Comparative morphological, physiological, and biochemical traits in sensitive and tolerant maize genotypes in response to salinity and pb stress

Abstract

Salinity and lead are two important abiotic stresses that limit crop growth and yield. In this study, we assayed the effect of these stresses on tolerant and sensitive maize genotypes. Four-week-old maize plants were treated with 250 mM sodium chloride (NaCl) and 250 µM lead (Pb). Our results show that NaCl or Pb treatment of the sensitive genotype caused a significant reduction in the root length, plant height, total fresh and dry weights, as well as chlorophyll, and carotenoid content. Salt stress led to a significant decrease in the relative water content, shoot and root length, fresh and dry weight as well as leaf area and K⁺ content but increase Na⁺ content. Both NaCl and Pb stresses increased the antioxidant enzyme activity, proline content, malondialdehyde, and hydrogen peroxide levels. Principal component analysis (PCA) accounted for 69.8% and 16.5% of the total variation among all the variables studied. PCA also suggested a positive correlation between hydrogen peroxide, malondialdehyde, peroxidase, catalase, ascorbate peroxidase levels, and Na⁺ content and a negative correlation between K⁺ content, chlorophyll content, relative water content, leaf area, root length, plant height, and total fresh, and dry weights. Together, these results suggest that the salt-tolerant maize genotype is more suitable for adapting to Pb stress compared to the salt-sensitive genotype.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-82173-5.

Crops are sessile organisms that struggle under harsh environmental conditions. Abiotic stress influences the growth rate, cellular metabolism, productivity, and yield¹. For instance, salt stress affects both vegetative and reproductive stages and causes substantial losses of yield^2,3. Biochemical changes associated with salt stress include activation of enzymes associated with biosynthesis of antioxidants, generation of reactive oxygen species (ROS), and disruption of photosynthetic pathways⁴. Consequently, soil salinization is universally recognized as a severe agricultural hazard⁵. Salinity stress has a noteworthy impact on plant morphology and physiology, as it causes osmotic stress and disrupts plant water relations and ionic balance. Inevitably, this leads to ionic toxicity in plant metabolic processes⁶.

Plants require a certain amount of some heavy metals (HMs) for their growth and reproduction. However, an excess of these metals can have negative effects on plant growth, development, reproduction, and metabolic processes as they become toxic. A higher concentration of HMs can lead to oxidative stress and sudden plant death^7,8. HM stress also affects soil microorganisms associated with the decomposition of organic matter and thereby lower nutrient availability⁹.

HMs stress can negatively affect the function of proteins and enzymes, and interfere with the replacement of essential metal ions by biomolecules. This interference can damage membrane integrity and cause alterations in important metabolic processes, including homeostasis, respiration, and photosynthesis¹⁰. Furthermore, heavy metal stress can elevate the generation of reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂), hydroxyl radicals (OH), and superoxide radicals (O₂), which can trigger oxidative stress and lead to lipid peroxidation^11,12.

Lead (Pb) is considered one of the most hazardous HMs that can contaminate soil due to natural weathering, mining, and smeltin^13,14. Once inside the plant cells, Pb disrupts the hormonal balance, compromises cell membrane integrity and permeability, inhibits enzymes with the sulfhydryl group, interferes with mineral nutrition, and reduces water content resulting in the creation of reactive oxygen species¹⁵. High levels of Pb in the soil can have a significant impact on plant health, leading to decreased root length, leaf chlorosis, and inhibited growth¹³. Pb can induce oxidative stress in plants by disrupting the antioxidant defense system and by causing an excessive production of ROS. The tolerance of plants to Pb toxicity can depend on the production of antioxidant enzymes that help in scavenging ROS, as well as on the accumulation of osmoprotectants like soluble sugars, proline, and glycine betaine¹⁶. Plants grown in Pb-stressed soil may suffer negative impacts on their photosynthetic processes, as lead can disturb the chloroplast structure and impedes biosynthesis of important photosynthetic pigments such as chlorophyll, carotenoids, and plastoquinone¹⁷.

Maize (Zea mays L.) with a production of 1210 million tons (https://www.fao.org/faostat/en/#data/QCL) can be grown on a range of soils and climates. Maize is a major cereal crop worldwide used for food, feed, and bioenergy¹⁸. While maize is a C4 plant that can withstand moderate salinity levels, its early growth phases are vulnerable to salinity stress. Overall, both heavy metal and salinity stresses can greatly affect the growth and yield of maize^19,20.

Agriculture in dry areas faces environmental challenges such as water scarcity, high temperatures, salinity, and heavy metal pollution from industrial activities, affecting plant growth²¹. Salinity and heavy metals, especially Pb, are two important stresses in these areas that the plant faces²². There are some studies to show maize can consider as an accumulator plant for the phytoremediation for Pb and salt polluted soils^23,24.

This study identified tolerant and sensitive maize genotypes among nine hybrid varieties and characterized their morphological, physiological, and biochemical traits under salinity and Pb stress. It was decided to investigate the impact of salinity and lead on proline, antioxidant enzyme activities, chlorophyll content, and ions accumulation in maize, as a potential candidate for phytoremediation of salinized and Pb-contaminated soils.

