3.1. Shoot and root dry weight
As an essential element, plants demand low quantities of Ni for optimal growth and development. However, higher concentrations of Ni cause toxicity and can lead to several harmful changes in plant physiology and anatomy 48. In the present study, high excess Ni doses can negatively affect plant growth; therefore, with increasing Ni concentration, a significant decrease was detected in the shoot and root DW of both populations of A. inflatum. The highest reduction in the shoot DW with 29.8% of M population and 21.6% and 24.4% in roots DW were observed in M and NM populations exposed to 400 µM Ni, respectively, compared to control (Table 1). Accordingly, Rathor et al. (2014) demonstrated a significant decline in dry matter yield in Zea mays plants due to the accumulation of elevated doses of Ni 49. Another study showed that Ni at lower concentrations (0–5 mg Ni/L) induced a meaningful increment in dry matter yield, but at higher concentrations (6–25 mg/L) caused toxic effects and led to a significant decrease in dry matter yield 50. However, the reduction in biomass of plants exposed to Ni stress is related to plant metabolism, photosynthesis, water relations, transpiration, disturbance in the absorption of the nutrient, and oxidative damage caused by Ni stress 51. Based on Table 1, using SA and JA alone or in combination in Ni-stressed plants in both populations exhibited different effects on plant mass. Using of SA augmented shoot and root dry matter of M plants, also root DW of NM plants under Ni treatments. In contrast, JA decreases the shoot DW of both populations under Ni treatments. The SA plays a main role in the regulating of cell growth, seed germination, seedling development, and ion uptake and transport 29. JA influences root growth by preventing the primary root, forming of lateral roots, regenerating the root, and reducing adventitious root formation 52. As shown in Table 1, the application of SA in high doses (200 µM) caused the highest DW in shoots (30.3%) and roots (27.5%) of M population plants under the highest Ni concentration relative to the stressed plants under 400 µM Ni alone. A similar result in the same treatment was displayed in the root region of the NM population (46.03%). Similarly, in strawberry plants, applying of Ni (150–300 mg/L) with SA (2 mM) increased the DW and FW of shoots and roots 53. Unlike SA, JA in the presence of high Ni concentrations did not induce significant alterations in shoot biomass of both populations. In contrast to this result, Azeem (2018) indicated that in Z. mays plants, JA at 6–10 µM increased the seedling emergence, leaf number, and shoot length alone and in combination with Ni (8 µM) 54. According to this study, JA via elevating the action of antioxidant enzymes ameliorated the damaging impacts of oxidative stress on biomass production, growth, and protein amounts in Ni-stressed plants. However, JA treatment increased root biomass in plants against 400 µM Ni stress in both populations to plants against the same dose of Ni. The highest DW of the M population was detected in the presence of 400 µM Ni + 50 and 200 µM SA + 10 µM JA. In this condition, the shoots and roots DW were 1.16–1.23 and 1.13–1.31 times, respectively, more than the stressed plants under 400 µM Ni alone. However, an enhancement in biomass by simultaneous application of SA with JA was observed only in the roots of the NM population under high Ni toxicity. For example, the highest increase in root DW (40.4%) was detected in 400 µM Ni + 200 µM SA + 5 µM JA treatments concerning the stressed-plants under 400 µM Ni alone. Based on Table 1, the maximum ratio R/S was detected in plants exposed to 400 µM Ni + 10 µM JA by 1.49- and 1.84-fold increment, in M and NM populations respectively, compared to untreated plants. In the same situation, the highest enhancement of this ratio was detected in the treatment of 400 µM Ni + 200 µM SA only in the NM population (1.84). Also, using SA and JA externally in plants against high Ni concentration only boosted the R/S ratio in the NM population by 1.54 compared to the control plant. Similarly, Zaid et al. stated that biomass of Brassica juncea L. decreased significantly in the presence of Ni stress particularly at 150 µM, relative to the nitrated control. Still, co-application of Ni with SA augmented shoot and root biomass 36. It has been informed that the exogenous usage of JA at 0, 1, and 60 µM doses in sunflower seedlings growth media decreased the primary root development and reduced the number of lateral roots 55. However, JA and SA act as essential signaling agents to adjust the defensive response in plants against abiotic stress, which can be due to the nature, exposure time, and intensity of stress in plants 29,52.
Table 1
Changes in DW the presence of SA and JA in two parts of populations (NM and M) of A. inflatum plants stressed with various doses of Ni.
Ni | SA | JA | M plant species | NM plant species |
Shoot Dry weight (g) | Root Dry weight (g) | Root/Shoot Ratio (%) | Shoot Dry weight (g) | Root Dry weight (g) | Root/Shoot Ratio (%) |
