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. 2023 Feb 3;29(2):173–184. doi: 10.1007/s12298-023-01281-0

Investigation of the combined effects of cadmium chloride, silver nitrate, lead nitrate, methyl jasmonate, and salicylic acid on morphometric and biochemical characteristics of St. John’s wort

Ahmad Abdollahi 1, Nader Farsad-Akhtar 1, Elham Mohajel Kazemi 1, Maryam Kolahi 2,✉
PMCID: PMC9981836  PMID: 36875733

Abstract

Hypericum perforatum L., is a sprawling, leafy herb that grows in open, disturbed areas, known as St. John’s wort, has a variety of secondary metabolites that can be used for medicinal and therapeutic purposes. Heavy metals have become the most dangerous pollutants in the environment. The effect of cadmium chloride, lead nitrate, silver nitrate, methyl jasmonate, and salicylic acid was studied on several morphometric and biochemical features of St. John’s wort simultaneously using the Taguchi statistical method. The results showed cadmium chloride and lead nitrate reduced the morphometric and biochemical properties of St. John’s wort while salicylic acid compensated for the adverse effects of heavy metals. Simultaneously, use of salicylic acid and silver nitrate with cadmium chloride and lead nitrate reduced the toxic effects of these metals on morphometric properties. Methyl jasmonate improved growth characteristics at low levels and inhibited at higher levels. Also, according to the results, salicylic acid could reduce the effects of heavy metals on the biochemical properties, while silver nitrate acts like heavy metals, especially at higher levels. Salicylic acid reduced the harmful effects of these heavy metals and at all levels was able to create a better induction effect on St. John’s wort. These elicitors mainly changed the adverse effects of heavy metals by strengthening the pathways of the antioxidant system in St. John’s wort. The research assumptions were validated, which suggests that the Taguchi method could be considered in an optimum culture of medicinal plants under different treatments such as heavy metals and elicitors.

Keywords: Biochemical, Elicitor, Heavy metals, Morphometric, St. John's wort, Taguchi

Introduction

Hypericum perforatum L. known as St. John’s wort belong to the Hofarqoon family and has a variety of secondary metabolites that can be used for medicinal and therapeutic purposes (Naghdibadi et al. 2005). St. John’s wort is an important medicinal plant with many secondary metabolites such as terpenes, terpenoids, phenolic chemicals, and aliphatic derivatives (Crockett 2011). This plant has been used as a preservative in the food industry to reduce spoilage and enhance food shelf life, as well as medical and therapeutic usesbesides having applications in cosmetics due to their superior antibacterial properties (Crockett 2011). Research showed St. John’s wort to be an effective treatment for premature ejaculation with low side effects (Falahatkar et al. 2009). Environmental contaminants such as heavy metals can be found in all industrial regions. Heavy metals toxicity and their bioaccumulation in food chains, are  most pressing environmental and health issues (Lasat 2002). The cadmium is one of the most hazardous soil pollutants. The adverse effects of this element on biological activities in soil, plant metabolism, and human and animal health prompted researchers to the behavior of cadmium in the environment, as well as its effects on plant physiology (Majer et al. 2002). Cadmium is easily absorbed by plant roots and its toxicity is 20 times higher than other heavy metals. (Dinakar et al. 2009). St. John’s wort accumulates high amounts of cadmium (1.087 mg/Kg dry weight of stem), so it can be classified as a cadmium accumulator plant (Zandavifard et al. 2017). Lead is another heavy metal causes environmental pollution and lead to reduced development, growth, and reproduction of plants (Ghelich et al. 2014). Use of plant growth regulators is one of the strategies to increase resistance in plants under stress ( Mansour et al. 2020). Various methods used to increase the production of secondary metabolites in medicinal plants, which include the use of elicitors, adding precursors, optimizing the culture environment, hairy root culture, and metabolite engineering. Elicitors induce biosynthesis and accumulation of secondary metabolites and defense responses in plants (Rasouli et al., 2018; Bahabadi and Sharifi 2013). Researchers use elicitors such as methyl jasmonate and salicylic acid to increase the production of secondary metabolites through physiological and developmental changes in medicinal plants. Salicylic acid has essential roles in physiological processes such as growth, development, respiration, ethylene synthesis, opening and closing of stomata, and induction of flowering ( Mansour et al. 2020). Salicylic acid, as an antioxidant compound, can increase plant resistance to abiotic stresses such as ultraviolet, drought, heavy metals, salinity, and high temperatures (Padash et al. 2016). It’s one of the antioxidant compounds that has been used in recent years to increase the resistance of plants to stress (Rajabi et al. 2016). Methyl jasmonate, as a volatile aromatic compound, regulates growth and developmental processes and responses to abiotic stresses in plants (Kohanmoo et al. 2016). The properties of silver nitrate, such as availability and water-solubility, make it suitable for external plant growth regulator. It has been proven that silver nitrate is a potent inhibitor of ethylene activity is widely used in plant tissue culture (Alirezaei et al. 2017). Silver nitrate has been used as an abiotic elicitor, including effects on enzyme activity, gene expression, and production of secondary metabolite in plants (Alirezaei et al. 2017).