Materials and methods

Pre-experiment to select tolerant and susceptible genotypes

Nine maize hybrid genotypes (BK 74, Dominate, MAY, Hektor, AGN 720, KSC 704, KSC 703, BC 678, and Simon), collected from different seed companies in Iran, were tested for salinity tolerance. The germination of maize seeds was examined at 0, 100, and 200 mM NaCl concentrations. The collected seeds were sterilized with 5% sodium hypochlorite for 30 min and washed several times with sterile distilled water. Five sterilized seeds were placed in each Petri dish. To determine salinity tolerance, the number of non-germinated seeds and the radicle and plumule weights of all seedlings were examined when ~ 75% of the seedlings in the Petri dishes reached a length of 1 cm. The most tolerant and susceptible genotypes were selected based on their ability to germinate under salinity stress.

Germination percentage: The emergence of plumules was recorded daily and used as the germination index. The germination percentage was calculated daily using the following formula:

1

Salinity tolerance index: (STI): After 10 days, root and shoot dry weights were measured for each seedling and STI was calculated using the following formula:

2

The experiment was arranged in a completely randomized design with four replications under laboratory conditions. Data were subjected to statistical analysis using ANOVA, a statistical package available in SAS 9.1 (SAS Institute Inc. Cary). Significant differences between treatments were determined using Tukey’s test (p < 0.05).

Application of salinity and lead stresses

Based on the results of the germination test, we selected BC 678 and Dominate, as tolerant and sensitive maize genotypes, respectively. The seeds were disinfected with 2% sodium hypochlorite for 5 min and then rinsed with distilled water. Then, they were sown into pots, and the seedlings were later replanted in plastic pots. These pots were filled with a mixture of clay and sand in a 2:1 ratio, respectively. The experiment was carried out in a greenhouse at the research station of Shiraz University, located at 29°50′ N, 52°46′ E, with an altitude of 1810 m above sea level. The temperatures were maintained at 15 °C during the night and 30 °C during the day throughout the experiment. Four-leaf-seedlings were subjected to 0 and 250 mM of NaCl and 0 and 250 µM Pb to evaluate salt-tolerant and -sensitive maize genotypes under salinity and Pb stresses. The levels of NaCl and Pb were gradually increased by 100 mM and 100 µM per day, respectively, until reaching the final concentration for each treatment. No additional nutrients or fertilizers were added. Harvesting was carried out after 8 days to measure various parameters.

Measurement of morphological and physiological characters

Roots and shoots were separated, then root length (RL) and shoot length (SL) were measured (cm). Total fresh weight (TFW) was determined. The plants were then oven-dried at 70 °C for 72 h, and the total dry weight (TDW) was determined. Fully expanded young leaves were used for leaf physiological and biochemical measurements. To determine relative water content (RWC), for each replicate, 10 samples of leaf discs (10 mm in diameter) were taken and fresh weight measured. Then, turgid weight (TW) was determined by flotation on water for 4 h. After that, the samples were dried at 75 °C for 24 h to measure the dry weights (DW)²⁵. The RWC was calculated using the following formula:

3

FW = Fresh Leaf weight; TW = Turgid leaf weight; DW = Dry leaf weight.

The chlorophyll (a and b) and carotenoids contents of leaf tissues were determined by following formula²⁶:

4

5

where V is the volume of the solution and W is the leaf weight.

6

7

Proline content

The proline content was measured by the method of Bates²⁷. About 0.1 g of leaves was homogenized in 500 µl of 3% (w/v) aqueous sulfosalicylic acid and the homogenate was centrifuged at 13,000 rpm for 5 min. The supernatant was used to determine proline content. The reaction mixture consisted of 100 µl of supernatant, 100 µl of 3% (w/v) sulfosalicylic acid, 200 µl of acid ninhydrin, and 200 µl of glacial acetic acid, which was boiled at 100 °C for 1 h, and the reaction terminated in an ice bath. The proline was extracted using 1 ml toluene, and the absorbance read at 520 nm.

Measurements of antioxidant enzyme activities

To measure enzyme activity, 0.5 g of fresh leaves were ground in liquid nitrogen to fine powder and homogenized in 5 mL extraction buffer (pH 7.8) containing potassium phosphate (50 mM), EDTA (1 mM) and polyvinyl-poly-pyrrolidone (2%). The homogenate was then centrifuged at 15,000 rpm for 30 min at 4 °C, and the supernatant was used to evaluate the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX). All enzyme activities were measured at 25 °C using a UV-B spectrophotometer (UV-B 2501; Shimadzu, Japan). SOD activity was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT) at OD₅₆₀ using the method described by Giannopolitis and Ries²⁸. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of NBT reduction. Pütter’s method was used to measure POD activity, and the absorbance was read at OD₄₃₆²⁹. CAT activity at OD₂₄₀ was measured according to the Aebi’s method and described as µM of H₂O₂ degraded min^− 1 mg^− 1 protein³⁰. APX activity at OD₂₉₀ was measured using the method described by Nakano and Asada³¹.