0 | 0 | Control | 0.315 ± 0.010cd | 0.074 ± 0.003cd | 23.4 ± 0.61g − n | 0.248 ± 0.006g − m | 0.045 ± 0.004s − v | 17.9 ± 1.35s − x |
5 | 0.217 ± 0.046fi | 0.065 ± 0.003g − k | 30.8 ± 6.90a − d | 0.218 ± 0.007g − o | 0.025 ± 0.003z − c | 11.4 ± 1.29y |
10 | 0.270 ± 0.017b | 0.063 ± 0.003j − k | 23.4 ± 1.38g − n | 0.248 ± 0.008g − k | 0.033 ± 0.003z − b | 13.3 ± 1.59xy |
50 | 0 | 0.356 ± 0.008ab | 0.074 ± 0.004cd | 20.8 ± 1.12g − o | 0.247 ± 0.004g − m | 0.036 ± 0.006z − b | 14.2 ± 1.89k − s |
5 | 0.233 ± 0.071b − f | 0.054 ± 0.002o − p | 25.3 ± 10.2f − i | 0.170 ± 0.005h − t | 0.039 ± 0.002v − y | 23.0 ± 1.56hi |
10 | 0.183 ± 0.041b − k | 0.053 ± 0.003o − q | 29.7 ± 6.52b − f | 0.181 ± 0.009h − r | 0.045 ± 0.004s − v | 24.6 ± 3.18g − l |
200 | 0 | 0.325 ± 0.014abc | 0.073 ± 0.003de | 22.5 ± 1.73g − n | 0.217 ± 0.008l − o | 0.034 ± 0.006z − a | 15.6 ± 2.95u − y |
5 | 0.259 ± 0.021def | 0.062 ± 0.005jk | 23.7 ± 0.18 a−d | 0.131 ± 0.008l − t | 0.040 ± 0.004v − y | 30.4 ± 1.79bc |
10 | 0.310 ± 0.033cde | 0.074 ± 0.004cd | 24.2 ± 3.90 g−m | 0.167 ± 0.010l − t | 0.035 ± 0.003z − b | 21.0 ± 2.32j − r |
100 | 0 | 0 | 0.284 ± 0.010def | 0.069 ± 0.004efg | 24.2 ± 0.90 g−m | 0.261 ± 0.006f − n | 0.048 ± 0.004st | 18.1 ± 1.54p − w |
5 | 0.224 ± 0.039def | 0.066 ± 0.002f − i | 30.0 ± 3.97bcd | 0.253 ± 0.010f − n | 0.038 ± 0.002x − b | 15.0 ± 1.04w − y |
10 | 0.214 ± 0.010e − i | 0.068 ± 0.003f − i | 31.7 ± 0.98abc | 0.222 ± 0.008k − p | 0.041 ± 0.002u − x | 18.3 ± 1.30p − w |
50 | 0 | 0.290 ± 0.016d − g | 0.076 ± 0.003bcd | 26.3 ± 2.51d − i | 0.231 ± 0.007i − p | 0.038 ± 0.003x − b | 16.2 ± 0.81t − x |
5 | 0.278 ± 0.045d − g | 0.060 ± 0.003klm | 22.1 ± 4.80j − q | 0.213 ± 0.005l − q | 0.052 ± 0.004opq | 24.2 ± 1.19g − l |
10 | 0.280 ± 0.030d − h | 0.065 ± 0.004ghi | 23.3 ± 2.38g − n | 0.179 ± 0.005l − r | 0.034 ± 0.003z − b | 18.6 ± 1.81l − t |
200 | 0 | 0.301 ± 0.017d − h | 0.073 ± 0.004de | 24.1 ± 1.00g − m | 0.243 ± 0.011i − m | 0.042 ± 0.005u − x | 17.4 ± 2.641s − x |
5 | 0.296 ± 0.034d − h | 0.066 ± 0.001f − j | 22.5 ± 2.36g − n | 0.190 ± 0.002l − r | 0.051 ± 0.002pq | 26.8 ± 0.51d − h |
10 | 0.339 ± 0.007a − h | 0.078 ± 0.003bc | 22.9 ± 0.99h − p | 0.231 ± 0.006j − p | 0.039 ± 0.003w − a | 16.6 ± 1.37vw |
400 | 0 | 0 | 0.221 ± 0.007e − h | 0.058 ± 0.004lmn | 26.3 ± 2.58d − i | 0.226 ± 0.005j − p | 0.034 ± 0.005z − a | 15.1 ± 2.14uv |
5 | 0.190 ± 0.017e − j | 0.062 ± 0.002jk | 32.8 ± 2.63ab | 0.231 ± 0.005i − s | 0.051 ± 0.002pqr | 22.1 ± 1.02j − q |
10 | 0.199 ± 0.033e − j | 0.069 ± 0.002e − h | 35.0 ± 5.75a | 0.191 ± 0.007n − s | 0.045 ± 0.002stu | 23.6 ± 1.102g − m |
50 | 0 | 0.313 ± 0.009c − h | 0.064 ± 0.004jk | 20.3 ± 1.51k − s | 0.212 ± 0.010n − s | 0.054 ± 0.002nop | 25.6 ± 0.75e − i |
5 | 0.308 ± 0.025def | 0.056 ± 0.003mno | 18.3 ± 2.29p − w | 0.177 ± 0.005o − s | 0.049 ± 0.003qrs | 27.6 ± 2.05c − g |
10 | 0.386 ± 0.055a | 0.072 ± 0.002def | 18.9 ± 2.81k − t | 0.174 ± 0.004o − t | 0.035 ± 0.002z − b | 19.8 ± 1.32k − s |
200 | 0 | 0.317 ± 0.011c − h | 0.080 ± 0.002ab | 25.3 ± 1.44f − i | 0.192 ± 0.004o − s | 0.063 ± 0.002jk | 32.9 ± 0.95ab |
5 | 0.309 ± 0.048d − h | 0.080 ± 0.005 ab | 26.2 ± 4.73d − i | 0.213 ± 0.006o − s | 0.057 ± 0.004mno | 26.5 ± 2.45d − h |
10 | 0.363 ± 0.043a | 0.084 ± 0.001a | 23.3 ± 2.37g − n | 0.194 ± 0.005o − s | 0.043 ± 0.003t − w | 22.1 ± 0.65j − q |
All values in the table are presented as mean ± SE (n = 3). |
3.2. Photosynthetic pigments contents
The destructive effect of Ni toxicity on physiological processes, including photosynthetic pigments, has been proven. Excessive amounts of Ni might directly destroy the photosynthetic machine of leaves via different routes. Excessive Ni can smash epidermal and mesophyll cells, disrupt thylakoid membranes and grana structures of chloroplasts, reduce grana size, and raise the number of lamellae in stressed areas. These alterations diminish chlorophylls, carotenoids, and xanthophylls amounts 16,56. In fact, oxidative stress induced by elevating doses of Ni decreases the electron transport in the photosynthetic electron transport chain, disorder in the activity of reaction centers (P680, and P700), and delays chlorophyll synthesis 57. As expected, augmenting Ni level led to a declining tendency in chlorophyll a and b content in both studied species of A. inflatum. The Ni toxicity (at 400 µM) led to a decline in chlorophyll a concentration by 17.1% and 32.5%, and chlorophyll b content by 10.1% and 30.9% in the M and NM plants of A. inflatum, respectively, in comparison with the untreated plants (Fig. 1). In line with this study, Ni at concentrations of 50 µM and 100 µM meaningfully declined the chlorophyll pigments (a, b and total) in cotton plants relative to the untreated plants 58. Also, chlorophyll a and b contents in Z. mays plant leaves decreased after 13 days of plant growth under 100- and 200-mM Ni concentrations 59. In confirmation of our results, Srivastava et al. showed that Ni considerably reduced the chlorophyll a/b ratio at concentrations of 0.1 and 1 mM, indicating that chlorophyll a was greater sensitive to Ni than chlorophyll b 60.
