3.1. Impact of Pb and OA + SA's on root length, shoot length and relative water content of Zea mays Plants
Table 2, Fig. 1 provides specifics on how exposure to 0.5 mM Pb affects maize plant root length. The root lengths of plants treated exclusively with Pb were substantially shorter than those of the control (p < 0.0001): 1.23 cm ± 0.02 (15 days), 2.26 cm ± 0.02 (30 days), and 3.56 cm ± 0.02 (45 days). The highest mean root lengths were shown by the combination of OA and SA (25 mg/l each), which substantially outperformed other treated groups (p < 0.0001) at 15 days (3.14 cm ± 0.02), 30 days (4.25 cm ± 0.04), and 45 days (5.35 cm ± 0.03). According to these findings, Pb exposure shortened maize roots; however, the combination of OA and SA lessened this effect and improved root growth in comparison to Pb treatment alone.
Table:-2 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on root length (cm.) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 1: Showing effect of Pb (0.5 µM) on root length of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
The shoot length results of maize plants treated with a 0.5 mM dose of Pb are shown in Table 3, Fig. 2. The mean shoot lengths for the control group were 15.6 cm ± 0.15 (15 days), 17.4 cm ± 0.15 (30 days), and 18.5 cm ± 0.25 (45 days). On the other hand, Pb treated plants showed noticeably shorter shoot lengths than the control: 11.4 cm ± 0.15 (15 days), 12.4 cm ± 0.1 (30 days), and 13.6 cm ± 0.15 (45 days) (p < 0.0001). The combination of OA and SA (25 mg/l) produced the longest mean shoot lengths (p < 0.0001), measuring 14.1 cm ± 0.15 (15 days), 15.6 cm ± 0.15 (30 days), and 17.2 cm ± 0.25 (45 days).These results suggest that Pb exposure has a deleterious effect on Zea mays plant shoot length. In contrast to the Pb-alone group, the addition of both OA and SA to Pb-treated plants seemed to lessen the adverse effects and encourage greater shoot growth.
Table:-3 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on shoot length (cm.) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 2: Showing effect of Pb (0.5 µM) on shoot length of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
The specific results about the relative water content of maize plants treated with Pb at a dose of 0.5 mM are shown in Table 4, Fig. 3. When compared to the control group, the plants treated alone with Pb showed significantly lower relative water content (p < 0.0001), with mean values of 124% ± 2.1 (15 days), 128.7% ± 1.5 (30 days), and 131.7% ± 2.1 (45 days). On Pb-stressed plants, combining OA + SA (25 mg/l each) produced the highest mean relative water content (p < 0.0001) of all treated groups: 144.7% ± 1.5 (15 days), 152.7% ± 2.5 (30 days), and 155.7% ± 1.5 (45 days). These findings suggest that Zea mays plant relative water content decreased as a result of Pb exposure and the combination application of OA and SA to Pb-treated plants seemed to lessen the decline and preserve a greater relative water content.