Determination of oxidative stress by malondialdehyde, and hydrogen peroxide contents

Lipid peroxidation was determined by measuring malondialdehyde (MDA) content, as described by Cakmak and Horst³² with minor modifications. Briefly, 0.25 g of fresh leaves were ground in liquid nitrogen to fine powder and homogenized in 5 ml of trichloroacetic acid, TCA (0.1%), then centrifuged at 12,000 rpm at 4 °C for 15 min. Then, 250 µL of supernatant was added to 1 mL extraction buffer (0.5% thiobarbituric acid, TBA, and 20% trichloroacetic acid, TCA). The mixture was boiled at 90 °C for 30 min, cooled to room temperature. The MDA content was calculated by subtracting OD₆₀₀ value from OD₅₃₂ value and using Lambert–Beer law with a molar absorption coefficient of 155 mM^− 1 cm^− 1.

The hydrogen peroxide (H₂O₂) content was determined according to method described by Loreto and Velikova³³. Briefly, 0.25 g of fresh leaves were ground in 5 mL of TCA (0.1% w/v), and then centrifuged (12000 rpm) for 15 min. After that, 500 µL of supernatant was mixed with 500 µL of potassium phosphate (10 mM) (pH 7.0) and 1 mL of potassium iodide (1 M), then vortexed and read at OD₃₉₀. The concentration of H₂O₂ was evaluated using standard curve analysis.

Analysis of Na+, K+, and Pb in plant tissues

The leaf and root samples were dried at 70 °C for 48 h, and then finely ground to measure the levels of cations (Na⁺, K⁺, and Pb). Around 0.5 g of each sample was ashed at 580 °C for 4 h (for leaf) and 8 h (for root) and then digested with 5 mL of 2 N HCl. The resulting solutions were filtered and diluted with boiled distilled water to a final volume of 50 mL³⁴. Na⁺ and K⁺ levels were measured using a flame photometer (PFP7, Jenway, Staffordshire, UK), while Pb levels were determined using an atomic absorption spectrometer (AA-670, Shimadzu, Kyoto, Japan).

Statistical analyses

A factorial experiment based on a completely randomized design with three replications was carried out in the greenhouse. The main factors were genotypes (C), salinity stress (S), and Pb stress (L). Data were analyzed with ANOVA and significant differences between treatments were determined using Tukey’s test (p < 0.05). Statistical analyses were conducted using SAS 9.1 (SAS Institute Inc., Cary). Principal component analysis (PCA) and correlation analysis were performed by ggplot2 and factoextra packages R 4.0.4 Statistical Software.

Results

Response to salt stress

To test relative tolerance of maize seeds to salt stress we treated different genotypes with low (100 mM) or high (200 mM) concentrations of NaCl. The germination percentage of maize seeds was not significantly affected upon treatment with 100 mM NaCl except for Dominate genotype that was selected as a sensitive genotype (Fig. 1). Low concentration of NaCl caused a significant reduction in the root dry weight in Dominate genotype but not BC 678 genotype (Fig. 1b). Treatment with 100 mM NaCl did not affect the shoot dry weight (Fig. 1c). At 100 mM NaCl, we observed 13.6% and 64.9% reduction in STI for BC 678 and Dominate genotypes, respectively (Fig. 1d).

Fig. 1.

Seed germination traits in seedlings of nine maize genotypes under different salinity levels (0, 100, and 200 mM NaCl). (a) Germination percentage, (b) Root dry weight, (c) Shoot dry weight, (d) Salt tolerance index. Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

High concentration of NaCl (200 mM) caused a significant reduction in germination parameters as compared to the water treated plants. Dominate and BC 678 genotypes showed 90% and 36.3% reduction in germination, respectively, at 200 mM NaCl (Fig. 1a). Treatment with 200 mM NaCl also caused a significant reduction in root (95.7%) and shoot (86%) dry weights of Dominate genotype (Fig. 1b and c). In comparison, BC 678 showed relatively less reduction in root (39.2%) and shoot (27.7%) dry weights (Fig. 1b and c). A 32.3% and 93.5% reduction in STI was observed for BC 678 and Dominate genotypes at 200 mM NaCl, respectively (Fig. 1d). Based on these results, Dominate and BC 678 were selected as sensitive and tolerant genotypes, respectively.

Morphological and growth parameters

To evaluate the effect of stress on tolerant and sensitive genotypes, various morphological and physiological characters were measured. All plants survived throughout the experimental period (i.e., 100% survival rate), even in salinity and lead stress conditions. Shoot length, root length, dry and fresh weights, and leaf area generally decreased in both stresses (Fig. 2a-e). Although, both stresses caused a reduction in shoot length in sensitive and tolerant genotypes, but the highest reduction of shoot length (61.5%) was seen for Dominate genotype subjected to salt stress (Fig. 2a). In general, salinity stress reduced shoot length more than lead stress (Fig. 2a). A significant reduction in the root length was also seen in both genotypes under salt and lead (Pb) stresses (Fig. 2b). Although, the susceptible genotype showed a greater reduction (48.4%) in root length under salinity stress compared to the tolerant one (38.7%), there was no difference between susceptible and tolerant genotypes under lead stress (Fig. 2b). These results suggest that salinity stress has more drastic effect on root length compared to lead stress (Fig. 2b).

Fig. 2.

Morphology and growth traits in seedlings of BC 678 and Dominate genotypes under salinity and Pb stress. (a) shoot length (b) root length, (c) dry weight, (d) fresh weight, (e) leaf area. Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

Under salinity conditions, dry and fresh weight dramatically decreased in Dominate (78.3 and 88.6%), while in BC 678, the reduction was smaller (65.1 and 81.4%) (Fig. 2c and d). Lead stress also showed a significant reduction in dry and fresh weights, although the reduction was milder (Fig. 2c and d). As shown in Fig. 2e, salinity stress markedly reduced the leaf area in both tolerant and susceptible genotypes (78.02 and 82.30%, respectively), while the reduction under lead stress was not prominent.