The presence of SA or JA in both populations' growth media, especially at higher doses, led to a drop in the amount of chlorophyll a and b compared to control group. In this investigation, different impacts of SA and JA on chlorophyll content were evident in two A. inflatum populations against Ni toxicity. In both populations exposed to 400 µM Ni, SA, especially at high concentration (200 µM), caused a slight increase in chlorophyll content with respect to plants exposed to 400 µM Ni only (Fig. 1). One of the critical roles of SA is the regulation of photosynthetic pigments (chlorophyll and carotenoid), photosystem II (PSII) and the performance of carbonic anhydrase and Rubisco enzymes against HMs stress conditions 61. It was also found that the co-treatment of SA and NO reduced Ni toxicity by improving photosynthetic apparatus, antioxidant enzymes' action, maintaining nutrients homeostasis and reducing MDA formation 62. However, JA, especially at high concentration (10 µM) in M plants exposed to 400 µM Ni, showed a reducing trend of chlorophyll content. In contrast, the NM population treated with SA or JA alone with 400 µM Ni resulted in an augmenting trend of chlorophyll content compared to plants under Ni stress alone (Fig. 1). External application of JA improved the chlorophyll amount and development of Glycine max seedlings exposed to Ni stress 57. Overall, the highest amounts of chlorophyll a and b in both populations were attended in the treatment of 400 µM Ni + 200 µM SA + 5 µM JA, which led to an enhancement in the amount of chlorophyll a (of 1.69- and 1.88 times) and chlorophyll b (of 1.49- and 1.06 times) in M and NM populations, respectively, compared to the plants treated with Ni (400 µM) alone (Fig. 1). Accordingly, Kamran et al. reported that high doses of external JA alone inhibited the growth and photosynthesis rate in choy sum plants. Whereas, JA (5, 10, and 20 µM) mitigated chromium (Cr) toxicity (150 and 300 µM) and improved gas exchange and chlorophyll contents with respect to the Cr-alone treatment plants. In this study, the useful impact of JA was attributed to the protective roles of JA on the performance of the photosynthetic machine, biosynthesis of chlorophyll, guiding of stomata, and transport rates of CO2. These authors stated that reducing in Cr amounts in plant cells could be a significant reason for JA-induced stimuli on photosynthesis 39. Furthermore, matched outcomes were detected in mustard plants in which Ni stress at concentrations of 50, 100, and 150 µM meaningfully decreased the amounts of chlorophyll by 22.15, 30.95, and 40.64%, respectively. In this study, the application of exogenous SA to Ni-stressed plants promoted chlorophyll amounts by 34.2%, 47.2% and 41.8% in the (50, 100 and 150 µM) Ni + SA treatments with respect to plants treated with Ni alone 36, which confirmed the obtained results in the current work. In another study, the amount of photosynthetic pigments was significantly decreased in lead (Pb)-stressed maize plants as compared to non-exposed plants. Meanwhile, the exogenous usage of JA, SA, proline alone, and co-treatment with Pb significantly enhanced these pigment content in Pb-stressed and control maize plants. This was attributed to the protecting task of JA in enhancing the photosynthetic apparatus and uptake of key minerals against stress to Pb and the role of SA in falling chlorophyll disruption and MDA increase and electrolyte leakage in treated plants 63.
3.3. Plant Ni concentrations
To survey the impact of SA and JA on Ni uptake and potential phytoremediation, we determined the amount of Ni accumulated in the shoots and roots of Ni-exposed M and NM populations. As shown in Tables 2 and 3, with increasing doses of Ni in the plant root environment, the amount of Ni uptake by the plant roots in both populations showed a considerable increase. Thus, at 400 µM Ni treatment, the Ni doses in the ground part of the M and NM populations were 832 and 886 µg/g DW, respectively, which was 16.3- and 25.6 times higher than ground part of control plants. The uptake of Ni by ground part of plants occurred through two principal mechanisms, active transport system and passive diffusion pathway. Different factors such as plant species, Ni form, Ni amount in the rhizosphere, soil pH, organic matters in the soil, and plant metabolism affect Ni transport 48. Furthermore, the uptake of Ni ions can be regulated by amino acids because these compounds act as chelators that form complexes with metal ions such as Ni, thus enhancing Ni uptake in plants 64. The uptake of divalent cations in roots usually happens through apoplastic binding and symplastic uptake systems 65. One study reported that the ratio of apoplastic to symplastic absorption in Ni-hyperaccumulator and non-hyperaccumulator plants was 85–95% to 5–15%, indicating that the symplastic route is very restricted for both plants 66. Due to the rise in Ni uptake by the plant's roots, the rate of Ni transport from the root-to-shoot of these plants enhanced, therefore, excessive Ni accumulation was detected in the aerial parts of M and NM populations. After uptake, Ni is transferred simply into the xylem and then guided to aerial parts of plants 67, then in there, Ni is redistributed through the phloem to stem, leaves, and other parts of plants 48. The maximum Ni concentrations in the shoots of M and NM populations under Ni (400 µM) stresses were 1392 and 1382 µg/g DW, respectively, which was 88.2- and 71.6 times more than the aerial parts of untreated plants. The use of SA and JA alone and/or in combination in plants under Ni stress led to various accumulation patterns of Ni in root and shoot of M and NM populations. Therefore, Ni uptake in root of the M population treated with the highest amounts of Ni, SA, and JA, i.e. 400 µM Ni + 200 µM SA and also 400 µM Ni + 10 µM JA was 12.6% and 38.4% respectively, with respect to plants treated with 400 µM Ni alone. However, in root of the NM population, the uptake of Ni at the highest dose of Ni and SA increased (6.24%), while it decreased (approximately 7.33%) against the highest dose of Ni and JA relative to the treated plants with 400 µM Ni alone. Furthermore, in M population plants treated with of 400 µM Ni + 200 µM SA + 10 µM JA, a higher Ni uptake (38.2%) was recorded in the root. While in the Ni-treated NM population root, the interaction of JA and SA led to the reduction in Ni uptake with the minimum amount at the treatments of 100 µM Ni + 50 µM SA + 5 µM JA and then at 400 µM Ni + 50 µM SA + 5 µM JA comparison with Ni treatments alone. Similar to our results, in soybean plants compared with leaves, a higher amount of Ni accumulated in plant roots against Ni stress with or without JA-priming. JA-priming in this plant eventuated in a lesser Ni accumulation in different parts of the plant, thereby lessening the damaging impacts of Ni and augmenting the growth yield of soybean. This was attributed to the stimulation role of JA in the fabrication of OAs, for example, citrate and malate in root's exudates or thiol compounds to Ni sequestration 68. On the other hand, Khalid et al. (2023) revealed that foliar 100 µM SA spray led to a decline in mercury (Hg) accumulation in the aerial parts, root, and fruit of Capsicum annum L. exposed to various doses of Hg (0, 50, 100 and 150 µM). Likewise, they confirmed that SA is important in controlling signaling molecules in developing and mediating plants' response to HM stress 69.
Table 2
Changes in Ni levels and phytoremediation factors (BCF, BAC and TF) in the presence of SA and JA in the M population of A. inflatum stressed with various doses of Ni.
