9%, respectively, over control plants (Fig. 3 A and B). The activity of catalase (CAT) and glutathione reductases (GR) increased by 3.8%, 2.73%, and 3.4% and 2.03%, respectively, in IC8 and NC2 under 150 µM of Zn relative to control seedlings. Conversely, CAT and GR activity in IC8-B under 150 µM of Zn was shown to be much lower than in the control (2.5% and 1%, respectively). In addition, CAT and GR decreased slightly in IC8 and NC2 with the increasing concentration of Zn, but still higher than in control (Fig. 3 C and D). Also, the increasing concentration of Zn resulted in a significant increase ( p < 0.05) in the levels of proline, soluble sugars, and total protein in the shoots of chickpea cultivars. The content of proline increased by 456.8%, 341.4%, and 321.3%, respectively, in IC8, NC2, and IC8-B treated with 150 µM of Zn, compared to the control (Fig. 3 E). In contrast, the percentage increase of soluble sugars in IC8, NC2, and IC8-B at 150 µM of Zn was 119%, 90.6%, and 64.5%, respectively (Fig. 3 F). Additionally, the percentage increase of total protein in IC8, NC2, and IC8-B at 150 µM of Zn was, 56%, 47.4%, and 29.3%, respectively (Fig. 3 G). Overall, osmolytes were more pronounced at 150 µM of Zn, while control plants possessed the lowest active substances in all cultivars. Comparatively, the trend of osmolytes increase was in the order of IC8 > NC2 > IC8-B. Multivariate statistical analysis of different variables Pearson's correlation revealed significant variation (positive or negative) in growth, physiological, and biochemical attributes in chickpea cultivars treated with Zn, where a positive correlation exists among the closely related variables in the same quadrant (Fig. 4 A). The statistics of correlation in growth parameters with each other ( p < 0.05 and 0.01) and with chlorophyll contents were positive. However, a negative correlation existed with Zn (r + s). Moreover, stress biomarkers (H 2 O 2 , EL, and MDA), antioxidants (SOD, POD), and osmolytes (Pro, SS, and Prot) were negatively correlated with growth (TPH, TDW), and physiological indices (R-S. R, and SWC). In addition, CAT and GR have no significant correlation with all parameters. Subsequently, positive correlations exist within antioxidants, stress biomarkers, and osmolytes while negatively correlated with growth and physiological attributes. Crucially, all parameters positively correlated with Zn content in the root and shoot, while a negative correlation was found with chlorophyll. The principal component analysis (PCA) reveals significant variations in growth and physio-biochemical features in chickpea cultivars exposed to Zn applications. A two-dimensionally constructed diagram of PCA showed two different variability percentages of the principal component (PC), such as PC1 and PC2, at 61.7% and 24.4%, respectively (Fig. 4 B). Growth and physiological parameters revealed smaller angles with each other (< 90°) and with CAT and GR activity, thereby indicating their positive relationship. Additionally, a positive relationship exists between osmolytes, ROS, SOD, POD, and Zn (r + s). At the same time, no significant relation was shown with CAT and GR activity along with growth and physiological indices. The indicators with the maximum contribution to PC1 had a 64.1% variance difference, while in PC2, there was approximately 35.1% variation. Metabolic assay Forty-six metabolites were identified in the shoot of three chickpea cultivars, belonging to different groups, including amino acids (14), organic acids (13), amines (6), sugars (4), alcohols (4), and others (5). The PLS-DA of the scores plot for 30 metabolites with discrimination (VIP > 1) indicated that cultivar IC8 expressed the higher concentration of metabolites with 53.3%, followed by NC2 (30%) and IC8-B (16.6%) (Fig. 5 A). Subsequently, sPLS-DA was performed to better understand the relationship and closeness of IC8, NC2, and IC8-B treated with Zn stress. The scores plot of sPLS-DA suggested that cultivars IC8 and NC2 were remarkably close in response to Zn stress and closely overlapping each other, while a contrasting pattern of IC8-B was observed relative to them (Fig. 5 B). The scoring plot of sPLS-DA revealed 75% and 29.6% of percentage difference in component 1 and 2, respectively. Simultaneously, to complement our observation regarding the tolerance abilities and toxicity of chickpea cultivars, a hierarchical clustering analysis (HCA) was used (Fig. 5 C). The samples of chickpea cultivars were analyzed three times to observe the metabolic abundance and enrichment in the shoots. The accumulation of primary metabolites in each replicate of the tested chickpea cultivars showed an apparent fluctuation in their abundance and expression in shoots. However, to further rectify the expression of metabolites in IC8, NC2, and IC8-B, visualization was performed for 46 metabolites with sPLS-DA (Fig. 5 D). In total, IC8 and NC2 revealed the highest expression and abundance of metabolites with 47% and 32.6%, respectively, followed by IC8-B with 19.5%. The results presented above provide tentative evidence that IC8 and NC2 shoots exhibit significant levels of responsive metabolites, possibly contributing to their tolerance. In contrast, the partial or intermediate expression of metabolites in IC8-B indicates its sensitivity under Zn stress. To further understand the effects of Zn stress in shoot of IC8, NC2 and IC8-B, the pathway enrichment analysis of metabolites was conducted using KEGG database. As shown in Fig. 6 A and D, 16 pathways were significantly enriched in the shoot, whereas 9 pathways were depressed. Among them, phenylalanine, tyrosine, and tryptophan metabolism were more enriched, while porphyrin and glycolysis (gluconeogenesis) pathways were significantly affected in the shoot. Discussion Zinc (Zn) is a micronutrient that is necessary for the growth and development of plants. However, elevated concentrations can cause severe toxicity by disturbing the physiological and metabolic processes (Garg and Singh 2018 ). The present findings indicated that cultivars IC8, NC2, and IC8-B varied in response to Zn stress, such as the plant height and biomass were decreased under high Zn supply. The decline in growth parameters may attributes to a significant Zn buildup in various plant parts that impede the uptake of nutrients and cause damage to the ultrastructure of plant tissues (Cherif et al. 2010 ). In contrast, the high ratio of R/S in plants exposed to HM stress suggests its strong affinity for the uptake of nutrients from its surroundings and leads to more aerial plant biomass (Agathokleous et al. 2019 ). In the current work, cultivars IC8 and NC2 revealed higher biomass and R/S ratio than IC8-B, suggesting strong tolerance abilities, while IC8-B was sensitive under high Zn stress. Also, a significant decrease in SWC was noticed in all cultivars under high Zn supply. In plants, the reduction of water content maybe due to the high Zn uptake in aerial parts or reduced root biomass and surface area (Garg and Singh 2018 ). Also, Zn may affect the plant water status by limiting the hydraulic conductivity and reducing xylem tissue available for water conduction (De Silva et al. 2012 ). Similar to our results, Bonnet et al. ( 2000 ) reported the decrease of water content in ryegrass ( Lolium perenne ). TI is critical in determining plants' tolerance capacity to HM stress. In this study, the tolerance capacity of the root was less (TIr 60) compared with IC8-B (TIs 60 were reported to be tolerant (Lux et al., 2004 ). The accumulation of metals varies significantly within plant species and cultivars and is influenced by edaphic factors (Chen et al. 2011 ). Callegari et al. ( 2022 ) reported that the root is the first point of connection to metal toxicity and accounts for more uptake. In this study, IC8-B and NC2 showed a higher Zn storage in the root than in IC8, while in the shoot, the uptake was higher in IC8 and NC2 than in IC8-B. Impa et al. ( 2013 ) attribute the increased Zn contents in the shoots of tolerant species primarily to better root development matching. In IC8-B, the mechanism of Zn restriction in the root may weaken the root's ultrastructure, ultimately decreasing the above-ground biomass. The immobilization of Zn in the root happens through symplastic flow to the vacuole, thereby showing a substantial storage area for Zn excess (Kabir et al. 2017). The TF values of all cultivars were less than 1, indicating that none of them could effectively transfer Zn from their roots to shoots. However, the highest TF was noticed in IC8 than the other ones. Dinh et al. ( 2015 ) stated that the decrease of TF in Zn-treated plants relative to control could be associated with the retention of Zn in their roots. Decreased photosynthetic pigment is the leading indicator of Zn toxicity in plants with obvious chlorosis symptoms (Cherif et al. 2010 ). In this study, chlorophyll content decreased in all cultivars with increased Zn concentrations. The decline in chlorophyll may be related to the blockage of photo-electron transport and disorganization of chloroplasts or fewer thylakoid and grana and under Zn excess (Sidhu et al. 2020 ). It has been demonstrated that HM stress increases the production of singlet oxygen (O 2 ), superoxide radical (O 2 •− ), hydroxyl radical ( • OH), and H 2 O 2 , hence increasing the accumulation of ROS. Subsequently, ROS production increased lipid peroxidation, which caused oxidative cell damage (Huihui et al. 2020 ). Generally, MDA is considered an essential indicator of oxidative damage, which is the ultimate breakdown product of lipid peroxidation (Kumar et al. 2021 ). Our data illustrated