Proline, RWC, and chlorophyll content

Generally, proline content increased under the salinity and Pb stresses, although this enhancement was not significant under Pb stress. BC 678, tolerant genotype, (110.6% and 30.3%) showed greater accumulation of proline than Dominate, sensitive genotype, (100.7% and 10.9%) in response to salinity and Pb stresses, respectively (Fig. 3a). As shown in Fig. 3b, Dominate genotype under salinity stress had the highest reduction in RWC (68.5%), while the reduction was less in the tolerant genotype BC 678 (26.7%). These results also showed that the Pb stress had no effect on RWC in any of the genotypes (Fig. 3b). There was no significant reduction in chlorophyll a, b, and total chlorophyll in the tolerant genotype under salinity or Pb stress, while both stresses significantly decreased chlorophyll a b and total chlorophyll in the sensitive genotype (Fig. 3c-e). Salinity or Pb stress had no effect on carotenoids content in the tolerant genotype BC 678, but the Pb stress caused a reduction in the carotenoids levels in the sensitive genotype. The salt stress had no significant effect on carotenoids levels in the sensitive genotype (Fig. 3f).

Fig. 3.

Proline, relative water content (RWC), and Chlorophyll content in leaves of seedlings of BC 678 and Dominate genotypes under salinity and Pb stresses. (a) proline, (b) relative water content (RWC), (c) chlorophyll a (chl a), (d) chlorophyll b (chl b), (e) total chlorophyll (total chl), (f) carotenoid. Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

Antioxidant enzymes activities

Both salinity and Pb stresses had a significant effect on the antioxidant enzyme activities (SOD, POD, APX, and CAT) in the leaves of tolerant and sensitive genotypes (Fig. 4a-d). Our results showed a significant increase in the SOD activity in Dominate (214.7% and 82.4%) and BC 678 (271.3% and 110%) genotypes under salinity and Pb stress, respectively. Under both stresses, the increase in the tolerant genotype was significantly greater than that in the sensitive genotype (Fig. 4a). The results also indicated that salinity stress increased SOD activity significantly higher than Pb stress in both genotypes (Fig. 4a). Under the same conditions, the activity of POD was increased only under salinity stress in the tolerant genotype (72%), and in the rest of the cases, no significant increase was observed (Fig. 4b). The activity of APX increased in response to both stresses in both genotypes (Fig. 4c). APX activity in the sensitive genotype (171% and 67.7%) was significantly less than tolerant genotype (291.7% and 158.3%) under salinity and Pb stress, respectively (Fig. 4c). In comparison, CAT activity increased under salinity and Pb stress in both tolerant (122.2% and 66.7%) and sensitive (57.1% and 28.6%) genotypes, respectively. This increase, however, was less significant under Pb stress in the sensitive genotype (Fig. 4d). Generally, our results showed that salinity stress increased all antioxidant enzymes activities greater than Pb stress.

Fig. 4.

Activities of anti-oxidative enzymes in leaves of seedlings of BC 678 and Dominate genotypes under salinity and Pb stresses. (a) superoxide dismutase (SOD), (b) peroxidase (POD), (c) ascorbate peroxidase (APX), (d) catalase (CAT). Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

Malondialdehyde (MDA), and hydrogen peroxide contents

The MDA content was measured because its levels are used as a marker for oxidative stress suggestive of damage to the cell membrane. Compared with the control, the MDA content increased under both salt and Pb stresses in both genotypes, although this increase under salinity stress was dramatic than the Pb stress (Fig. 5a). The results also indicated that the MDA content in the sensitive genotype (373.7% and 57.5%) was greater than that of the tolerant genotype (287.6% and 8.2%) under salinity and Pb stress, respectively (Fig. 5a). Likewise, the H₂O₂ content significantly increased in the sensitive genotype in both stresses, while the increase of H₂O₂ content was not significant in tolerant genotype (Fig. 5b).

Fig. 5.

Malondialdehyde, and Hydrogen Peroxide Contents in leaves of seedlings of BC 678 and Dominate genotypes under salt and Pb stresses. (a) malondialdehyde (MDA), and (b) hydrogen peroxide (H₂O₂). Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

Na+, K+, and Pb in plant tissues

Compared to the control, Na⁺ content in leaves increased under salinity stress in both genotypes, but did not change under lead stress. The Na⁺ cotent in leaves was not significantly different between resistant and sensitive genotypes under salt stress (Fig. 6a). Leaf K⁺ content decreased under salinity stress in both genotypes, and also decreased in the resistant genotype under Pb stress, while did not change in the sensitive genotype. Also, the results showed that there was no significant difference in K⁺ content of leaves in sensitive (41.77%) and resistant (30.99%) genotypes under salt stress conditions (Fig. 6b). The K⁺/Na⁺ ratio in leaves decreased under salt stress in both genotypes, and also decreased in the susceptible genotype under lead stress, while did not change much in the resistant genotype. The K⁺/Na⁺ ratio in leaves was not significantly different between sensitive (96.42%) and resistant (92.68%) genotypes under salt stress (Fig. 6c). Pb content in the leaves increased significantly under Pb stress in the resistant genotype but not in the sensitive genotype. The content of Pb in leaves increased in the resistant genotype under salt stress but decreased in the sensitive genotype. The results also indicated an increase in the content of Pb in leaves (61.67%) in the resistant genotype and 12.47% in the sensitive genotype compared to the control (Fig. 6d).