Plant species | Ni | SA | JA | Shoot Ni concentration (µg g− 1 DW) | Root Ni concentration (µg g− 1 DW) | BCF | BAC | TF |
M plant species | 0 | 0 | Control | 15.8 ± 2.89 y | 50.9 ± 4.03 s | 0 ± 0 q | 0 ± 0 t | 0.308 ± 0.03 v |
5 | 6.90 ± 0.76 y | 45.2 ± 3.89 s | 0 ± 0 q | 0 ± 0 t | 0.153 ± 0.006 w |
10 | 7.16 ± 0.49 y | 50.5 ± 4.34 s | 0 ± 0 q | 0 ± 0 t | 0.144 ± 0.02 w |
50 | 0 | 27.3 ± 3.45 xy | 41.4 ± 5.04 s | 0 ± 0 q | 0 ± 0 t | 0.671 ± 0.14 l−o |
5 | 21.3 ± 2.32 y | 41.4 ± 5.04 s | 0 ± 0 q | 0 ± 0 t | 0.517 ± 0.015 q−t |
10 | 23.5 ± 1.55 xy | 54.4 ± 3.72 s | 0 ± 0 q | 0 ± 0 t | 0.432 ± 0.002 q−v |
200 | 0 | 23.5 ± 1.55 xy | 54.4 ± 3.72 s | 0 ± 0 q | 0 ± 0 t | 0.432 ± 0.002 q−v |
5 | 38.6 ± 0.87 xy | 60.1 ± 1.44 s | 0 ± 0 q | 0 ± 0 t | 0.643 ± 0.03 l−p |
10 | 31.5 ± 1.10 xy | 55.5 ± 2.22 s | 0 ± 0 q | 0 ± 0 t | 0.57 ± 0.04 n−t |
100 | 0 | 0 | 383 ± 8.87 m−o | 602 ± 18.0 l | 6.025 ± 0.185 d | 3.837 ± 0.089 ed | 0.638 ± 0.03 l−p |
5 | 636 ± 34.5 j−k | 577 ± 13.9 ml | 5.774 ± 0.139 ed | 6.36 ± 0.345 b | 1.101 ± 0.034 w |
10 | 565 ± 50.7 l | 538 ± 34.5 no | 5.38 ± 0.345gf | 5.655 ± 0.508 b | 1.058 ± 0.16 d−g |
50 | 0 | 317 ± 11.5 p−o | 553 ± 19.7 m−o | 5.528 ± 0.197 ef | 3.169 ± 0.116 g−i | 0.575 ± 0.042 n−t |
5 | 148 ± 15.8 u−w | 798 ± 9.78ij | 7.98 ± 0.098 c | 1.481 ± 0.158 r | 0.186 ± 0.018 w |
10 | 329 ± 21.2 n−r | 839 ± 19.7 h | 8.386 ± 0.197 b | 3.29 ± 0.212 hg | 0.392 ± 0.017 v |
200 | 0 | 248 ± 8.87 r−t | 563 ± 10.0 mn | 5.628 ± 0.1 ef | 2.477 ± 0.089 k−m | 0.44 ± 0.008 q−v |
5 | 244 ± 7.88 r−t | 877 ± 28.0 g | 8.773 ± 0.289 a | 2.437 ± 0.079 mn | 0.279 ± 0.019 v−w |
10 | 380 ± 9.37 m−p | 895 ± 6.00 g | 8.952 ± 0.617a | 3.802 ± 0.094 d−f | 0.426 ± 0.029 q−v |
400 | 0 | 0 | 1392 ± 173a | 832 ± 29.1 hi | 2.08 ± 0.073 op | 3.481 ± 0.435 e−g | 1.671 ± 0.151 b |
5 | 1269 ± 125 b | 1018 ± 13.0 e | 2.546 ± 0.034 j | 3.175 ± 0.313 g−i | 1.249 ± 0.14 cd |
10 | 1163 ± 154 c | 1350 ± 45.8 a | 3.377 ± 0.115 ij | 2.907 ± 0.386 ij | 0.865 ± 0.144 h−j |
50 | 0 | 1132 ± 52.7 c | 945 ± 15.1 f | 2.363 ± 0.038 mn | 2.831 ± 0.132 i−k | 1.198 ± 0.037 c−e |
5 | 601 ± 195kl | 1269 ± 16.2 c | 3.172 ± 0.041 ij | 1.503 ± 0.49 t | 0.476 ± 0.161 q−t |
10 | 771 ± 3.45 e−g | 1310 ± 39.8b | 3.276 ± 0.1 ij | 1.929 ± 0.009 n−q | 0.59 ± 0.021 l−r |
200 | 0 | 853 ± 39.9 ef | 952 ± 38.0 f | 2.38 ± 0.098 mn | 2.134 ± 0.1 mn | 0.899 ± 0.074 g−j |
5 | 669 ± 0.99 i−k | 1152 ± 34.3 d | 2.88 ± 0.086 k | 1.674 ± 0.003 p−r | 0.582 ± 0.019 n−s |
10 | 822 ± 31.8 e−g | 1345 ± 16.9 ab | 3.364 ± 0.043 ij | 2.056 ± 0.08 on | 0.611 ± 0.016 l−q |
All values in the table are presented as mean ± SE (n = 3). |
Table 3
Changes in Ni levels and phytoremediation factors (BCF, BAC and TF) in the presence of SA and JA in the NM population of A. inflatum stressed with various doses of Ni.