a considerable rise in H 2 O 2 , EL, and MDA contents. However, IC8-B has higher oxidative stress indices than IC8 and NC2, suggesting high sensitivity under high Zn stress. Likewise, Singh et al. ( 2020 ) found that severe stress of Cr increased MDA and H 2 O 2 contents in sensitive chickpea genotypes. To avoid cell damage and detoxify the ROS, plants have a highly developed system of antioxidants, including SOD, POD, CAT, GR, and several others (Al Mahmud et al. 2017 ). Our results documented that the activities of antioxidants were higher in IC8 and NC2 relative to IC8-B. SOD provides the first line of protection against the harmful levels of ROS using the metal-catalyzed Habere-Weiss-type reaction, eliminates O 2 •− and reduces the potentially detrimental impact of • OH production (Gill et al. 2011 ). Similarly, CAT and GR decreased the overproduction of ROS caused by H 2 O 2 and HM excess (Ghuge et al. 2023 ). In addition, GR maintains the reduced environment within the plant cell by transforming oxidized glutathione (GSSG) into reduced glutathione (GSH) using the ascorbate-glutathione cycle (Madhu et al. 2022 ). Notably, IC8-B showed a significant decline in CAT and GR under high Zn supply compared to control, which may be anticipated to severe oxidative stress, as described previously (Wang et al. 2009 ). Tripathi et al. ( 2015 ) also demonstrated a substantial decline in the activity of CAT and GR under Zn stress. Plants also have a powerful defense system of osmolytes against stress biomarkers. Proline functions as an antioxidant and osmoprotectant in plants to ameliorate the oxidative damage induced by ROS (Spormann et al. 2023 ). Soluble sugars and proteins have been demonstrated to detoxify HM and sustain biological molecules and membranes in tolerant cultivars (Kumar et al. 2022 ). Overall, the antagonistic response of antioxidants and their production in plants may be affected by the crop species, cultivars, developmental stages, time, and level of exposure to HM stress (Wang et al. 2009 ). In the present work, IC8 and NC2 contained considerably more proline, soluble sugars, and total protein than IC8-B. Metabolite profiling is an efficient tool for finding metabolites and metabolic pathways in studying plant stress responses. Under environmental stress, several metabolites, including organic acids, amino acids, sugars, and sugar alcohols, can act as signal transduction molecules, osmoprotectants, and antioxidants (Shulaev et al. 2008 ). In this study, IC8 and NC2 revealed up-regulation, or higher expression of metabolites, while IC8-B exhibited down-regulation or partially expressed. Previous studies have shown that amino acids and their derivatives trigger the defense system of plants during growth and development and serve as substrates for various metabolites that respond to different abiotic factors (Batista-Silva et al. 2019 ). Particularly histidine plays a crucial role in maintaining the balance of Zn in the root and facilitating its storage and transportation through the xylem in Thlaspi caerulescens shoots (Salt et al. 1999 ). Similarly, asparagine may reduce the toxicity of Zn in Deschampsia cespitosa by establishing an intracellular compound with it (Smirnoff and Stewart 1987 ). Moreover, tryptophan is an essential amino acid for plants to grow normally. It helps to open the stomata, regulates ions transport, and reduces HMs toxicity (Sadak et al. 2021). Remarkably, our findings showed significant enrichment of tryptophan metabolism in the pathway of tyrosine, phenylalanine, and tryptophan biosynthesis under Zn stress in chickpea cultivars (Fig. 6 ). Allantoin has been identified as a compound that enhances plant tolerance, especially under HM stress (Nourimand and Todd 2017 ). It makes up 90% of all the nitrogenous compounds mobilized inside legumes' plant tissue (Kaur et al. 2021 ). Therefore, incrementing allantoin content under HM stress may enhance plant tolerance. Similarly, glutamic acid is the precursor of chlorophyll (Wang et al. 2023 ) which is up-regulated in cultivar IC8. Figure 5 C, and B revealed similar results in IC8 and NC2 chickpea cultivars regarding histidine, tryptophan, asparagine, allantoin and glutamic acid. Moreover, a series of other critical amino acids like methionine, glycine, alanine, and proline, etc., are up-regulated in the shoots of IC8 and NC2, implying their tolerance abilities under Zn stress compared to IC8-B. Organic acids are the main phytomolecules in the related network, accumulating more in IC8 than in NC2 and IC8-B. Sidhu et al. ( 2019 ) demonstrated that organic acids are crucial to HM tolerance in plants. Sarret et al. ( 2002 ) indicated that Arabidopsis halleri exhibited a high amount of malic acid, with Zn preponderantly combined with malic acid in the shoot. Similarly, benzoic acid is crucial for the growth of plants and increases their tolerance to abiotic stresses (Widhalm and Dudareva 2015 ). These results suggested that high accumulations of organic acids can chelate HM and rapidly detoxify them in shoots. Likewise, the accumulation of sugar molecules in shoots is associated with alleviating abiotic stress (Hassan et al. 2023 ). Sugars like sucrose and hexoses increase stress tolerance by down-regulating genes linked to stress and up-regulating-controlling genes related to growth (Salam et al. 2023 ). Similar results were investigated in the present work, where sugar molecules (sucrose) are up-regulated in IC8, while no expression was observed in NC2 or IC8-B. Moreover, indoleacetic acid, naringenin, cysteamine, xanthine, spermidine, etc., were also up-regulated in the tested chickpea cultivars. However, the overall expression of targeted metabolites was higher in IC8 (47.8%) and NC2 (32.6%) than in IC8-B (19.5%), indicating the tolerant and sensitive capacities of IC8, NC2, and IC8-B, respectively, in response to Zn stress. Conclusions Globally, HM pollution is a significant risk to agriculture safety and has become a major environmental issue. In the present study, Zn supplementation (50, 100, and 150 µM) significantly influenced the growth, physiological, and biochemical traits of the experimented cultivars (IC8, NC2, and IC8-B) in hydroponics. In particular, high Zn exposure (150 µM) severely affected the growth and biomass, R/S ratio, SWC, and chlorophyll content. Moreover, differential responses were observed among the tested cultivars concerning the tolerance indices, Zn content analysis, oxidative stress markers, osmolytes, and antioxidant enzymes. The increased ROS likely results from suppressed growth parameters and low photosynthetic pigment contents. Hence, high Zn levels significantly promoted the production of H 2 O 2 , EL, and MDA and elicited oxidative stress in chickpea cultivars. The growth and physio-biochemical characteristics response confirmed that IC8 and NC2 exhibited better Zn tolerance than IC8-B. The relative tolerance potential of IC8 and NC2 may be attributed to different adaptive strategies, such as a well-developed profile of responsive metabolites, such as histidine, asparagine, tryptophan, allantoin and others. Similarly, antioxidative defense enzymes (SOD, POD, CAT and APX), and soluble components, such as proline, soluble sugars and total protein may contributed to the tolerance capabilities of IC8 and NC2. In contrast, the low tolerance abilities of IC8-B based on growth, physio-biochemical, and metabolic responses confirmed it to be more sensitive when subjected to Zn excess. Hence, cultivar IC8-B maybe utilized as a control cultivar under Zn stress to evaluate other chickpeas' tolerance capacity. Besides, we suggested IC8 and NC2 as promising candidates for Zn-contaminated soil. However, more in-depth molecular exploration of these responses is required to understand the mechanisms behind Zn toxicity and tolerance and how they relate to diverse metabolic processes. These findings not only enhance our understanding of Zn-mediated stress in chickpeas but also offer valuable insights for optimizing stress adaptation strategies in diverse cultivars, paving the way for improved agricultural practices and crop resilience. The findings also stipulate new metabolites understanding for the response of Zn-treated chickpeas and perhaps contribute to a better kind of stress adjustment in other chickpea cultivars. Declarations Authorship contributions: Shakir Ullah and Xingfan Li: Designed and performed the experiments. Uzma Salam: Conceived and designed the experiment. Ahmed A. Elateeq: Analyzed the data. Ilbong Ri: Methodology and investigation. Shakir Ullah: Writing-original draft and validation. Shakir Ullah and Ahmed A. Elateeq: Writing-review and editing. Dewen Li: Resources and conceptualization. Xiaorui Guo: Contributed reagents and materials. Zhonghua Tang: Supervision, and funding. Mahmoud F. Seleiman: Validation, and Funding. All authors read and approved the final manuscript. Funding: This work was supported by "The Key Resource and Development Project of Heilongjiang Province, China (JD22A008)", and Researchers Supporting Project King Saud University, Riyadh, Saudi Arabia (No. RSPD2024R751). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5188964","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":364519853,"identity":"e7b50109-92dd-4d23-af57-1933a0461a83","order_by":0,"name":"shakir ullah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie2PMUvDQBiGPwk4RVw/CeR+gXAlEJz6M5y/Q+gtpxQKkikEBH+CCAH7F1oCTg4HASd/QOEcLAXnK12cxEsouCS1boL3cMMdvA/vewAez18EIQCC5gAc2Azj5qLf9lUCfDlLWoV+UmCrwNFtJorvZzesvFnx5VN+dXxXryweopye10vXMoxPi26Fvz4nRO/1BM0oxXGIl3Mz4k65SFLdoyAlmrQWhaEUEJ1SUqNo8dijsHu5cUoupkZubMhRDkppdyqwUG6YDsTMKI4hIbFI7W7hCzXhpGsxN+o6Qo2DWaTGmnj/X9yw6uTDDXswslqvP3PGSllZmw3jPqWjt03yfeNtb/GbtMfj8fwHvgAHqmiYfUGNFQAAAABJRU5ErkJggg==","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"shakir","middleName":"","lastName":"ullah","suffix":""},{"id":364519854,"identity":"999cbf5f-8495-423e-8013-3045c282dc13","order_by":1,"name":"Xingfan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xingfan","middleName":"","lastName":"Li","suffix":""},{"id":364519855,"identity":"de69430e-1796-4baf-944d-027e9f528e49","order_by":2,"name":"Uzma Salam","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Uzma","middleName":"","lastName":"Salam","suffix":""},{"id":364519856,"identity":"c3aa7030-c0a5-49cc-a677-0f90367b5588","order_by":3,"name":"Ahmed A. Elateeq","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"A.","lastName":"Elateeq","suffix":""},{"id":364519857,"identity":"6c4a939e-892b-48f1-8eb2-e854d4d1c763","order_by":4,"name":"Ilbong Ri","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ilbong","middleName":"","lastName":"Ri","suffix":""},{"id":364519858,"identity":"d6224c0c-f448-4181-9d07-22b20e26c932","order_by":5,"name":"Dewen Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dewen","middleName":"","lastName":"Li","suffix":""},{"id":364519859,"identity":"a2ede8d5-0989-402d-9b67-2283742031cf","order_by":6,"name":"Mahmoud F. Seleiman","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"F.","lastName":"Seleiman","suffix":""},{"id":364519860,"identity":"efce6521-855a-4e9f-bec3-a2886a33f202","order_by":7,"name":"Xiaorui Guo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaorui","middleName":"","lastName":"Guo","suffix":""},{"id":364519861,"identity":"b964ca2b-c482-4d24-99b8-d56f7c39ec34","order_by":8,"name":"Zhonghua Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhonghua","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2024-10-01 17:31:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5188964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5188964/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66546870,"identity":"4f42c109-fca7-4077-9976-5345d80e327a","added_by":"auto","created_at":"2024-10-14 08:34:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":194468,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different Zn concentrations on morphology (A), dry weight (B, C and D), and physiological traits [root to shoot ratio (R/S) (E), shoot water content (SWC) (F) and tolerance indices of root (TIr) (G) and shoot (TIs) (H)] of three chickpea cultivars (IC8, NC2, and IC8-B). Bars represent the standard error (SE) of three replicates. Significant differences are shown with different letters (p \u0026lt; 0.05) according to DMRT\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/721cbe3d433721287ba64ffd.png"},{"id":66546437,"identity":"57921e10-cc36-40f3-ae7d-4ec7dbcbddc8","added_by":"auto","created_at":"2024-10-14 08:26:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56467,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different Zn concentrations on chlorophyll [chl a (A), chl b (B), and total chl (C)] contents and reactive oxygen species (ROS) [H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (D), EL (E), and MDA (F)] contents of three chickpea cultivars (IC8, NC2, and IC8-B). Bars represent the standard error (SE) of three replicates. Significant differences are shown with different letters (p \u0026lt; 0.05) according to DMRT\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/91cc7279b1e5ebdab48b109f.png"},{"id":66546434,"identity":"9553c4ba-3418-4312-9a3a-248760c22e7b","added_by":"auto","created_at":"2024-10-14 08:26:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49111,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different Zn concentrations on the activities of antioxidants; SOD. (A), POD (B), CAT (C) and GR (D) and soluble substances; proline (E), soluble sugars (F), and total protein (G) of three chickpea cultivars (IC8, NC2, and IC8-B). Bars represent the standard error (SE) of three replicates. Significant differences are shown with different letters (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) according to DMRT\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/075c21fd81002ae654b8b9d8.png"},{"id":66546439,"identity":"6e04affb-eace-438d-b2e9-f37f168c7f97","added_by":"auto","created_at":"2024-10-14 08:26:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66342,"visible":true,"origin":"","legend":"\u003cp\u003ePearson's correlation (A) and principal component analysis (PCA) (B) for total plant height (TPH), total dry weight (TDW), shoot water content (SWC), root-to-shoot ratio (R/S. R), total Zn content, Zn (r + s), chlorophyll (Chl), malondialdehyde (MDA), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), electrolyte leakage (EL), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductases (GR), proline (Pro), soluble sugars (SS), and total proteins (TP) in three chickpea cultivars (IC8, NC2, and IC8-B). In Pearson correlation, the red and blue circles indicated a positive and negative correlation. Significant correlations were marked by * at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and ** at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Abbreviations in PCA are the same as mentioned in the legend in correlation analysis. Different colored dots in PCA represent the concentrations of Zn (Ck, 50, 100, and 50 µM)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/62a321845da3907d1cb073ee.png"},{"id":66546436,"identity":"8fceb3ef-b3bf-4f53-9daf-866552773e43","added_by":"auto","created_at":"2024-10-14 08:26:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146564,"visible":true,"origin":"","legend":"\u003cp\u003eMultivariate statistical analysis of metabolites in the shoot of chickpea cultivars (IC8, NC2, and IC8-B). VIP score plot of PLS-DA discriminatory metabolites (VIP\u0026gt; 1) (A) and the corresponding scores plot of sPLS-DA (B). The colored rectangles on the right of VIP scores (red, blue, and yellow) indicate the levels of metabolite up- and down-regulation in each chickpea cultivar. Hierarchical cluster analysis (heat map) (C) and VIP scores plot of PLS-DA (D) of 46 primary metabolites affected by Zn treatments in chickpea cultivars. The color range from top to bottom (red to blue) in the heat map scale represents the enrichment of metabolites in each cultivar. Red rectangles represent the up-regulation of expression, and blue represents the down-regulation\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/e710af5e6e134906614e1537.png"},{"id":66546440,"identity":"87e2a216-ecba-4d96-8e71-49b977921042","added_by":"auto","created_at":"2024-10-14 08:26:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1187405,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG\u003cstrong\u003e \u003c/strong\u003epathway analysis for enrichment ratio (A) and overview of enriched metabolites (B). The enrichment ratio is calculated as the number of hits within a particular metabolic pathway divided by the expected number of hits. See the MetaboAnalyst 6.0 website: \u003ca href=\"https://www.metaboanalyst.ca/MetaboAnalyst/Secure/enrichment/OraView.xhtml.\"\u003ehttps://www.metaboanalyst.ca/MetaboAnalyst/Secure/enrichment/OraView.xhtml.\u003c/a\u003e A big dot and higher graph with red color represent a more significant enrichment of pathways metabolism.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/438358518b574ce73d2a7fbd.png"},{"id":74834841,"identity":"1d92839a-5462-44bb-9ae6-00d3c7800f13","added_by":"auto","created_at":"2025-01-27 11:26:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2331790,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5188964/v1/9a6f1587-eead-4b44-a187-98d42c5824c7.pdf"}],"financialInterests":"","formattedTitle":"Assessment of zinc toxicity and tolerance in chickpea (Cicer arietinum L.) cultivars using physiological, biochemical and metabolomic responses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWorldwide, heavy metals (HM) seriously threaten agriculture security and has become an environmental problem (Aithani and Kushawaha \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Zinc (Zn) is a critical contaminant of the terrestrial and aquatic environment released from anthropogenic and natural sources (Khudsar et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Although, Zn is an essential trace element for all living organisms, however, at elevated levels (\u0026gt;\u0026thinsp;50 mg/L), it inhibits the normal ionic homeostasis system, provokes reactive oxygen species (ROS) production, and increases the level of membrane lipid peroxidation (Andrade et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, Zn toxicity causes chlorosis in young leaves, hindering photosynthesis and adversely affecting the nutritional status and Calvin cycle (Fariduddin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hence, the capacity of plants to effectively regulate the uptake, transportation, and scattering of Zn within their cells, organs, and intracellular compartments is essential for preserving their vital functions (Zlobin \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo counteract the toxic effects of HM, plants developed several adoptive strategies, including changing the cells' biochemical, physiological, and molecular status (Chen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Besides, plants overcome the toxicity of HM by compartmentalization, vacuolization, excluding, and accumulation or hyperaccumulation (Giannakoula et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, plants have a well-developed system of enzymatic and non-enzymatic antioxidants to detoxify HM stress (Al Mahmud