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. 1A). 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. 1B, 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. 1E). 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. 1F). 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. 1G). 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. 1H).
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 ± 1.34f | 14.65 ± 1.58h | 0.47 ± 0.030a |
50 | 926.24 ± 9.78e | 154.37 ± 14.20f | 0.17 ± 0.014d |
100 | 2367.27 ± 54.74d | 342.84 ± 3.79c | 0.15 ± 0.003d |
150 | 2866.70 ± 64.01b | 645.56 ± 16.48a | 0.23 ± 0.008c |
NC2 | Control | 39.22 ± 0.26f | 11.81 ± 0.94h | 0.30 ± 0.020b |
50 | 967.55 ± 16.71e | 145.48 ± 1.80f | 0.15 ± 0.003d |
100 | 2567.84 ± 128.3c | 336.73 ± 4.64c | 0.13 ± 0.008de |
150 | 2997.20 ± 76.63ab | 415.36 ± 3.22b | 0.14 ± 0.005de |
IC8-B | Control | 47.41 ± 2.32f | 5.66 ± 0.98h | 0.12 ± 0.029de |
50 | 984.08 ± 4.77e | 122.61 ± 5.92g | 0.12 ± 0.003de |
100 | 2665.53 ± 64.74c | 258.95 ± 10.71d | 0.09 ± 0.003ef |
150 | 3106.80 ± 65.77a | 186.51 ± 7.42e | 0.06 ± 0.005f |
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. 2A, 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 (H2O2, 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 H2O2 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. 2D 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. 2F).
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. 3A 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. 3C 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. 3E). 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. 3F). 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. 3G). 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. 4A). 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 (H2O2, 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. 4B). 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. 5A). 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. 5B). 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. 5C). 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. 5D). 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. 6A 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.