Plant species | Ni | SA | JA | Shoot Ni concentration (µg g− 1 DW) | Root Ni concentration (µg g− 1 DW) | BCF | BAC | TF |
NM plant species | 0 | 0 | Control | 19.3 ± 2.50 xy | 34.6 ± 3.74 s | 0 ± 0 q | 0 ± 0 t | 0.568 ± 0.134 n−t |
5 | 56.5 ± 13.9 xy | 46.1 ± 4.07 s | 0 ± 0 q | 0 ± 0 t | 1.226 ± 0.283 c−e |
10 | 64.8 ± 15.0 w−y | 50.8 ± 5.69 s | 0 ± 0 q | 0 ± 0 t | 1.31 ± 0.451 c |
50 | 0 | 31.9 ± 5.50 xy | 30.8 ± 1.18 s | 0 ± 0 q | 0 ± 0 t | 1.036 ± 0.181 e−h |
5 | 16.9 ± 0.50 xy | 30.8 ± 1.18 s | 0 ± 0 q | 0 ± 0 t | 0.549 ± 0.028 n−t |
10 | 15.1 ± 4.90 y | 36.5 ± 4.036 s | 0 ± 0 q | 0 ± 0 t | 0.413 ± 0.126 q−w |
200 | 0 | 15.1 ± 4.90 y | 36.5 ± 4.04 s | 0 ± 0 q | 0 ± 0 t | 0.413 ± 0.126 q−w |
5 | 40.4 ± 0.60 xy | 53.5 ± 1.80 s | 0 ± 0 q | 0 ± 0 t | 0.756 ± 0.026 j−m |
10 | 29.4 ± 0.35 xy | 35.2 ± 3.01 s | 0 ± 0 q | 0 ± 0 t | 0.841 ± 0.07 i−k |
100 | 0 | 0 | 405 ± 11.1 mn | 520 ± 19.4 o | 5.202 ± 0.194 g | 4.049 ± 0.111 d | 0.78 ± 0.05 j−l |
5 | 752 ± 101 g−i | 474 ± 10.0 p | 4.74 ± 0.10 h | 7.518 ± 1.01 a | 1.59 ± 0.247 b |
10 | 661 ± 22.3 j−k | 336 ± 26.0 q | 3.36 ± 0.26 ij | 6.613 ± 0.223 b | 1.98 ± 0.221 a |
50 | 0 | 296 ± 2.70 p−r | 353 ± 29.5 q | 3.535 ± 0.295 i | 2.961 ± 0.027 h−i | 0.842 ± 0.063 i−k |
5 | 267 ± 38.0 r−s | 242 ± 16.0 r | 2.42 ± 0.160 mn | 2.674 ± 0.382 j−l | 1.116 ± 0.233 d−f |
10 | 266 ± 23.2 q−r | 254 ± 25.5 r | 2.545 ± 0.255 lm | 2.658 ± 0.232 j−l | 1.046 ± 0.014 e−h |
200 | 0 | 204 ± 17.8 s−u | 346 ± 38.0 q | 3.462 ± 0.386 i | 2.044 ± 0.178 no | 0.599 ± 0.115 l−r |
5 | 164 ± 14.2 t−v | 259 ± 24.0 r | 2.59 ± 0.24 lm | 1.642 ± 0.142 q−r | 0.635 ± 0.004 l−p |
10 | 105 ± 12.6 v−x | 273 ± 23.0 r | 2.73 ± 0.23 kl | 1.05 ± 0.126 s | 0.384 ± 0.014 t−v |
400 | 0 | 0 | 1382 ± 21.8 a | 886 ± 19.5 g | 2.217 ± 0.049 np | 3.456 ± 0.055 fg | 1.56 ± 0.01 b |
5 | 872 ± 30.6 e | 777 ± 62.0 j−k | 1.943 ± 0.155 p | 2.18 ± 0.077 mn | 1.13 ± 0.13 c−f |
10 | 890 ± 16.0 e | 821 ± 8.50 hi | 2.054 ± 0.022op | 2.226 ± 0.04 mn | 1.084 ± 0.031 d−g |
50 | 0 | 823 ± 38.4 e−g | 952 ± 11.0 f | 2.38 ± 0.028 mn | 2.058 ± 0.097 no | 0.865 ± 0.031 h−j |
5 | 982 ± 4.00 d | 743 ± 9.50 k | 1.859 ± 0.024 p | 2.455 ± 0.01 r | 1.321 ± 0.012 c |
10 | 806 ± 111 f−h | 818 ± 37.5 hi | 2.047 ± 0.094 op | 2.016 ± 0.277 n−p | 0.991 ± 0.181 f−i |
200 | 0 | 689 ± 15.2h − i | 945 ± 21.5 f | 2.364 ± 0.054 mn | 1.724 ± 0.038 o−r | 0.73 ± 0.001 g−j |
5 | 421 ± 7.30 m | 803 ± 2.00 h−j | 2.008 ± 0.063 op | 1.054 ± 0.019 s | 0.526 ± 0.026 q−t |
10 | 346 ± 44.8 m−q | 900 ± 21.0 g | 2.25 ± 0.053 no | 0.864 ± 0.112 s | 0.385 ± 0.059 t−v |
All values in the table are presented as mean ± SE (n = 3). |
As shown in Tables 2 and 3, external usage of SA in combination with JA or alone decreased Ni uptake in the shoot of Ni-stressed M and NM populations. In contrast, exogenous JA led to an increase in Ni absorption in the stressed plant shoot. The maximum reduction of Ni uptake in the aerial parts of M and NM populations was detected in the groups of 400 µM Ni + 200 µM SA (38.7% and 50.1%), and 400 µM Ni + 10 µM JA (16.5% and 35.6%), respectively, in compared to plants treated with 400 µM Ni. Besides, a sharp reduction in Ni accumulation in the shoots of both populations was perceived with increasing doses of Ni under the effect of simultaneous treatment with SA and JA. Accordingly, the lowest shoot Ni accumulation was related to the 400 µM Ni + 50 µM SA + 5 µM JA in the stressed-M plants (with 56.8%) and 400 µM Ni + 200 µM SA + 10 µM JA in the stressed-NM plants (with 75%). Accordingly, Ali et al. (2018) explained that amount of Cd in the aerial parts of Brassica napus increased in Cd stress conditions, but exogenous application of JA significantly decreased Cd accumulation in stressed plants. This study revealed that reduction in HMs absorption in reaction to external usage of stress phytohormones such JA, SA, abscisic acid (ABA), and brassinolide could be attributed to accumulation/ exudation of organic compounds 68. Also, in another study, the mitigation of Cd ions uptake and protection of plants against HMs stress were attributed to the endogenous and exogenous SA in plants. SA pre-soaking inhibited the accumulation of Cd in the shoot as a result of a considerable reduction in roots BCF and Cd transport, which were calculated using the TF and BAC in shoots, respectively 70. Nevertheless, the findings of the current study indicated the role of SA and JA in Ni absorption and accumulation in stressed plants, which may indicate the formation of Ni- organic or amino acids complexes to the chelation and sequestration of Ni in the root and thus tolerance of plants against Ni toxicity.
3.4. Phytoremediation potential
Phytoremediation is an internal mechanism for the absorption of HMs and deposition in their tissues, which allows the removal of HMs from contaminated sites at a higher rate and subsequent harvesting at a lower cost 71. Generally, three main factors TF (shoot-to-root ratio of HMs), BCF (root-to-soil ratio of HMs), and BAC (shoot-to-soil ratio of HMs) are employed to estimate the phytoremediation potential. Plants are expected to be appropriate for phytoremediation if the values of these factors are higher than one (TF > 1, BCF > 1, and BAC > 1) 9,43. The means of TF, BCF, and BAC values in different parts of M and NM populations of A. inflatum were shown in Tables 2 and 3, which help to investigate the phytoremediation ability of two populations by explaining the characteristics of Ni accumulation and transport behaviors in them. The values of BCF and BAC of both populations under different Ni doses were higher than one (BAC > 1, BCF > 1), while the TF value was higher than 1 in high Ni concentration. The plants' roots had the highest BCF value at 100 µM Ni concentration [M population (6.025), NM population (5.202)]; however, with the increase of Ni doses to 400 µM, a trend of decrease in BCF value was observed in M (2.08) and NM (2.217) populations of A. inflatum. Likewise, this decreasing trend was detected with the rise of Ni dose to 400 µM in BAC value with 9.3% and 14.6% in M and NM populations, respectively, in comparison with groups treated with Ni (100 µM) alone. In addition, the TF value was less than 1 (TF < 1) in both populations at 100 µM Ni, reaching higher than 1 when the Ni dose increased to 400 µM [M population (1.671), NM population (1.56)]. Since the BCF, TF, and BAC values for Ni are greater than 1, two populations of A. inflatum could be used as Ni-hyperaccumulator plants for phytoremediation of Ni-contaminated soil. Similarly, it was found that the accumulation and translocation of Ni in various parts of Alyssoides utriculata found that BCF and TF are intensely more than 1 and this plant is a suitable candidate for Ni phytoremediation from serpentine soils 72. Also, in an investigation performed by Sajad et al. (2020) to identify plants capable of phytoremediation of Ni from sixty-one sites in Pakistan, it was found that most plant species did not belong to the Ni hyperaccumulators group. Founded on the results of estimating the Ni doses in the soil, root and shoot, also analyzing the values of BCF, TF, and BAC, most of the species have the ability to Ni phytoremediation, for example, Xanthium strumarium (BCF: 89.97) suggested to phytostabilization and Bryophyllum daigremontianum (with TF: 2.37 and BAC: 198.11) suggested to phytoextraction 73. Overall, the accumulation of HMs in plants relies on the outer environment and inner physiological characteristics, including HMs bioavailability, physicochemical characteristics of soil, biodiversity of microbes in soil, root exudation, temperature, and the existence of transporters 74.