et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Initially, plant cell walls play an important role as they contain specific polysaccharides that chelate or bind with HM metal ions and protect their entry into the cell (Yu et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It has been shown that glutathione and phytochelatins enhanced the resistance of non-hyperaccumulators plants to cadmium (Cd) and Zn stress (Jin et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After getting into the cell, a high amount of Zn is also chelated by organic acids, like citrate and malate (Kupper et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, plant cells secure themselves against Zn toxicity by making proteins, such as metallothioneins, which bind them in the organelles by sequestration or exporting (Palmiter \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Besides, plants protect themselves from Zn toxicity by regulating Zn transporters, like ZRT/IRT-like protein (ZIP), metal tolerance protein (MTP), and Zn-induced facilitator (ZIF) (Kaur 2021). Likewise, a specific group of proteins called \"plant defense proteins type I\" (PDF1s) has been discovered and characterized because of the role they play in making plants resistant to Zn (Mirouze et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Similarly, plants exposed to Zn excess can develop anti-oxidative defense system, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) to scavenge ROS (Fariduddin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, stressed plants defend their cells from oxidative damage by producing high levels of osmolytes like soluble sugar and proline (Kumar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been demonstrated that plant metabolites contribute to metal stress resistance. Amino acids, such as histidine, methionine, and asparagine, can chelate HM ions, including Zn, in plant cells and xylem fluid, eventually enhancing plant tolerance (Ghuge et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Proline and total amino acids increase in the leaves when there is an excess of Zn in the soil, suggesting their responsive role in maintaining osmotic pressure (Spormann et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, phenolic compounds chelate Zn into the vacuole by sequestering or excluding it from the membrane space via transporters, which is essential to their homeostasis (Vega et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, metabolomics profiling was performed to understand the role of metabolites as stress responses in chickpea cultivars under Zn stress. Chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) is a valuable legume crop, rated third after beans and peas globally. The seeds of it are a rich source of protein, carbohydrates, and fats (Ullah et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Besides, it has significant health benefits and helps to reduce diabetic, cardiovascular, and cancer risks (Merga and Haji \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are limited reports on the toxicity of Zn in chickpeas in a hydroponic environment. Hence, the current study aims to broadly explore the relative tolerance of chickpea cultivars, and examine their physiological, biochemical, and metabolomics responses under various Zn levels.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant material and cultivation\u003c/p\u003e \u003cp\u003eThe seeds of chickpea (\u003cem\u003eC. arietinum\u003c/em\u003e L.) cultivars, [ICCV89310 (IC8), NC234 (NC2), and ICCV89323-B (IC8-B)] were obtained from the Crop Genetic Resources Institute, Xinjiang Academy of Agricultural Sciences, Xinjiang, China. Before germination, seeds were sanitized with 75% ethanol and 6% NaClO solution and washed several times with distilled water. After soaking for 24 h, the seeds were sown in plastic trays containing vermiculite and incubated in a germinator for 12 days. The growing chamber was climate-controlled, with a day/night temperature of 22\u0026ndash;25 \u003csup\u003eo\u003c/sup\u003eC, 70% relative humidity, and a 14/10 h light/dark cycle. Uniform-sized seedlings were then allowed to grow for 8 days in Hoagland nutrient solution (6 plants/pot). After an initial growth of 20 days, Zn was applied to the nutrient solution as ZnSO\u003csub\u003e4\u003c/sub\u003e in different concentrations (0, 50, 100, and 150 \u0026micro;M). Seedlings were exposed for one week under Zn stress in Hoagland nutrient solution. The concentrations of Zn were selected based on earlier reports (Andrade et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Cherif et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The nutrient solution was changed every four days.\u003c/p\u003e \u003cp\u003eMeasurement of growth and physiological indices\u003c/p\u003e \u003cp\u003eAfter a week of Zn application, plants were collected and divided into roots and shoots. The lengths of root and shoot, as well as fresh weight (FW), were determined. For dry weight (DW), plant tissues were oven-dried at 120\u0026deg;C for 20 min and remained for three days at 60\u0026deg;C to measure their constant biomass.\u003c/p\u003e \u003cp\u003eDried samples of root and shoot were crushed into powder, weighed (0.3 g), and digested with an acid mixture (HNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;HClO\u003csub\u003e4\u003c/sub\u003e) to determine the content of Zn using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima-8300 DV; Perkin-Elmer, Inc., Waltham, MA, USA). The translocation factor (TF) of Zn from root to shoot and shoot water content (SWC) was calculated using the following formulas:\u003c/p\u003e \u003cp\u003eTF = [Zn]\u003csub\u003eshoot\u003c/sub\u003e/[Zn]\u003csub\u003eroot\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eSWC= (FW-DW)/FW x 100.\u003c/p\u003e \u003cp\u003e The growth tolerance index (TI) of root and shoot was measured separately based on DW, according to Chen et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). TIr and TIs represent the tolerance indices of root and shoot, respectively.\u003c/p\u003e \u003cp\u003eTIr\u0026thinsp;=\u0026thinsp;root biomass \u003csup\u003e(Zn)\u003c/sup\u003e / root mean biomass \u003csup\u003e(control)\u003c/sup\u003e x 100,\u003c/p\u003e \u003cp\u003eTIs\u0026thinsp;=\u0026thinsp;shoot biomass \u003csup\u003e(Zn)\u003c/sup\u003e / shoot mean biomass \u003csup\u003e(control)\u003c/sup\u003e x 100.\u003c/p\u003e \u003cp\u003eDetermination of chlorophyll contents\u003c/p\u003e \u003cp\u003eThe chlorophyll contents were quantified following the method explained by Fang et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In brief, 100 mg of fresh leaves were ground, and their extract was combined with 4 mL of a 2:1 acetone and ethanol mixture. The chlorophyll a and b absorbance levels were 665 and 649 nm, respectively.\u003c/p\u003e \u003cp\u003eDetermination of oxidative stress markers (MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and EL)\u003c/p\u003e \u003cp\u003eMalondialdehyde (MDA) content in the leaves was estimated as depicted by Hasanuzzaman and Fujita. (2013), using TBA (thiobarbituric acid) as the reactive agent. The absorbance of a colorful supernatant was measured at 532 nm and 600 nm. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was determined according to Yu et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), utilizing 0.1% TiCl\u003csub\u003e4\u003c/sub\u003e in 20% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (v/v) at 410 nm. Electrolytic leakage (EL) was identified according to Callegari et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) using the following formula:\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEL (%) = (EC1/EC2) \u0026times; 100\u003c/h2\u003e \u003cp\u003eEnzyme assays\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) activity was assayed using the reduction method of nitroblue tetrazolium solution (NBT) (Zhang et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), whereas peroxidase (POD) activity was detected using the guaiacol method of Afzal et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The absorbances were monitored at 560 and 470 nm, respectively. Catalase (CAT) and glutathione reductases (GR) were assayed corresponding to the protocols of Li et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and the absorbance was read at 240 and 340 nm, respectively.\u003c/p\u003e \u003cp\u003eOsmolytes detection\u003c/p\u003e \u003cp\u003eProline (Pro) content was measured using the acid ninhydrin method (Bates et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1973\u003c/span\u003e), and the absorbance was read at 520 nm. Soluble sugars (SS) were determined following the method Irigoyen et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) described, and the absorbance was detected at 625 nm. Total protein (Prot) content was assayed using the Bradford reagent (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Bovine serum albumin was standard, and the absorbance was measured at 595 nm.