Table:-4 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on relative water content (%) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 3: Showing effect of Pb (0.5 µM) on relative water content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
3.2. Effect of Pb Stress and Protective Effects of OA + SA on Chlorophyll content, Chlorophyll-a, Chlorophyll-b Zea mays Plants
The control group showed a mean chlorophyll content of 14.1 µg g-1 FW (15 days), 17.2 µg g-1 FW (30 days), and 19.3 µg g-1 FW (45 days) with no additional chemicals given (Table 5, Fig. 4). The plants treated exclusively with Pb showed considerably lower levels of chlorophyll, with averages of 7.4 µg g-1 FW (15 days), 8.7 µg g-1 FW (30 days), and 9.8 µg g-1 FW (45 days) (p < 0.0001). Applying OA and SA (25 mg/l each) simultaneously to Pb-stressed plants resulted in a significant increase in chlorophyll content, which was the highest mean of the treated groups (p < 0.0001). The average chlorophyll content was 13.9 µg g-1 FW at 15 days, rising to 15.2 µg g-1 FW at 30 days and peaking at 18 µg g-1 FW at 45 days. These results imply that Zea mays plant chlorophyll content was lowered as a result of Pb exposure. In contrast to Pb treatment alone, the combination application of OA and SA to Pb-treated plants seemed to considerably mitigate this drop and preserve a higher chlorophyll content. Table 6 presents the results for the amount of chlorophyll-a in maize plants treated with 0.5 mM Pb. Plants with Pb treatment showed a considerably (p < 0.0001) lower level of chlorophyll-a than the control group; the means were 0.071 µg g-1 FW (15 days), 0.09 µg g-1 FW (30 days), and 0.10 µg g-1 FW (45 days). The combination application of OA and SA (25 mg/l each) to Pb-stressed plants produced the highest mean chlorophyll-a content, which was statistically significant (p < 0.0001). The average concentration of chlorophyll-a was 0.210 µg g-1 FW after 15 days, rising to 0.29 µg g-1 FW at 30 days, and peaking at 0.36 µg g-1 FW at 45 days. These findings suggest that Zea mays plant chlorophyll-a content decreased as a result of Pb exposure. In contrast to Pb treatment alone, the simultaneous application of OA and SA to Pb-treated plants greatly reduced this decline and maintained a greater chlorophyll-a concentration. Table 7 displays the chlorophyll-b content results for maize plants treated with a 0.5 mM Pb concentration. The chlorophyll-b content of plants that were just treated with Pb, on the other hand, was significantly (p < 0.0001) lower than that of the control, with averages of 0.034 µg g-1 FW (15 days), 0.04 µg g-1 FW (30 days), and 0.05 µg g-1 FW (45 days). Interestingly, simultaneous application of OA and SA to Pb-stressed plants resulted in a considerable (p < 0.0001, p < 0.0001, p < 0.002) increase in the amount of chlorophyll-b. Among all treatment groups, the combined application of OA and SA (25 mg/l each) to Pb-stressed plants produced the highest mean chlorophyll-b concentration. The average concentration of chlorophyll-b was 0.057 µg g-1 FW at 15 days, rising to 0.10 µg g-1 FW at 30 days and reaching its maximum at 0.14 µg g-1 FW at 45 days. These results imply that Pb-stressed plants' chlorophyll-b content was significantly increased when they were exposed to both OA and SA together.
Table:-5 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on chlorophyll content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 4: Showing effect of Pb (0.5 µM) on chlorophyll content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
Table:-6 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on Chl-a content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 5: Showing effect of Pb (0.5 µM) on Chl-a content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
Table:-7 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on Chl-b content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 6: Showing effect of Pb (0.5 µM) on Chl-b content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
3.3. Effects of Pb Stress and Protective Effects of OA + SA on Anthocyanin, Carotenoid, Xanthophyll content and soluble sugars in Zea mays Plants
Table 8 presents findings about the anthocyanin content in maize plants treated with a 0.5 mM concentration of Pb. The plants treated alone with Pb showed a significant (p < 0.0001) decrease in anthocyanin content, with averages of 7.8 µg g-1 FW (15 days), 9.7 µg g-1 FW (30 days), and 11.9 µg g-1 FW (45 days). The simultaneous treatment of 25 mg/l of OA and SA to plants stressed by Pb produced the greatest mean anthocyanin concentration (p < 0.0001) among all treated groups. The average anthocyanin concentration was 21.8 µg g-1 FW at day 15, increased to 23.7 µg g-1 FW at day 30, and reached its high at 29.4 µg g-1 FW at day 45. These results imply that the anthocyanin content of Zea mays plants was significantly increased when Pb-stressed plants were exposed to both OA and SA together.