Fig. 6.

Na⁺, K⁺, K⁺/Na⁺, and Pb Contents in leaf and root of seedlings of BC 678 and Dominate genotypes under salt and Pb stresses. (a) Na⁺ in leaf, (b) K⁺ in leaf, (c) K⁺/Na⁺ in leaf, (d) Pb in leaf, (e) Na⁺ in root, (f) K⁺ in root, (g) K⁺/Na⁺ in root, and (h) Pb in root. Different letters indicate significant differences between the salinity levels (S) within each genotype and between genotypes (C), across different salinity levels, respectively, at P < 0.05 level. Bars with similar letters are not different at P < 0.05 according to Tukey test. ns: not significant, *P < 0.05, **P < 0.01.

Compared to the control, Na⁺ content in the roots increased under salinity stress in both genotypes while did not change under Pb stress. The sensitive genotype showed a significantly higher increase in root Na⁺ content (323.02%) compared to the resistant genotype (241.79%) (Fig. 6e). Root K⁺ concentration decreased under salinity stress in both genotypes, while did not change much underPb stress. The sensitive genotype (36.11%) showed a significant decrease in root K⁺ content compared to the resistant genotype (25.51%) (Fig. 6f). The ratio of K⁺/Na⁺ in the roots decreased under salt stress in both genotypes, while did not change much under Pb stress. There was no significant difference in the K⁺/Na⁺ ratio between the sensitive (84.81%) and resistant (78.23%) genotypes under salt stress conditions (Fig. 6g). Moreover, the content of Pb in roots increased significantly in both resistant and sensitive genotypes under Pb stress. Under salt stress, the concentration of root Pb decreased in the resistant genotype but remained unchanged in the sensitive genotype compared to the control. The results also showed an increase in the content of Pb in roots in the resistant (45.78%) and sensitive (19.90%) genotypes compared to the control (Fig. 6h).

Principal component, Pearson correlation, and cluster analysis

Principal component analysis (PCA) showed correlations between different variables under salinity and Pb stress (S1 Table). The significantly correlated variables were placed very closely and in the same quadrant. Variable plot analysis showed 86.3% of variance was explained by PC1 (69.8%) and PC2 (16.5%) (Fig. 7). Based on the biplot, SOD, MDA, CAT, Na⁺ in root and leaf, K⁺ in leaf, chlorophyll content, carotenoids, H₂O₂, and APX had a stronger impact on the model (Fig. 7). Four variables including proline, Chl b, Carotenoids and H₂O₂ separated Dominate genotype, as the sensitive genotype, under both stresses from BC 678 genotype (Table S1). Also, high content of proline could be used to separate the tolerant genotype under salinity stress. Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids also positively correlated and grouped together. The plot showed Dominate genotype under both stresses had low chlorophyll and carotenoids. Based on these results, it can be concluded that Dominate had the same behavior under salinity and lead stresses, but BC 678 did not (Fig. 7). Also, according to P2 axe it can be concluded that Dominate genotype is a sensitive genotype to salinity and Pb stress, and BC 678 is tolerant to salt and Pb stresses and based on P1 axe with the highest cumulative variation can be separated salinity stress from control (Fig. 7).

Fig. 7.

PCA biplot analysis between biochemical parameters and antioxidant with plant biomass parameters under various salt increments. Root length, plant height, total fresh weight, total dry weight, leaf area, chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (T. chl), carotenoids, superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APX), relative water content (RWC), proline content, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), Na⁺ in leaf (Na leaf), K⁺ in leaf (K leaf), K⁺/Na⁺ in leaf (K/Na leaf), Pb in leaf (Pb leaf), Na⁺ in root (Na root), K⁺ in root (K root), K⁺/Na⁺ in root (K/Na root), and Pb in root (Pb root).

Pearson’s correlation of antioxidants and other parameters with plant biomass was analyzed (Fig. 8). Proline, as an osmolyte in plants, had the highest correlation with the increase of antioxidant enzymes, SOD, and CATactivities and low correlation with POD activity, but was not correlated with APX activity (Fig. 8). Plant biomass parameters including total dry weight, total fresh weight, plant height, root length, and leaf area was highly correlated and had a strong correlation with the content of chlorophyll, content of K⁺, K⁺/Na⁺ ratio and RWC. These parameters negatively correlated with the content of Na⁺, MDA and H₂O₂ contents, and antioxidant enzyme activities (Fig. 8).

Fig. 8.

Pearson’s correlation between biochemical parameters and antioxidant with plant biomass parameters under various conditions. Root length, plant height, total fresh weight, total dry weight, leaf area, chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (T. chl), carotenoids, superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APX), relative water content (RWC), proline content, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), Na⁺ in leaf (Na leaf), K⁺ in leaf (K leaf), K⁺/Na⁺ in leaf (K/Na leaf), Pb in leaf (Pb leaf), Na⁺ in root (Na root), K⁺ in root (K root), K⁺/Na⁺ in root (K/Na root), and Pb in root (Pb root).