\u003c/p\u003e \u003cp\u003eAnalysis of primary metabolites\u003c/p\u003e \u003cp\u003ePrimary metabolites were extracted from shoot tissues using the method of Yang et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with minor changes. In total, 90 mg samples were homogenized with 540 \u0026micro;L cold methanol and 60 \u0026micro;L internal standard (L-2-chloro-phenylalanine, 0.3 mg/mL). Following a 30 min ultrasonication period, 600 \u0026micro;L of water and 300 \u0026micro;L of chloroform were added to the samples. After vortexing (2 min) and sonicating (30 min), the samples were centrifuged (10 min) at 12000 rpm and 4\u0026deg;C. Next, the supernatant of 800 \u0026micro;L was moved to a glass sample container and dried using a vacuum (15000 rpm for 8 h at 40\u0026deg;C). The dried residue was redissolved by adding 400 \u0026micro;L of methoxyamine (15 mg/mL in pyridine). Then, samples were vortexed (2 min) and incubated (90 min) at 37\u0026deg;C. The solution was treated with 400 \u0026micro;L of N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) (15 mg/mL in 1% trimethylchlorosilane) and 60 \u0026micro;L of n-hexane successfully. Subsequently, the collected solution was injected into a GC-MS (Agile 7890A-5975C, Agilent Technologies, Inc., Santa Clara, U.S.A.). The separation process was conducted using a non-polar DB-5 capillary column (30 m \u0026times; 250 \u0026micro;m ID, J\u0026amp;W Scientific, Folsom, U.S.A.) in conjunction with a constant flow rate of 1 mL/min and helium as a carrier gas. The initial programming temperature for the gas chromatography (GC) was 50\u0026deg;C, subsequently increasing by 8\u0026deg;C/min. The oven temperature was scaled from 125\u0026deg;C, 15\u0026deg;C/min to 170\u0026deg;C, 4\u0026deg;C/min to 210\u0026deg;C, 10\u0026deg;C/min to 270\u0026deg;C, 5\u0026deg;C/min to 305\u0026deg;C, and remained constant for 5 min. The electron impact (EI) ion source was maintained at 70 eV, and the acquisition rate was 20 spectra/sec for a scanning range of 50 to 500 m/z.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe results underwent statistical analysis using Duncan's Multiple Range Test (DMRT) at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All collected data were analyzed for variance (ANOVA) using the COSTAT computer package version 6.4 (CoHort software in Monterey, California, USA). Pearson's correlation and principal component analysis (PCA) were conducted to examine the correlation between morpho-physiological and biochemical indicators using OriginPro-2024. Hierarchical clustering analysis (HCA), partial least squares-discriminant analysis (PLS-DA), and sparse partial least squares-discriminant analysis (sPLS-DA) were performed using MetaboAnalyst 6.0 to analyze the data. Subsequently, KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/MetaboAnalyst/Secure/enrichment/OraView.xhtml\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca/MetaboAnalyst/Secure/enrichment/OraView.xhtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to construct metabolic pathway. The data represent the average of at least three replicates per treatment under the same conditions (n\u0026thinsp;=\u0026thinsp;3). The values of the results were displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEffects of Zn on growth and physiological parameters\u003c/p\u003e \u003cp\u003eAll chickpea cultivars (IC8, NC2, and IC8-B) responded differently to the applied Zn levels in the growth medium. The decrease in root and shoot length was concentration-dependent in all cultivars compared to control plants. For instance, the length of root and shoot was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in IC8 (7.34 and 14.3 cm) than in NC2 (4.4 and 11.3 cm) and IC8-B (2.9 and 6.8 cm) at excess of Zn (150 \u0026micro;M). The decrease of root and shoot length in IC8, NC2, and IC8-B was 425.3%, 706.6%, 832.1%, and 719.7%, 729.9%, and 451.3%, respectively, relative to the control. Additionally, the FW of the three chickpea cultivars was examined and decreased significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 150 \u0026micro;M of Zn, total FW decreased 560.1%, 534.3%, and 686.5% for IC8, NC2, and IC8-B, respectively, compared to control plants (Table S1).\u003c/p\u003e \u003cp\u003eAfter three days of Zn exposure, the symptoms of toxicity were noticed. The visual observations revealed that IC8-B exhibited more toxicity symptoms of shoot and root compared to cultivars NC2 and IC8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The phytotoxicity trend at 150 \u0026micro;M of Zn was IC8-B\u0026thinsp;\u0026gt;\u0026thinsp;NC2\u0026thinsp;\u0026gt;\u0026thinsp;IC8\u0026thinsp;\u0026gt;\u0026thinsp;control plants. The decrease in DW was more prominent in the root compared to the shoot. Under high concentration of Zn, IC8 recorded the highest DW (0.15, 1.64, and 1.79 g), followed by NC2 (0.10, 1.35, and 1.45 g), and IC8-B (0.04, 1.09, and 1.13 g), receptively, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, and D). Considering the growth attributes, IC8 was potentially a more tolerant cultivar than NC2, while IC8-B was more sensitive, especially at 150 \u0026micro;M of Zn exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, several physiological indices were considered to investigate the overall health of cultivars IC8, NC2, and IC8-B seedlings. An elevated concentration of Zn in the growth medium led to a substantial decrease (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the root-to-shoot ratio (R/S). Under Zn excess, the R/S ratios of IC8, NC2, and IC8-B were 0.09, 0.07, and 0.03, respectively. No statistical difference in R/S was observed between the IC8 and NC2 cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the content of water in shoots (SWC) was lowered (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the excessive dose of Zn (150 \u0026micro;M). In contrast, no such change was noticed at 50 and 100 \u0026micro;M relative to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Substantially, Zn at 150 \u0026micro;M was highly detrimental and disturbed the SWC more severely in NC2 and IC8-B than in cultivar IC8. All cultivars significantly declined the tolerance index (TI) values under Zn stress relative to control. Zn at 150 \u0026micro;M was more acute and reduced the TI of root (TIr), where IC8 showed the highest value (48.90), while IC8-B exhibited the lowest value (12.97) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Similarly, TI of shoot (TIs) were significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in IC8 compared to NC2 and IC8-B at 150 \u0026micro;M of Zn. The values of TIs for cultivars IC8, NC2, and IC8-B ranged from 90\u0026ndash;100%, 75\u0026ndash;92%, and 57\u0026ndash;93%, respectively. Exceptionally, cultivar NC2 accounts for a higher TIs value (74.70) than IC8-B at 150 \u0026micro;M of Zn (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eBesides, to better understand the phytotoxicity and tolerance of IC8, NC2, and IC8-B, Zn content was studied in the root and shoot tissues of seedlings. Generally, the amount of Zn is enhanced in the root and shoot with increasing Zn levels in the nutrient solution. The content of Zn was higher in the root than in the shoot. The root of cultivar IC8-B seedlings exhibited high Zn content (47.41\u0026ndash;3106.80 \u0026micro;g/g DW), followed by NC2 (39.22-2997.20 \u0026micro;g/g DW), while IC8 possessed the lowest Zn content (31.16\u0026ndash;2866.70 \u0026micro;g/g DW). Conversely, high Zn content was recorded in the shoots of cultivars IC8 (645.56 \u0026micro;g/g DW) and NC2 (415.36 \u0026micro;g/g DW), while IC8-B showed the lowest Zn content (186.51 \u0026micro;g/g DW) under high Zn treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, the values of translocation factor (TF) decreased in all cultivars with the application of Zn in growth medium. However, IC8 and NC2 revealed comparatively higher values of TF than IC8-B at 150 \u0026micro;M of Zn. Besides, the highest TF values were recorded in control plants for IC8 and NC2, followed by IC8-B (0.47, 0.30, and 0.12), respectively. Considerably, the transfer of Zn (TF) was more significant at 50 and 100 \u0026micro;M of stress levels (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZn content analysis and translocation factor (TF) of three chickpea cultivars grown hydroponically at various concentrations of Zn for 25 days\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCultivars\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatments (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZn content in root (\u0026micro;g/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZn content in shoot (\u0026micro;g/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eIC8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e926.24\u0026thinsp;\u0026plusmn;\u0026thinsp;9.78\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e154.37\u0026thinsp;\u0026plusmn;\u0026thinsp;14.20\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2367.27\u0026thinsp;\u0026plusmn;\u0026thinsp;54.74\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e342.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2866.70\u0026thinsp;\u0026plusmn;\u0026thinsp;64.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e645.56\u0026thinsp;\u0026plusmn;\u0026thinsp;16.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eNC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e967.55\u0026thinsp;\u0026plusmn;\u0026thinsp;16.71\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2567.84\u0026thinsp;\u0026plusmn;\u0026thinsp;128.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e336.73\u0026thinsp;\u0026plusmn;\u0026thinsp;4.64\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2997.20\u0026thinsp;\u0026plusmn;\u0026thinsp;76.63\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e415.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eIC8-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e984.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122.61\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2665.53\u0026thinsp;\u0026plusmn;\u0026thinsp;64.74\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e258.95\u0026thinsp;\u0026plusmn;\u0026thinsp;10.71\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3106.80\u0026thinsp;\u0026plusmn;\u0026thinsp;65.