According to Tables 2 and 3, external usage of SA and JA alone or in combination in plants against various concentrations of Ni, with rising Ni doses, a reducing trend was observed in the BCF and BAC indexes of both populations of A. inflatum; however, BCF and BAC values of these treatments were greater than one. As an exception, exogenous JA use, especially at a lower concentration (5 µM) in plants under Ni (100 µM) stress, led to an enhancement in the BAC value of the M population (6.36) by 1.7 times and the NM population (7.518) by 1.9 times comparison with Ni (100 µM) treatment [M population (3.837), NM population (4.049)]. Also, the highest BFC value was recorded in the M population of A. inflatum in simultaneous treatments of 200 µM SA + 10 µM JA exposed to 100 µM (8.952) and 400 µM (3.364) of Ni in comparison with plants against Ni [100 (6.025) and 400 (2.08) µM] only. Unlike the roots, the BAC value displayed a decreasing trend in these treatments. Nevertheless, all these values are higher than one. Similarly, in the NM population treated with 400 µM Ni + 200 µM SA + 10 µM JA, the BFC value exhibited a slight increase, while in this treatment, the BAC value (0.864) decreased significantly with 74.9% compared to the plants under 400 µM Ni stress (3.456), that showed this value was less than one (BAC < 1). Besides, the TF values incremented in the presence of JA, especially at the higher dose (10 µM) in 100 µM Ni-treated plants in both populations (TF > 1), whereas at the same dose of Ni, the existence of SA led to a discount in the two populations (TF < 1). Generally, treatment with SA and JA in plants in the presence of 400 µM Ni resulted in a reduction in the potential of Ni transport to the aerial parts of the plant (TF value) in both populations. Therefore, the decreasing trend of TF values was shown in 400 µM Ni + 200 µM SA treatment [M population (0.899) and NM population (0.865)], and 400 µM Ni + 10 µM JA treatment [M population (0.73) and NM population (1.084)], in comparison to the plants under Ni (400 µM) stress [M population (1.671) and NM population (1.56)]. Similarly, simultaneous treatment of SA and JA in plants under 400 µM Ni resulted in the minimum Ni transport from the root-to-shoot of M and NM population plants (TF < 1). The minimum TF values were detected in 400 µM Ni + 200 µM SA + 10 µM treatment in the NM population (0.385), and 400 µM Ni + 50 µM SA + 5 µM treatment in the M population (0.476). In approving our results, Kazemi et al. (2010) informed that the exogenous use of SA in B. napus L. exposed to Ni toxicity caused a discount of Ni accumulation in the shoot. They suggested that SA probably prevents the accumulation of Ni in the aerial parts by reducing the root uptake and also reducing root-to-shoot Ni translocation 75. Likewise, it was found that the callus cultures of two wheat genotypes, tetraploid (Durum-97) and hexaploid (Shafaq-06), which were under Cd toxicity, raised Cd accumulation in the callus with the augmentation of Cd doses in the medium. The usage of 0.5 mM SA led to decreased BCF value to less than 1 in higher concentrations of Cd 76. Similarly, seed priming of Linum usitatissimum L. with SA reduced the amount of Cd in the root and shoot of this plant, and as a result, the values of BCF, BAC, and TF were reduced to less than one. In this way, SA prevented the toxicity of Cd in the plant by preventing the absorption of Cd and its transport to the shoot by xylem 70. In confirmation of our results, Coelho et al. (2020) found that the use of JA resulted in the accumulation of arsenic (As) above 1000 µg/kg DW and enhanced BCF value (281) in Lemna valdiviana. They stated that JA leads to an enhancement in As accumulation in plants by modulating ROS homeostasis and signaling and promoting the antioxidant system 77. On the contrary, the decreasing trend of Cd accumulation with significantly decreased BCF and TF values was observed in chickpea (Cicer arietinum) plants treated with JA and gibberellic acid (GA3) alone and/or in combination 78. It has also been proven that exogenously JA treatment can disrupt the absorption, transport (root-to-shoot), and accumulation of HMs by suppressing the transporter genes involved in the absorption of HMs by root from soil such as As (Lsi1, Lsi2, and Lsi6 genes) and Cd (AtHMA4 and AtHMA2 genes), as well as transporter genes related to HMs xylem loading such as Cd (AtIRT1 gene), in plants 79. As mentioned by our results, in the existence of SA and JA, in plants under high Ni concentration, the BCF value had an increasing trend compared to the BCF of plants exposed to 400 µM Ni alone. In contrast, the values of BAC and TF exhibited a decreasing trend. Accordingly, probably SA and JA can prevent the accumulation of Ni in the aerial parts of plant by stimulating the uptake, sequestration, and precipitation of Ni in special organelles of root cells, such as vacuoles 80, as well as disrupting the mechanisms of Ni translocation from root to shoot.
3.5. Citric acid (CA) and malic acid (MA) concentrations
Organic acids (OA) have multiple roles in plants; for example, MA, CA, and oxalate induce HMs tolerance by moving them via the xylem and sequestrating them in vacuoles 81. According to Kocaman (2023), increasing the content of malonic acid and MA (up to 1 unit) in the plants can lead to binding (chelation) of 0.7 and 0.5 units of Ni 64. In addition, OAs are chelated by their carboxyl groups with cationic HMs, thus reducing the amount of free toxic active ions. After entering, cationic HMsmay stimulate OA channels or interact with a receptor protein and induce effective genes in the synthesis of OA. Occasionally, in the root surface, OA-HM complexes can arrive at cells through diffusion or by active transport via particular ligand/transporter channels and after that may sequester or accumulate in intracellular parts such as the vacuoles 82.