Cluster analysis was applied to the measured parameters using the Pearson distance method to group the samples and variables (Fig. 9). The results indicated that in the first step, salinity stress separated from Pb stress and controls, and in the second step, Pb stress and controls are located in two different subgroups (Fig. 9). The measured parameters also associated in two main groups; biomass parameters were placed in one group together with RWC, K⁺ content, K⁺/Na⁺ ratio, and pigments, and another group included proline, H₂O_2, Na⁺ content and antioxidant enzymes activities (Fig. 9). In this grouping, Na⁺ content, MDA, H₂O₂, and APX and SOD, CAT and POD activities grouped while proline grouped separately (Fig. 9).

Fig. 9.

Cluster analysis of biochemical parameters, antioxidant and plant biomass parameters under various condition. Root length, plant height, total fresh weight, total dry weight, leaf area, chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (T. chl), carotenoids, superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APX), relative water content (RWC), proline content, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), Na⁺ in leaf (Na leaf), K⁺ in leaf (K leaf), K⁺/Na⁺ in leaf (K/Na leaf), Pb in leaf (Pb leaf), Na⁺ in root (Na root), K⁺ in root (K root), K⁺/Na⁺ in root (K/Na root), and Pb in root (Pb root).

Discussion

The current study was carried out to unravel the effects of salinity and Pb stress on maize by exploring different morphological, physiological, and biochemical attributes. The success of crop production on land with high salt content depends heavily on the germination of seeds, which is a crucial stage for the growth of seedlings. Salinity stress during germination can delay the start of germination, reduce the rate, and cause uneven germination³⁵. It is important to note that germination and early growth are more vulnerable to salinity than later stages of development³⁶. Salinity affects seed germination by lowering the osmotic potential of the soil solution, causing toxicity to the embryo, or altering protein synthesis³⁷. Although, during germination, the root is the first organ to be exposed to salinity stress, shoots are more sensitive to the stress³⁸. Our results also showed shoots were more affected than roots under salinity stress in both susceptible and tolerant genotypes. However, germination parameters in the tolerant genotype were significantly better than sensitive ones (Fig. 1). These results indicated that the germination test may be used as an easy and low-cost method for screening tolerant genotypes under abiotic stress.

Our results indicate that plant growth and biomass were decreased to a great extent at 250 mM NaCl which was consistent with previous works showing maize is sensitive to abiotic stress³⁹. The decline in plant biomass at elevated levels of salinity may be due to osmotic stress, oxidative stress⁴⁰, imbalance of ions⁴¹ reducing shoot growth by suppressing leaf initiation and expansion, reducing internode growth, accelerating leaf abscission⁴², reducing gas exchange and chlorophyll contents⁴⁰.

In the case of Pb, we found that plant growth was affected by 250 µM concentration, which was consistent with an earlier study⁴³. Pb toxicity has been shown to inhibit root growth, and causes blackening and chlorosis of roots⁴⁴. High concentration of Pb can also lead to cell death⁴⁵. Additionally, Pb inhibits seed germination and retards seedling growth, leading to decreased germination percent, germination index, root/shoot length, tolerance index, and dry mass of roots and shoots⁴⁶. Different studies showed high concentration of Pb reduced root, shoot, and leaf growth as well as fresh and dry biomass in different plants^43,47.

Rodríguez Coca et al.,⁴⁸ reported that salinity does not have a direct effect on plant growth but affects turgor, photosynthesis, and enzyme activity which can be extended to other stresses. Salinity has a two-phase effect on plant growth: first, it reduces growth by decreasing soil water potential (osmotic phase), and second, it causes injury in leaves due to a rapid increase in salt concentration in the cell walls or cytoplasm when vacuoles can no longer sequester incoming salts (ionic phase).

The accumulation of salt in old leaves has been found to hasten their death, leading to a decrease in the supply of carbohydrates and growth hormones to the meristematic regions, ultimately hindering growth. This reduction in photosynthesis rate and excessive salt uptake limits plant growth and affects the production of specific metabolites that impede growth⁴⁸.

The anatomy of the root system plays a crucial role in determining root performance, which enables plants to acquire water and nutrients⁴⁹. While a proliferated root system is advantageous for plants, some studies have shown that species with small roots can be more beneficial for shoot development⁵⁰. Also, in cereal crops, genotypes with a greater root volume may exhibit a greater salt tolerance. This has been documented in other cereal species such as emmer wheat⁵¹. Our results indicate that although salinity stress reduced root growth, but the root length of the tolerant genotype was longer than in the susceptible genotype under salinity stress while, there was no difference between these genotypes under Pb stress (Fig. 2b). It has been shown that the larger and more developed roots of plants may lead to a higher accumulation of reserves, improving plant resistance to saline situations⁵⁰. It was previously shown that salinity reduces plant growth through osmotic and toxic effects, causing sodicity, which reduces root growth, and decreases water movement through the root⁵².