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e186.51\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe data is shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;3). Values in columns with the same characters imply a non-significant variation depending on DMRT at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEffects of Zn on chlorophyll contents and oxidative damage\u003c/p\u003e \u003cp\u003eAdding Zn to the growth medium considerably impacts the chlorophyll (chl) contents. The toxicity of Zn reduced chl a, chl b, and total chl in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and C). Cultivar IC8, NC2, and IC8-B revealed non-significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the control medium, while total chl and chl b were slightly increased at 100 \u0026micro;M of Zn. High Zn stress was more harmful in IC8-B compared to IC8 and NC2. In contrast, cultivars NC2 and IC8 showed the lowest reduction of chlorophyll contents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxidative stress indices (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, EL, and MDA) significantly enhanced in the shoots of IC8, NC2, and IC8-B under different Zn levels related to control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest increase in oxidative stress markers was observed at 150 \u0026micro;M of Zn. At the same time, the lowest contents were noticed in control seedlings. Among the studied cultivars, IC8-B documented the highest contents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and EL (32.4 \u0026micro;mol/g FW and 58.5%), followed by NC2 (23.02 \u0026micro;mol/g FW and 49.1%) and IC8 (20.97 \u0026micro;mol/g FW and 46.3%), respectively, at 150 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E). A similar trend was observed in MDA content under control and Zn-treated plants. The content of MDA in cultivars IC8-B, NC2, and IC8 increased by 44.2%, 37.02%, and 35.4%, respectively, at 150 \u0026micro;M of Zn related to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eEffects of Zn on antioxidants and osmolytes contents\u003c/p\u003e \u003cp\u003eDifferent enzymatic and non-enzymatic activities and their ratios were assessed under different Zn treatments to examine the defense system of chickpea cultivars. Compared to control plants, the activity of SOD and POD were stimulated significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by Zn supplementation in all cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Cultivars IC8, NC2, and IC8-B treated with 150 \u0026micro;M of Zn showed an intensification in the activity of SOD and POD by 9.1% and 8.6%, 7.6% and 7.3%, and 8.1% and 6.9%, respectively, over control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The activity of catalase (CAT) and glutathione reductases (GR) increased by 3.8%, 2.73%, and 3.4% and 2.03%, respectively, in IC8 and NC2 under 150 \u0026micro;M of Zn relative to control seedlings. Conversely, CAT and GR activity in IC8-B under 150 \u0026micro;M of Zn was shown to be much lower than in the control (2.5% and 1%, respectively). In addition, CAT and GR decreased slightly in IC8 and NC2 with the increasing concentration of Zn, but still higher than in control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlso, the increasing concentration of Zn resulted in a significant increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the levels of proline, soluble sugars, and total protein in the shoots of chickpea cultivars. The content of proline increased by 456.8%, 341.4%, and 321.3%, respectively, in IC8, NC2, and IC8-B treated with 150 \u0026micro;M of Zn, compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In contrast, the percentage increase of soluble sugars in IC8, NC2, and IC8-B at 150 \u0026micro;M of Zn was 119%, 90.6%, and 64.5%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, the percentage increase of total protein in IC8, NC2, and IC8-B at 150 \u0026micro;M of Zn was, 56%, 47.4%, and 29.3%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Overall, osmolytes were more pronounced at 150 \u0026micro;M of Zn, while control plants possessed the lowest active substances in all cultivars. Comparatively, the trend of osmolytes increase was in the order of IC8\u0026thinsp;\u0026gt;\u0026thinsp;NC2\u0026thinsp;\u0026gt;\u0026thinsp;IC8-B.\u003c/p\u003e \u003cp\u003eMultivariate statistical analysis of different variables\u003c/p\u003e \u003cp\u003ePearson's correlation revealed significant variation (positive or negative) in growth, physiological, and biochemical attributes in chickpea cultivars treated with Zn, where a positive correlation exists among the closely related variables in the same quadrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The statistics of correlation in growth parameters with each other (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 0.01) and with chlorophyll contents were positive. However, a negative correlation existed with Zn (r\u0026thinsp;+\u0026thinsp;s). Moreover, stress biomarkers (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, EL, and MDA), antioxidants (SOD, POD), and osmolytes (Pro, SS, and Prot) were negatively correlated with growth (TPH, TDW), and physiological indices (R-S. R, and SWC). In addition, CAT and GR have no significant correlation with all parameters. Subsequently, positive correlations exist within antioxidants, stress biomarkers, and osmolytes while negatively correlated with growth and physiological attributes. Crucially, all parameters positively correlated with Zn content in the root and shoot, while a negative correlation was found with chlorophyll.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe principal component analysis (PCA) reveals significant variations in growth and physio-biochemical features in chickpea cultivars exposed to Zn applications. A two-dimensionally constructed diagram of PCA showed two different variability percentages of the principal component (PC), such as PC1 and PC2, at 61.7% and 24.4%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Growth and physiological parameters revealed smaller angles with each other (\u0026lt;\u0026thinsp;90\u0026deg;) and with CAT and GR activity, thereby indicating their positive relationship. Additionally, a positive relationship exists between osmolytes, ROS, SOD, POD, and Zn (r\u0026thinsp;+\u0026thinsp;s). At the same time, no significant relation was shown with CAT and GR activity along with growth and physiological indices. The indicators with the maximum contribution to PC1 had a 64.1% variance difference, while in PC2, there was approximately 35.1% variation.\u003c/p\u003e \u003cp\u003eMetabolic assay\u003c/p\u003e \u003cp\u003eForty-six metabolites were identified in the shoot of three chickpea cultivars, belonging to different groups, including amino acids (14), organic acids (13), amines (6), sugars (4), alcohols (4), and others (5). The PLS-DA of the scores plot for 30 metabolites with discrimination (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1) indicated that cultivar IC8 expressed the higher concentration of metabolites with 53.3%, followed by NC2 (30%) and IC8-B (16.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Subsequently, sPLS-DA was performed to better understand the relationship and closeness of IC8, NC2, and IC8-B treated with Zn stress. The scores plot of sPLS-DA suggested that cultivars IC8 and NC2 were remarkably close in response to Zn stress and closely overlapping each other, while a contrasting pattern of IC8-B was observed relative to them (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The scoring plot of sPLS-DA revealed 75% and 29.6% of percentage difference in component 1 and 2, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimultaneously, to complement our observation regarding the tolerance abilities and toxicity of chickpea cultivars, a hierarchical clustering analysis (HCA) was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The samples of chickpea cultivars were analyzed three times to observe the metabolic abundance and enrichment in the shoots. The accumulation of primary metabolites in each replicate of the tested chickpea cultivars showed an apparent fluctuation in their abundance and expression in shoots. However, to further rectify the expression of metabolites in IC8, NC2, and IC8-B, visualization was performed for 46 metabolites with sPLS-DA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In total, IC8 and NC2 revealed the highest expression and abundance of metabolites with 47% and 32.6%, respectively, followed by IC8-B with 19.5%. The results presented above provide tentative evidence that IC8 and NC2 shoots exhibit significant levels of responsive metabolites, possibly contributing to their tolerance. In contrast, the partial or intermediate expression of metabolites in IC8-B indicates its sensitivity under Zn stress.\u003c/p\u003e \u003cp\u003eTo further understand the effects of Zn stress in shoot of IC8, NC2 and IC8-B, the pathway enrichment analysis of metabolites was conducted using KEGG database. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and D, 16 pathways were significantly enriched in the shoot, whereas 9 pathways were depressed. Among them, phenylalanine, tyrosine, and tryptophan metabolism were more enriched, while porphyrin and glycolysis (gluconeogenesis) pathways were significantly affected in the shoot.