Table:-8 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on anthocyanin content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 7: Showing effect of Pb (0.5 µM) on anthocyanin content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
The carotenoid content of maize plants treated with 0.5 mM Pb are presented in table 9. Plants that were treated with Pb showed a substantial (p < 0.0001) decrease in carotenoid content, with average values of 0.004 µg g-1 FW (15 days), 0.005 µg g-1 FW (30 days), and 0.006 µg g-1 FW (45 days). When Pb-stressed plants were treated with OA and SA (25 mg/l each), there was a significant (p < 0.0001) increase in carotenoid content, with mean values of 0.009 µg g-1 FW (15 days), 0.010 µg g-1 FW (30 days), and 0.013 µg g-1 FW (45 days). These results suggest that Zea mays plant carotenoid content was significantly reduced as a result of Pb exposure. In contrast to Pb treatment alone, the combination application of OA and SA had a considerable impact on the carotenoid concentration in Pb-stressed plants (Fig. 8).
Table:-9 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on carotenoid content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 8: Showing effect of Pb (0.5 µM) on carotenoid content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
When maize plants were exposed to Pb alone, there was a substantial (p < 0.0001) decrease in the mean xanthophyll content, with mean values of 0.10 µg g-1 FW (15 days), 0.12 µg g-1 FW (30 days), and 0.13 µg g-1 FW (45 days) compared to the control (Table 10, Fig. 9). Notably, when Pb-stressed plants were treated with the, mean values of xanthophyll content increased significantly (p < 0.0001). These results suggest that Zea mays plant xanthophyll content was significantly (p < 0.0001) reduced as a result of Pb exposure (Fig. 8). However, when OA and SA were added to Pb-treated plants, this reduction was partially reversed, leading to considerably (p < 0.0001) higher levels of xanthophyll than when Pb treatment was used alone.
Table:-10 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on xanthophyll content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 9: Showing effect of Pb (0.5 µM) on xanthophyll content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
Pb stress (0.5 mM Pb) caused a significant increase (p < 0.0001) in total carbohydrate content (Table 11, Fig. 10) with mean ± SEM values of 3.40 ± 0.53 after 15 days; 5.13 ± 0.24 after 30 days and 5.90 ± 0.02 after 45 days, respectively as compared to control (1.36 ± 0.56 after 15 days; 3.28 ± 0.09 after 30 days; 3.65 ± 0.13 after 45 days). The combination application of OA and SA (25 mg/l each) to Pb-stressed plants significantly (p < 0.0001) decreased the total carbohydrate content after 15 days (mean ± SEM; 4.62 ± 0.18) ; 30 days (mean ± SEM 5.70 ± 0.33) and 45 days (mean ± SEM 6.30 ± 0.30) respectively.
Table:-11 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on Total carbohydrate content content (µg g-1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 10: Showing effect of Pb (0.5 µM) on Total Carbohydrate content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
3.4. Stomatal Conductance in Maize Plants: Examining the Results of Pb Stress and the Protective Effects of OA + SA (Scanning Electron Microscope Analysis) on Zea mays Plants
Using SEM stomatal conductance in maize plants under Pb stress was examined over the course of 15 days, 30 days and 45 days (Fig. 11). SEM pictures of Pb-treated maize plants showed a constant decrease in stomatal conductance, suggesting a cumulative detrimental effect on gas exchange. On the other hand, Pb stressed maize plants treated with OA and SA in combination showed a significant increase in stomatal conductance. According to SEM pictures, stomatal pores began to widen after 15 days, kept doing so at day 30, and then significantly enlarged at day 45, indicating that the combined treatment was improving gas exchange capabilities.
Figure 11: Showing effect of Pb (0.5 µM) on stomatal conductance of plants and Pb stressed plants treated with combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days).
Pb treated maize plant – 15 days
Pb treated maize plant depicting closed stomata – 30 days plant
Pb treated maize plant depicting closed stomata – 45 days plant
Pb + OA + SA treated maize plants depicting open stomata- 15 days plants
Pb + OA + SA treated maize plants depicting open stomata- 30 days plants
Pb + OA + SA treated maize plants depicting open stomata- 45 days plants
3.5. Effects of Pb Stress and Protective Effects of OA + SA on SOD, GPOX, O 2− and H2O2 in Zea mays Plants
Pb has the ability to produce too many free radicals (ROS) and harm plant tissues through oxidative stress. One important defense mechanism in plants that reduces oxidative damage in contaminated tissues is the maintenance of the equilibrium between ROS, or free radicals, and the activation of the antioxidative system under heavy metal stress (Mittler., 2002). As a result, the production of ROS (superoxide ion and H2O2) and the possible function of antioxidant enzymes (SOD, GPOX) in response to oxidative stress in Zea mays treated with Pb were examined in the current work. These enzymes play a major role in the effective metabolism of free radicals, or ROS, and the products they produce. As a result, they closely regulate oxidative stress caused by heavy metals in plants.