The content of OA including MA and CA as Ni chelators that role in its transfer and as well as store in roots and shoots of M and NM populations of A. inflatum are displayed in Figs. 2 and 3. Interestingly, the CA and MA amounts in both populations' plant roots and shoots were raised due to an increment in Ni concentration in the plant growth medium. According to the results obtained from this experiment, in plant roots exposed to 400 µM Ni, the CA content was increased by 47.9% and 37.2% (Fig. 2), and as well as the MA content was augmented by 25.5% and 21.2% (Fig. 3), in the M and NM populations, respectively, in comparison to the untreated plant groups. As it is known, CA is synthesized in plants by citrate synthase enzyme. Compared to MA and oxalate, this OA has a greater tendency for metal ions such as Ni2+ and Cd2+, although, its chief role is to chelate Fe2 + 81. Furthermore, in the shoot regions of plants under 400 µM Ni stress, the content of CA and MA augmented by 17.0% and 37.0% in the M population and by 10.0% and 32.0% in the NM population, respectively, relative to the unexposed plants group. Similarly, Amari et al. (2016) demonstrated a significant rise of MA content in xylem sap in Ni-stressed B. juncea and Mesembryanthemum crystallinum. While CA content augmented in the xylem sap of B. juncea alone led to the accumulation of a greater Ni amount than M. crystallinum. Overall, these outcomes recommend that CA and MA may play a role in long-distance Ni transport and can also perform as intracellular chelators capable of binding Ni in the cytosol or subcellular parts, hence preventing the harmful activity of free HM ions 83. In contrast to our results, Pietrini et al. (2015) revealed that at higher Ni doses (150 µM), both oxalic and CA could not prevent the deleterious effects on Amaranthus plants at the physiology-biochemical points initiated by this metal. Although in Ni-exposed plants, the content of CA in plant leaves increased relative to the control, with the maximum amount against 25 µM NiCl2, in plant roots, the high amount of CA was observed only at 150 µM NiCl2. Also, the MA content has not changed in the root and shoot of Amaranthus plants exposed to Ni stress, indicating that this OA in this species may play a minor role as a metal chelator 84. Dresler et al. (2014) found that in maize seedlings exposed to 50 and 100 µM Cd and Cu stress, CA and MA (at 100 µM Cd) perhaps have a role in Cd transport and sequestration in old leaves, whereas MA is involved in the detoxification of Cu at 50 µM dose. This indicates that CA is one of the Cd chelators involved in the translocation of Cd to older parts of the plant 85.
A reducing trend in MA and CA contents in the existence of SA or JA at 400 µM Ni was observed in most shoots and roots of the M and NM plants against 400 mM Ni in comparison with plants stressed to 400 µM only. Nevertheless, the usage of exogenous SA at the highest dose (200 µM) created an increase only in the content of CA (1.1-fold) in two parts of the NM population plants exposed to 400 µM Ni compared to plants under Ni (400 µM)alone. The OAs contents in two plants' parts of the NM populations under different Ni doses and the simultaneous impact of SA and JA showed significant increases. Thus, in plants treated with 400 µM Ni + 200 µM SA + 10 µM JA, CA content increased by 1.54- and 1.96-fold, and MA content increased by 1.23- and 2.1-fold in roots and shoots, respectively, relative to the untreated plants. In the same treatment, the enhancing effect was observed only on the CA level, with a rise in the shoot (1.2-fold) and root (1.45-fold) of the M population to the untreated plants. In the M population, the maximum increase trend was determined by 1.9 times in root CA content and 1.6 times in shoot MA content in the treatment of 400 µM Ni + 50 µM SA + 5 µM JA compared to the control group. Also, the highest MA amount in M population plants was recorded in the roots under the influence of 400 µM Ni + 50 µM SA + 10 µM JA, with an increase of 1.2 times compared to untreated plants. However, concerning plants treated with 400 µM Ni alone, the maximum MA (29.6%) and CA (19.0%) content were observed at the treatments of 400 µM Ni + 200 µM SA + 10 µM JA in the shoot parts of NM plants. JA can induce the synthesis of OAs such as CA and MA or thiol compounds to sequester Ni in roots 38. Accordingly, tomato seedlings exposed to Pb stress indicated that Pb at 0.75 mM increased the content of CA (56.6%), MA (40.9%), fumaric acid (29.1%), and succinic acid (25.2%) over the unstressed plants. In these plants, the treatment of 100 nM JA + 0.75 mM Pb elevated CA (19.5%), MA (38.3%), fumaric acid (49.7%), and succinic acid (41.4%) in comparison to the seedlings exposed to Pb (0.75 mM) alone. It was found that the use of JA changed the expression of enzyme genes related to the metabolism of OAs in tomato seedlings under lead stress under toxic doses of lead, accordingly the amount of CA synthase and MA synthase enzymes decreased. Still, seedlings exposed to Pb (0.75 mM) with JA (100 nM) detected an up-regulation of the expression of fumarate hydratase and succinate dehydrogenase enzymes 86. In line with our findings, Kohli (2018) reported increased CA, MA, succinic acid, and fumaric acid contents in B. juncea under stress to Pb (0.75 mM). In this plant, the presence of SA (1 mM) and 24-epibrassinolide acid (EBL) (10− 7 M) alone led to a greater rise in the amounts of these OAs, as well as the simultaneous treatment with EBL + SA showed the greatest effect than these treatments alone in increasing the amounts of these compound in Pb-treated seedlings. For example, the authors demonstrated 123% and 141% increases in CA and MA contents, respectively, under treatment with Pb + EBL + SA in stressed plants 87. However, OAs such as CA and MA, similar to amino acids, have a role in Ni uptake through the formation of Ni-OA complexes by the roots and also cooperate with processes of root-to-shoot translocation and Ni precipitate in cell organelles such as vacuoles 48,80.