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eZinc (Zn) is a micronutrient that is necessary for the growth and development of plants. However, elevated concentrations can cause severe toxicity by disturbing the physiological and metabolic processes (Garg and Singh \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The present findings indicated that cultivars IC8, NC2, and IC8-B varied in response to Zn stress, such as the plant height and biomass were decreased under high Zn supply. The decline in growth parameters may attributes to a significant Zn buildup in various plant parts that impede the uptake of nutrients and cause damage to the ultrastructure of plant tissues (Cherif et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In contrast, the high ratio of R/S in plants exposed to HM stress suggests its strong affinity for the uptake of nutrients from its surroundings and leads to more aerial plant biomass (Agathokleous et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the current work, cultivars IC8 and NC2 revealed higher biomass and R/S ratio than IC8-B, suggesting strong tolerance abilities, while IC8-B was sensitive under high Zn stress.\u003c/p\u003e \u003cp\u003eAlso, a significant decrease in SWC was noticed in all cultivars under high Zn supply. In plants, the reduction of water content maybe due to the high Zn uptake in aerial parts or reduced root biomass and surface area (Garg and Singh \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, Zn may affect the plant water status by limiting the hydraulic conductivity and reducing xylem tissue available for water conduction (De Silva et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similar to our results, Bonnet et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported the decrease of water content in ryegrass (\u003cem\u003eLolium perenne\u003c/em\u003e). TI is critical in determining plants' tolerance capacity to HM stress. In this study, the tolerance capacity of the root was less (TIr\u0026thinsp;\u0026lt;\u0026thinsp;60) in all cultivars under Zn excess. In contrast, a much higher tolerance ability of shoot in IC8 (91.20%) and NC2 (74.69%) was noticed (TIs\u0026thinsp;\u0026gt;\u0026thinsp;60) compared with IC8-B (TIs\u0026thinsp;\u0026lt;\u0026thinsp;60). Plant species under HM stress with TI\u0026thinsp;\u0026gt;\u0026thinsp;60 were reported to be tolerant (Lux et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe accumulation of metals varies significantly within plant species and cultivars and is influenced by edaphic factors (Chen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Callegari et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the root is the first point of connection to metal toxicity and accounts for more uptake. In this study, IC8-B and NC2 showed a higher Zn storage in the root than in IC8, while in the shoot, the uptake was higher in IC8 and NC2 than in IC8-B. Impa et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) attribute the increased Zn contents in the shoots of tolerant species primarily to better root development matching. In IC8-B, the mechanism of Zn restriction in the root may weaken the root's ultrastructure, ultimately decreasing the above-ground biomass. The immobilization of Zn in the root happens through symplastic flow to the vacuole, thereby showing a substantial storage area for Zn excess (Kabir et al. 2017). The TF values of all cultivars were less than 1, indicating that none of them could effectively transfer Zn from their roots to shoots. However, the highest TF was noticed in IC8 than the other ones. Dinh et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) stated that the decrease of TF in Zn-treated plants relative to control could be associated with the retention of Zn in their roots.\u003c/p\u003e \u003cp\u003eDecreased photosynthetic pigment is the leading indicator of Zn toxicity in plants with obvious chlorosis symptoms (Cherif et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, chlorophyll content decreased in all cultivars with increased Zn concentrations. The decline in chlorophyll may be related to the blockage of photo-electron transport and disorganization of chloroplasts or fewer thylakoid and grana and under Zn excess (Sidhu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been demonstrated that HM stress increases the production of singlet oxygen (O\u003csub\u003e2\u003c/sub\u003e), superoxide radical (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e), hydroxyl radical (\u003csup\u003e\u0026bull;\u003c/sup\u003eOH), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hence increasing the accumulation of ROS. Subsequently, ROS production increased lipid peroxidation, which caused oxidative cell damage (Huihui et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Generally, MDA is considered an essential indicator of oxidative damage, which is the ultimate breakdown product of lipid peroxidation (Kumar et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our data illustrated a considerable rise in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, EL, and MDA contents. However, IC8-B has higher oxidative stress indices than IC8 and NC2, suggesting high sensitivity under high Zn stress. Likewise, Singh et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that severe stress of Cr increased MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents in sensitive chickpea genotypes.\u003c/p\u003e \u003cp\u003eTo avoid cell damage and detoxify the ROS, plants have a highly developed system of antioxidants, including SOD, POD, CAT, GR, and several others (Al Mahmud et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our results documented that the activities of antioxidants were higher in IC8 and NC2 relative to IC8-B. SOD provides the first line of protection against the harmful levels of ROS using the metal-catalyzed Habere-Weiss-type reaction, eliminates O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and reduces the potentially detrimental impact of \u003csup\u003e\u0026bull;\u003c/sup\u003eOH production (Gill et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Similarly, CAT and GR decreased the overproduction of ROS caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and HM excess (Ghuge et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, GR maintains the reduced environment within the plant cell by transforming oxidized glutathione (GSSG) into reduced glutathione (GSH) using the ascorbate-glutathione cycle (Madhu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, IC8-B showed a significant decline in CAT and GR under high Zn supply compared to control, which may be anticipated to severe oxidative stress, as described previously (Wang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Tripathi et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) also demonstrated a substantial decline in the activity of CAT and GR under Zn stress. Plants also have a powerful defense system of osmolytes against stress biomarkers. Proline functions as an antioxidant and osmoprotectant in plants to ameliorate the oxidative damage induced by ROS (Spormann et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Soluble sugars and proteins have been demonstrated to detoxify HM and sustain biological molecules and membranes in tolerant cultivars (Kumar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overall, the antagonistic response of antioxidants and their production in plants may be affected by the crop species, cultivars, developmental stages, time, and level of exposure to HM stress (Wang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the present work, IC8 and NC2 contained considerably more proline, soluble sugars, and total protein than IC8-B.\u003c/p\u003e \u003cp\u003eMetabolite profiling is an efficient tool for finding metabolites and metabolic pathways in studying plant stress responses. Under environmental stress, several metabolites, including organic acids, amino acids, sugars, and sugar alcohols, can act as signal transduction molecules, osmoprotectants, and antioxidants (Shulaev et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, IC8 and NC2 revealed up-regulation, or higher expression of metabolites, while IC8-B exhibited down-regulation or partially expressed. Previous studies have shown that amino acids and their derivatives trigger the defense system of plants during growth and development and serve as substrates for various metabolites that respond to different abiotic factors (Batista-Silva et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Particularly histidine plays a crucial role in maintaining the balance of Zn in the root and facilitating its storage and transportation through the xylem in \u003cem\u003eThlaspi caerulescens\u003c/em\u003e shoots (Salt et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Similarly, asparagine may reduce the toxicity of Zn in \u003cem\u003eDeschampsia cespitosa\u003c/em\u003e by establishing an intracellular compound with it (Smirnoff and Stewart \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Moreover, tryptophan is an essential amino acid for plants to grow normally. It helps to open the stomata, regulates ions transport, and reduces HMs toxicity (Sadak et al. 2021). Remarkably, our findings showed significant enrichment of tryptophan metabolism in the pathway of tyrosine, phenylalanine, and tryptophan biosynthesis under Zn stress in chickpea cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Allantoin has been identified as a compound that enhances plant tolerance, especially under HM stress (Nourimand and Todd \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It makes up 90% of all the nitrogenous compounds mobilized inside legumes' plant tissue (Kaur et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, incrementing allantoin content under HM stress may enhance plant tolerance. Similarly, glutamic acid is the precursor of chlorophyll (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) which is up-regulated in cultivar IC8. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, and B revealed similar results in IC8 and NC2 chickpea cultivars regarding histidine, tryptophan, asparagine, allantoin and glutamic acid. Moreover, a series of other critical amino acids like methionine, glycine, alanine, and proline, etc., are up-regulated in the shoots of IC8 and NC2, implying their tolerance abilities under Zn stress compared to IC8-B.\u003c/p\u003e \u003cp\u003eOrganic acids are the main phytomolecules in the related network, accumulating more in IC8 than in NC2 and IC8-B. Sidhu et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that organic acids are crucial to HM tolerance in plants. Sarret et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) indicated that \u003cem\u003eArabidopsis halleri\u003c/em\u003e exhibited a high amount of malic acid, with Zn preponderantly combined with malic acid in the shoot. Similarly, benzoic acid is crucial for the growth of plants and increases their tolerance to abiotic stresses (Widhalm and Dudareva \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These results suggested that high accumulations of organic acids can chelate HM and rapidly detoxify them in shoots. Likewise, the accumulation of sugar molecules in shoots is associated with alleviating abiotic stress (Hassan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Sugars like sucrose and hexoses increase stress tolerance by down-regulating genes linked to stress and up-regulating-controlling genes related to growth (Salam et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similar results were investigated in the present work, where sugar molecules (sucrose) are up-regulated in IC8, while no expression was observed in NC2 or IC8-B. Moreover, indoleacetic acid, naringenin, cysteamine, xanthine, spermidine, etc., were also up-regulated in the tested chickpea cultivars. However, the overall expression of targeted metabolites was higher in IC8 (47.8%) and NC2 (32.6%) than in IC8-B (19.5%), indicating the tolerant and sensitive capacities of IC8, NC2, and IC8-B, respectively, in response to Zn stress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eGlobally, HM pollution is a significant risk to agriculture safety and has become a major environmental issue. In the present study, Zn supplementation (50, 100, and 150 µM) significantly influenced the growth, physiological, and biochemical traits of the experimented cultivars (IC8, NC2, and IC8-B) in hydroponics. In particular, high Zn exposure (150 µM) severely affected the growth and biomass, R/S ratio, SWC, and chlorophyll content. Moreover, differential responses were observed among the tested cultivars concerning the tolerance indices, Zn content analysis, oxidative stress markers, osmolytes, and antioxidant enzymes. The increased ROS likely results from suppressed growth parameters and low photosynthetic pigment contents. Hence, high Zn levels significantly promoted the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, EL, and MDA and elicited oxidative stress in chickpea cultivars. The growth and physio-biochemical characteristics response confirmed that IC8 and NC2 exhibited better Zn tolerance than IC8-B. The relative tolerance potential of IC8 and NC2 may be attributed to different adaptive strategies, such as a well-developed profile of responsive metabolites, such as histidine, asparagine, tryptophan, allantoin and others. Similarly, antioxidative defense enzymes (SOD, POD, CAT and APX), and soluble components, such as proline, soluble sugars and total protein may contributed to the tolerance capabilities of IC8 and NC2. In contrast, the low tolerance abilities of IC8-B based on growth, physio-biochemical, and metabolic responses confirmed it to be more sensitive when subjected to Zn excess. Hence, cultivar IC8-B maybe utilized as a control cultivar under Zn stress to evaluate other chickpeas' tolerance capacity. Besides, we suggested IC8 and NC2 as promising candidates for Zn-contaminated soil. However, more in-depth molecular exploration of these responses is required to understand the mechanisms behind Zn toxicity and tolerance and how they relate to diverse metabolic processes. These findings not only enhance our understanding of Zn-mediated stress in chickpeas but also offer valuable insights for optimizing stress adaptation strategies in diverse cultivars, paving the way for improved agricultural practices and crop resilience. The findings also stipulate new metabolites understanding for the response of Zn-treated chickpeas and perhaps contribute to a better kind of stress adjustment in other chickpea cultivars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contributions:\u0026nbsp;\u003c/strong\u003eShakir Ullah and Xingfan Li: Designed and performed the experiments. Uzma Salam: Conceived and designed the experiment. Ahmed A. Elateeq: Analyzed the data.\u0026nbsp;Ilbong Ri: Methodology and investigation.\u0026nbsp;Shakir Ullah: Writing-original draft and validation. Shakir Ullah and Ahmed A. Elateeq: Writing-review and editing. Dewen Li: Resources and conceptualization. Xiaorui Guo: Contributed reagents and materials. Zhonghua Tang: Supervision, and funding.\u0026nbsp;Mahmoud F. Seleiman:\u0026nbsp;Validation, and Funding. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by \u0026quot;The Key Resource and Development Project of Heilongjiang Province, China (JD22A008)\u0026quot;, and Researchers Supporting Project King Saud University, Riyadh, Saudi Arabia (No. RSPD2024R751).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAfzal J, Hu C, Imtiaz M, Elyamine A, Rana M, Imran M, Farag M (2019) Cadmium tolerance in rice cultivars associated with antioxidant enzyme activities and Fe/Zn concentrations. 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Plant Physiol Biochem 162:327-335. https://doi.org/10.1016/j.plaphy.2021.03.003\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cicer arietinum, zinc toxicity, antioxidants, oxidative stress, metabolites.","lastPublishedDoi":"10.21203/rs.3.rs-5188964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5188964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground and Aims\u003c/em\u003e: Zinc (Zn) is an essential microelement that plants need for appropriate growth and development. However, high concentrations may hamper the physio-chemical and metabolic processes and weaken plant growth. This study aims to broadly explore the relative tolerance of chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) cultivars, and examine their physiological, biochemical, and metabolomics responses under various Zn levels.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods\u003c/em\u003e: Three chickpea cultivars: ICCV89310 (IC8), NC234 (NC2), and ICCV89323-B (IC8-B) were exposed to different Zn levels (Ck, 50, 100, and 150 µM) for one week in a hydroponic medium. Growth and physiological indices, oxidative stress markers, antioxidant enzymes activity, and osmolytes content were detected. Primary metabolites profile and accumulation of Zn were assessed using GC-MS and ICP-OES, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e: IC8 and NC2 cultivars exhibited more tolerance than IC8-B because of their high biomass and plant height, root-to-shoot ratio, shoot water, and chlorophyll contents under high Zn stress. Besides, Zn contents were higher in the root of IC8-B, while IC8 and NC2 showed high accumulation in the shoot. Under Zn stress, there was an increase in the concentrations of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), malondialdehyde (MDA), and electrolyte leakage (EL). Additionally, Zn supplementation positively regulated the activities of superoxide dismutase (SOD), peroxidase (POD), and osmolytes (proline, soluble sugars, and total protein), but catalase (CAT) and glutathione reductases (GR) were differential in response to Zn stress. Simultaneously, metabolomics profiling revealed forty-six responsive metabolites in IC8, NC2, and IC8-B, mainly consisting of organic acids, amino acids, amines, alcohols, and sugars.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusion\u003c/em\u003e: Cultivars IC8 and NC2 displayed superior tolerance to Zn stress compared to IC8-B, showcasing robust growth characteristics and biochemical responses. The relative tolerance potential of IC8 and NC2 may be attributed to different adaptive strategies, such as a well-developed profile of responsive metabolites, such as histidine, asparagine, tryptophan, allantoin and antioxidants. Hence, cultivar IC8-B maybe utilized as a control cultivar under Zn stress to evaluate other chickpeas' tolerance capacity. Besides, IC8 and NC2 can be suggested as promising candidates for Zn-contaminated soil.\u003c/p\u003e","manuscriptTitle":"Assessment of zinc toxicity and tolerance in chickpea (Cicer arietinum L.) cultivars using physiological, biochemical and metabolomic responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-14 08:26:39","doi":"10.21203/rs.3.rs-5188964/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa46e9e9-7768-451e-bde9-d5c36044bcc3","owner":[],"postedDate":"October 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T11:18:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-14 08:26:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5188964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5188964","identity":"rs-5188964","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}