A general decrease in fresh weight or dry weight was observed in all plant tissues subjected to salt stress, but it was especially noticeable in the aerial part (Fig. 2). Various studies have shown that plants respond to salt stress by reducing their total leaf area. This reduction in leaf growth is one of the earliest responses of salt-sensitive plants known as glycophytes^52,53. But, in our work, the reduction was observed in both susceptible and tolerant genotypes and there was no significant difference between them (Fig. 2e). High salt concentrations alters cell wall properties, leading to a decrease in leaf turgor and photosynthesis rate, which ultimately results in reduced total leaf area and stem growth⁵⁴. Consequently, the overall size of the aerial part of the plant and its height are also reduced.

Plants experience negative effects on their physiological and biochemical mechanisms when subjected to abiotic stresses. This results in nutrient uptake imbalances, changes in growth-inducing regulators, and inhibition of protein synthesis and photosynthesis, ultimately leading to reduced plant growth and yield⁵⁵. Proline, an organic osmolyte, is a highly effective and important compatible solute that acts as an osmoprotectant⁵⁶. Additionally, proline has antioxidant properties and can act as a molecular chaperone to protect biological macromolecule structures during abiotic stress, thereby conferring plant tolerance to environmental stresses⁵⁶. Applying Proline to salt-stressed rice plants has been shown to improve plant growth by reducing ROS detoxification caused by salt stress⁵⁷. Osmoprotectants such as free proline are formed under salt stress, which controls water-holding capacity and helps protect plants from osmotic stress⁵⁸. Increased proline under salinity stress plays a crucial role in the osmoregulatory function of plants⁵⁹. The present study showed that plants subjected to salinity and Pb stress exhibited higher levels of proline compared to control plants, which act as a compatible active solute to maintain membrane stability through osmotic adjustment⁶⁰. So, it is expected that tolerant genotypes will accumulate more proline than sensitive plants and our results are consistent with this notion. The RWC of plants under salinity stress was lower than the control, but it was not changed under Pb stress (Fig. 3b). Usually, abiotic stress decreased RWC which leads to turgor loss⁶¹. The extent of osmotic adjustment varied among plant species and was influenced by factors such as the level and kind of stress and plant age⁶².

It has been reported that abiotic stresses reduce the chlorophyll and carotenoids⁴⁰. Our results also indicated that Chl a, Chl b, total Chl, and carotenoids were reduced under lead and salinity stresses, although the reduction was not significant in the tolerant genotype (Fig. 3c-f). When plants are exposed to high levels of stresses such as Pb and salinity, it can result in various physiological issues such as destruction of chlorophyll molecules due to the damaging effects of various ROS⁶³. Additionally, the destruction of chlorophyll molecules may occur due to the disordering of grana and thylakoid membranes, as well as the replacement of essential nutrients with Pb⁶⁴. In many cases, chlorophyll synthesis is rather hampered than enhancement of chlorophyll destruction under saline conditions⁶⁵. Researchers have observed that salt-tolerant genotypes tend to maintain their chlorophyll content under salinity conditions, while salt-susceptible genotypes experience a decrease in chlorophyll levels. Therefore, chlorophyll content can serve as a biochemical marker to screen salt- and Pb- tolerant genotypes.

The enzymatic antioxidant defense system (APX, POD, CAT, and SOD) can be improved to regulate reactive oxygen species (ROS). Our results demonstrated an increase in the activity of antioxidant enzymes.

in both genotypes under both stresses, although we found the promising enhancement in their activities in the tolerant genotype under salinity and Pb stresses (Fig. 4). During the process of detoxifying ROS, various enzymes work together. SOD is involved in the first line of defense against ROS and also inhibits the production of OH radicals, thereby decreasing lipid peroxidation in cell membranes⁶⁶. Among all antioxidant enzymes, SOD plays the most crucial role in mitigating superoxide radicals⁴⁰. We found that SOD activity dramatically increased under both stresses. Superoxide radicals are converted into H₂O₂ and oxygen due to the activation of SOD⁶⁷. This increase in activity could be due to a rise in O₂⁻ content or direct interaction between Pb and SOD⁶³. Another enzyme, POD, is essential in removing H₂O₂ from seedling tissues and reducing oxidative damage⁶⁸. CAT and APX are also important enzymes in that their activities help to remove ROS and our results showed that their activities increased under Pb and salinity stress. It has been previously reported that the activity of these enzymes increased under Pb and salinity stresses which aided in detoxifying H₂O₂ by converting it into oxygen and water⁶⁹. Interestingly, the slope of increasing activity of these enzymes was almost the same in both genotypes and there was no difference between the susceptible and tolerant genotypes (Fig. 4). Thus, the activity of these enzymes is not a suitable biomarker to screen tolerant genotypes.

MDA and H₂O₂ are two important parameters that markedly increased under both stresses, especially salinity stress (Fig. 5). It has been previously reported that salinity stress substantially enhanced both of these parameters⁴⁰. MDA is a crucial physiological indicator used to evaluate the integrity and permeability of cell membranes, which can be disrupted by abiotic stress. The primary consequence of salinity stress is the accumulation of ROS in plant cells, leading to lipid peroxidation and subsequent damage to the cell membrane, resulting in increased MDA levels in leaves⁷⁰. Our results also indicated that Pb stress increased MDA more than salinity stress (Fig. 5a). It can be concluded that Pb had more destructive effects on the cell membranes than salinity. Both salinity and Pb caused oxidative stress by inducing the overproduction of H₂O₂ (Fig. 5b), which is consistent with earlier work suggesting that salinity⁴⁰ and Pb⁵⁸ increase H₂O₂ production and membrane damage. H₂O₂ is a potent oxidant that can cause severe toxicity to plants, particularly when it is converted into hydroxyl anions. Therefore, efficient and rapid detoxification of H₂O₂ is essential to prevent toxicity in plants. Various antioxidant enzymes are involved in mitigating H₂O₂ in different parts of the cell¹⁴. Also, we found both of these parameters increased more under both salinity and Pb stresses in the susceptible genotype than in the tolerant genotype. Hence, it can be suggested that the content of MDA and H₂O₂ in plants are two important parameters for selecting tolerant genotypes.