3.6. Histidine (His) concentration
Different amino acids such as His, alanine, asparagine, proline, arginine, methionine, glycine, serine, cysteine, lysine, and glutamic acid, are known to accumulate at high doses against HMs (Ni, Pb, Cr, Zn, Cd) stress to preserve plants from the toxic impacts of certain metals 88. To protect and tolerate the stressed plants, amino acids can act as compatible osmolytes, pH regulators and ROS detoxifiers as well as act as nitrogen or carbon stores for the synthesis of certain enzymes and precursors of various secondary metabolites for instance flavonoids and lignin 64. For example, His has been involved in the detoxifying of HMs in Ni-tolerant plants by its chelation 89. Therefore, in the current study, the His levels related to the roots and shoots of both populations of A. inflatum were analyzed, as shown in Fig. 4. As can be seen, high Ni doses enhanced the content of His in stressed plants. Its content was enhanced by 1.24- and 1.18-fold in the roots and incriminated by 1.59- and 1.34-fold in the shoots of the M and NM plants, respectively, with respect to the unstressed plants. It has been established His has a high tendency to link with metals. Likewise, the free amino acids and proteins with (metal's coordination residues) have a partly high correlation constant (Log K: 8.7) for Ni 90. Similarly, Ali et al. (2009) indicated the increasing concentration of His, serine, and cysteine in the xylem sap of B. napus cultivars exposed to various doses of Ni. In this research, the higher amount of His was attributed to the decontamination of Ni with increased binding to His, serine, and cysteine, and thus indicated Ni tolerance in B. napus91. Another study demonstrated an increase in the amount of His in the roots and leaf rosettes of Matricaria chamomilla against Ni stress, with a 4-fold increase in plant roots at 120 µM Ni 92. In addition, it was found that more His accumulated in Ni hyperaccumulator Alyssum lesbiacum than in non-accumulator Alyssum montanum under Ni stress condition 22. Furthermore, Callahan et al. (2007) reported a rise in the amount of His and nicotianamine in response to higher Ni concentrations in Thlaspi caerulescens as a serpentine population 93. According to Dalir, and Khoshgoftarmanesh’s hypothesis, Ni absorption in the presence of His may occur through two pathways: (I) Ni absorption action by the root may be individually from His absorption, however, the existence of free His around roots indirectly induces Ni absorption by chelating with Ni inside the apoplastic or symplastic spaces of the root. (II) Ni(His) ligands are directly absorbed by the root, and thus, His may enhance Ni absorption through specific His- or Ni(His)-transporters 90.
According to Fig. 3, the content of His in the shoots of two populations of A. inflatum under 400 µM Ni stress, in the presence of high doses of SA [in M population (14.0%), NM population (16.0%)] and JA [in M population (7.30%), NM population (24.5%)], exhibited a significant reduction over the plants stressed with Ni at a high dose. However, contradictory results were obtained in the root part of these plants. Besides, the simultaneous effect of SA and JA in combination at different concentrations in Ni-stressed plants caused a decrease in His content relative to the plants stressed with Ni alone. Although, compared to the unstressed plants, treatments of 400 µM Ni + 50 µM SA + 5 µM JA in M species roots by 1.13-fold and the treated group of 400 µM Ni + 200 µM SA + 10 µM JA in shoots of the M (1.36-fold) and NM (1.13-fold) populations and as well as the root of NM population by 1.2-fold caused an increase in the content of His. Similarly, the results obtained by Ahmad et al. (2021) indicated increasing proline and glycine betaine (GB) accumulation in chickpea plants under Cd stress supplemented with JA and GA3. They reported that the combination of JA and GA3 in stressed plants (Cd + JA + GA3) remarkably augmented proline and GB content by 6.08 times and 7.64 times increase, respectively, compared to control plants 78. Furthermore, in confirmation of our results, Bali et al. (2019) revealed that the treatment of tomato seedlings with 100 nM JA + 0.75 mM Pb reduced levels of His, cysteine, aspartic acid, isoleucine, leucine, and GABA (non-protein amino acid), while increased the content of other amino acids compared to plant stressed with 0.75 mM Pb alone. They pointed to the amino acids' role in nitrogen (N) metabolism that regulates the protein metabolism, re-mobilization of N, discount of nitrate and uptake of ammonium and nitrate 86. Likewise, Zanganeh et al. (2019) exhibited that the usage of SA on the seeds of the Z. mays plant, as a pre-treatment, led to a reduction in the amount of His in plant shoots and roots under Pb treatment, while the application of sodium hydrosulfide (NaHS) on the plant seeds caused a decline in the content of roots His and an augmentation in the level of His in shoots. However, according to the authors' results, SA accumulated other amino acids similar to alanine, tyrosine, valine, tryptophan, leucine, and isoleucine. They explained that the decrease of amino acid levels by pre-treatment with SA and NaHS could be considered as a contribution to the beginning of adaptive processes with the unfavorable impact of Pb. Amino acids play precursor roles in synthesizing stress-related proteins and defensive metabolites against plant stress. On the other hand in their study, the increased content of His was attributed to the production of de novo and N storage to inhibit ammonium toxicity and as a result the increase in the contents of His and arginine amino acids, which are appropriate for N storage owing to the existence of binary amides in their structure 94. These results were in line with our results, which observed that SA declined His levels in the shoots of both populations and root of the M population under Ni stress. However, the using exogenous phytohormones such as JA and SA can cause an increment in the content of endogenous amino acids in plants, resulting in improved plant tolerance to abiotic stress like HMs. This is achieved by enhancing plant metabolism through the regulation of membrane permeability, the activation of enzymatic process, the production of osmolytes, and the uptake of some ions 95.
3.7. Principal Component Analysis (PCA)
Utilizing PCA we investigate the interrelationships within a set of variables and elucidate them via diverse biplots. In our investigation of the correlation between variables and traits across two distinct populations of A. inflatum, we employed PCA independently for each population and collectively across both populations (Figs. 5, 6, 7). The first two principal components of PCA accounted for (57.7%), (57.6%) and (61.3%) of the variance when analyzing both populations collectively or when examining populations M and MN separately. Furthermore, PCA plots demonstrated a similar trend in both M and NM plants in response to the presence of SA and JA in plants under Ni stress. The PCA results revealed that in the M (Fig. 5) and NM (Fig. 6) populations, in the presence of SA and JA supplements under Ni toxicity, there was an approximate positive correlation between plant biomass, amount of Ni and the levels of His, MA, SA, and phytoremediation indices (TF, BAC, BCF). This pattern was also seen in the common PCA plot between the two populations (Fig. 7). Interestingly, in population M, the highest correlation between phytoremediation parameters and Ni levels of roots and shoots was observed. The pattern was also observed in the NM population. On the other hand, the lowest correlation value between chlorophyll (a, b) levels and phytoremediation parameters was detectable in all PCA plots. In total, the findings of this study and also the PCA plots showed that both populations (M, NM) follow a similar pattern in the presence of SA and JA alone or together (SA + JA) in reaction to high Ni doses. It can also be stated that although SA and JA, especially the presence of both of them together led to the activation of plant resistance mechanisms against Ni toxicity and decreased phytoremediation values (TF, BAC, BCF) in both populations (M, NM) exposed to high doses of Ni, however, the values of phytoremediation parameters were still higher than one. Finally, it is possible to confirm the effective role of both populations of A. inflatum as useful plants (Ni-hyperaccumulators) in the remediation of Ni from contaminated sites. Although there have been extensive debates about the role of SA and JA in HM stress conditions, according to our knowledge, no study has been documented on the impacts of these phytohormones simultaneously on phytoremediation parameters. Therefore, this study is an opening to clarify and evaluate the effective role of SA, JA, or/SA + JA in phytoremediation mechanisms in two populations of A. inflatum.