In comparison to the control group (1.68 ± 0.06 after 15 days; 2.01 ± 0.15 after 30 days; 2.20 ± 0.10 after 45 days), Pb stress resulted in a significant increase (p < 0.0001) in H2O2 generation (Table 12, Fig. 12) with mean ± SEM values of 2.48 ± 0.03 after 15 days; 2.64 ± 0.05 after 30 days; and 2.71 ± 0.02 after 45 days, respectively. When OA and SA (25 mg/l each) were applied together to Pb-stressed plants, the formation of H2O2 was considerably (p < 0.0001) reduced after 15 days (mean ± SEM; 2.24 ± 0.05); 30 days (mean ± SEM 2.31 ± 0.04); and 45 days (mean ± SEM 2.46 ± 0.08), respectively.
Table:-12 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on H2O2 content (µmol g-1 fresh weight (FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 12: Showing effect of Pb (0.5 µM) on H2O2content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
After 15, 30 and 45 days Pb treatment enhanced the production of superoxide ions (Table 13, Fig. 13) with mean ± SEM values of 2.24 ± 0.01, 2.26 ± 0.02, and 2.29 ± 0.02, respectively, compared to the control group's mean ± SEM values of 2.14 ± 0.03, 2.16 ± 0.01, and 2.19 ± 0.03. The superoxide ion level was significantly (p < 0.0001) reduced after 15 days (mean ± SEM; 2.17 ± 0.05); 30 days (mean ± SEM 2.22 ± 0.00); and 45 days (mean ± SEM 2.25 ± 0.00) of combined application of OA and SA (25 mg/l each) to Pb-stressed plants.
Table:-13 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on O2− content (µmol g − 1 FW) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 13: Showing effect of Pb (0.5 µM) on O2− content of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
SOD production was significantly increased (Table 14, Fig. 14) (p < 0.0001) by the combination application of OA and SA (25 mg/l each) to Pb-stressed plants with mean ± SEM value of 0.36 ± 0.02(15 days); 0.40 ± 0.01 (30 days); 0.48 ± 0.02 (45 days) as compared to control group 0.11 ± 0.01 (15 days); 0.40 ± 0.05 (30 days); 0.48 ± 0.02 (45 days) and Pb treated groups 0.19 ± 0.00 (15 days); 0.24 ± 0.01(30 days); 0.26 ± 0.00(45 days) treated group.
Table:-14 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on SOD activity (Unit mg-1 protein) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 14: Showing effect of Pb (0.5 µM) on SOD activity of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.
GPOX production was significantly increased (Table 15, Fig. 15) (p < 0.0001) by the combination application of OA and SA (25 mg/l each) to Pb-stressed plants with mean ± SEM value of 6.85 ± 0.50(15 days); 6.95 ± 0.51 (30 days); 6.99 ± 0.73 (45 days) as compared to control group 4.23 ± 0.33 (15 days); 5.03 ± 0.41 (30 days); 5.36 ± 0.63 (45 days) and Pb treated groups 5.12 ± 0.51 (15 days); 5.96 ± 0.62(30 days); 5.89 ± 0.56(45 days) treated group.
Table:-15 Effects of Exogenous application of Oxalic Acid (OA), salicylic acid (SA) on GPOX activity (Unit mg-1 protein) in maize plants under Pb toxicity (duration 15 days, 30 days, 45 days).
Figure 15: Showing effect of Pb (0.5 µM) on GPOX activity of plants treated with Oxalic Acid, Salicylic Acid and combination of both (Oxalic Acid and Salicylic Acid) (duration 15 days,30 days,45 days). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant values, which are defined as p values less than 0.05.