A high concentration of K⁺ ions and a low concentration of Na⁺ ions in the cytoplasm are necessary for enzyme processes in cells to function normally. Plants also protect themselves from salt damage by limiting the influx of sodium to their aerial parts⁷¹. A study has shown that salt stress increases sodium content and decreases K⁺ content in maize leaves⁷². Salinity reduces the ability to exchange potassium for sodium, leading to a decrease in sodium ion excretion, which causes more sodium ions to accumulate in cells⁷³. Sodium transport from roots to shoots occurs through the apoplast pathway⁷³, a Casparian band in endodermal root cells creates a barrier to the movement of ions from root to shoot⁷¹. Several studies have reported apoplast barrier enhancement as an effective approach to reducing sodium entry. For example, in a study, it was shown that extensive apoplast barriers in roots led to decreased sodium uptake, increased survival, and tolerance to salt stress in Oryza sativa L⁷⁴. Under Pb stress, the concentration of Pb in roots and leaves significantly increased compared to non-stressed plants (see Fig. 6d and h). This is because the root is the first organ to be exposed to Pb⁷⁵. Lead tends to remain in the root due to the binding of lead ions to ion exchange sites located in the cell wall and due to extracellular deposition. The accumulation of Pb in roots happens through binding with polysaccharides and organic acids in cell walls and xylem vessels. Therefore, Pb may become immobilized in roots⁷⁶. Excessive concentration of sodium has a negative effect on the metabolic and physiological function of the plant. Therefore, maintaining K⁺/Na⁺ homeostasis is essential for better growth⁷⁷. Sodium chloride improved Pb transport and its accumulation in plants under Pb stress, indicating that sodium chloride improves Pb uptake and Pb transport from root to shoot⁷⁸. In other words, moderate salinity may be beneficial for the transport and accumulation of Pb in plants⁷⁸.

Multivariate techniques like PCA are more suitable for detecting key patterns in data with complex variables⁷⁹. Graphical visualization of PCA loadings provides an easier and more intuitive way to explore the shared and contrasting physiological responses to salinity stress^80,81. The PCA analysis was used to examine the correlation between morphological, physiological, and biochemical traits under stress and non-stress conditions. The analysis revealed that tolerant genotypes exhibited elevated levels of osmoprotectant proline, K⁺/Na⁺ ratio, and antioxidants such as SOD, CAT, and APX under salt stress conditions, which aligns with findings from previous studies^82,83. The PCA analysis confirmed that CAT and APX activities are the key contributors to salt tolerance (CSR36 genotype) in rice⁸⁴.

We found that MDA, H₂O₂, and Na⁺ content had a greater repressive effect on the plant growth in the sensitive genotype compared to the tolerant genotype. Our results show a strong positive relationship between H₂O₂ generation and MDA amount (Fig. 8) based on the correlation analysis. Previous studies have demonstrated increased lipid peroxidation and decreased membrane stability index in plants under salinity stress, indicating a correlation between ROS/H₂O₂ generation and lipid peroxidation^85,86. Additionally, previous studies on various plant species have shown that lipid peroxidation in salt-sensitive genotypes is more than salt-tolerant genotypes^87,88. In our experiment, the Dominate genotype exhibited greater lipid peroxidation and oxidative stress.

Conclusion

The results of this study provide new insights into the effects of salinity and Pb stresses on the growth and development of maize plants. The findings indicate that the application of NaCl and Pb had significant effects on the growth parameters of the plants. Moreover, the study revealed that the chlorophyl and carotenoids content, physiological and morphological attributes, and antioxidant activity of maize were adversely affected by both stresses. However, salinity stress, particularly at a concentration of 250 mM NaCl, resulted in a more pronounced decline in most attributes compared to Pb stress. The data obtained from the study also suggests that the Dominate was more susceptible to the detrimental effects of salinity and Pb stress than BC 678. These findings have significant implications for the development of strategies to enhance the tolerance of maize plants to abiotic stresses, which is essential for improving crop productivity and food security. Future research in this area should focus on identifying the molecular mechanisms underlying the responses of maize plants to salinity and Pb stresses, which could facilitate the development of novel approaches for enhancing stress tolerance in crops. Overall, the findings of this study contribute to our understanding of the impacts of abiotic stresses on plant growth and have important implications for sustainable agriculture.

Electronic supplementary material

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Author contributions

E. Z. wrote the original draft. E.Z. and B.B. contributed to the acquisition of data and analysis. D.H., H.R., and A.M. participated in discussions and also reviewed and edited the manuscript. A.A. supervised the work, reviewed and edited the manuscript. All authors read and approved the final manuscript.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.