Among the most important multivariate techniques in analytical optimization, the Taguchi method implemented for this research with some factors and levels. Taguchi designs use orthogonal arrays, which estimate the effects of factors on the response mean and variation. Orthogonal arrays allow to investigate each effect independently from the others and may reduce the time and cost associated with the experiment when fractionated designs used. The Taguchi Method implementation in various industries such as aerospace, car manufacturing, shipbuilding, chemical, food, and pharmaceutical industries have widely used. In this research, due to many factors and levels, Taguchi method uses orthogonal arrays [partial factorial] to design, while in traditional methods such as factorial design, the number of experiments, cost, and time increased. The reasons for preferring this method include the number of fewer tests, investigating interactions, less cost and time compared to other plants, as well as the possibility of adjusting factors at different levels ( Siouki et al. 2017; Fotouhi et al. 2018).

The present study aimed to investigate the mutual effects of heavy metals cadmium chloride and lead nitrate and the growth elicitors methyl jasmonate, salicylic acid, and silver nitrate on the growth and antioxidant defense system activity of St. John’s wort plant using the Taguchi method for the first time. In this research, it has been tried to provide solutions to improve the damage caused by heavy metals in polluted environments using different elicitors on St. John’s wort with commercial value.

Materials and methods

Cultivation and treatment of plants

The seeds of St. John’s wort (Topaz cultivar) prepared by Isfahan Pakan Bazr Company. The seeds were sterilized and then cultured in 16 pots containing autoclaved cocuperlite according to Taguchi’s design. After seed germination, first seedlings were irrigated with 25% Hoagland solution, then 50% Hoagland solution, and completed Hoagland three times a week based on the field capacity. About four weeks, when the plants reached the four-leaf stage, treatment of plants with five factors, including solutions of CdCl2, Pb(No3)2, Ag(No3)2, C7H6O3, and C5H6(CH2CH = CHC2H5) CH2CO2CH3(= O) was done. The changes of all factors including, four levels of cadmium chloride (0, 0.25, 0.5, and 1 mM), lead nitrate (0, 12.5, 25, and 50 mg/L), silver nitrate (0, 0.25, 0.5, and 1 mM), salicylic acid (0, 0.125, 0.25, and 0.5 mM) and the methyl jasmonate (0, 0.125, 0.25, and 0.5 mM) performed once a week. Three replications considered for each of the treatments. Twenty days after applying the treatments, samples were taken for morphometric and biochemical studies.

Morphometric studies

Morphometric experiments conducted using a ruler, checkered paper, and digital scales to determine the length (roots, stems, and leaves), leaf area, and weight (fresh and dried).

Biochemical studies

Proline determined according to Bates et al. (1973). The reduction of hydrogen peroxide used to determine the catalase enzyme activity (Chance and Maehly,1955). Peroxidase enzyme activity performed according to Chance and Maehly’s (1955) method by guaiacol test and measurement of its conversion to tetraguaiacol. The Ascorbate peroxidase enzyme activity measured according to Boominathan and Doran’s (2002) method based on ascorbic acid oxidation and absorption reduction at 290 nm. The Superoxide dismutase enzyme activity assayed based on the inhibition of reduction of nitro blue tetrazolium (NBT) by enzymatic extract (Dhindsa et al. 1981). The specific activity of the enzymes expressed as a unit per milligram of protein (U mg− 1 protein). Heath and Packer’s (1968) method used to measure malondialdehyde (MDA) as a product of membrane lipid peroxidation. H2O2 assay performed according to Habibi et al. (2014) with slight modifications.

Experimental design by Taguchi method

Optimize the factors in this experiment, the Taguchi method was used to design experiments. This method, the effect of factors and levels entered into the software, and the software provides suggested and optimal experiments. In this study, 5 factors in 4 levels (according to Table 1) entered into Qualiteek-4 software, and the software suggested sixteen experiments (L16). Each experiment performed according to the conditions shown in Table 2. Therefore, each row represents the conditions of an experiment. Statistical analyzes were performed based on the Taguchi method using Minitab 19 software.

Table 1.

Factors and levels for optimization by the Taguchi method

Factors Level 1 Level 2 Level 3 Level 4 Unit
CdCl 2 0 0.25 0.5 1 mMol/L
Pb(NO3) 2 0 12.5 25 50 mg/L
AgNO 3 0 0.25 0.5 1 mMol/L
SA 0 0.125 0.25 0.5 mMol/L
MJ 0 0.125 0.25 0.5 mMol/L
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Table 2.

Design of sixteen experiments by the Taguchi method (L16)

Experiments CdCl2 (mMol/L) Pb(NO3)2 (mg/L) AgNO3 (mMol/L) SA (mMol/L) MJ (mMol/L)
1 0 0 0 0 0
2 0 12.5 0.25 0.125 0.125
3 0 25 0.5 0.25 0.25
4 0 50 1 0.5 0.5
5 0.25 0 0.25 0.25 0.5
6 0.25 12.5 0.5 0.5 0.25
7 0.25 25 1 0 0.125
8 0.25 50 0.5 0.125 0
9 0.5 0 0.5 0.5 0.125
10 0.5 12.5 1 0.25 0
11 0.5 25 0 0.125 0.5
12 0.5 50 0.25 0 0.25
13 1 0 1 0.125 0.25
14 1 12.5 0.5 0 0.5
15 1 25 0.25 0.5 0
16 1 50 0 0.25 0.125
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Results

Table 3 shows percentage of participation of each factor on the intended parameter and the best level of each factor on the parameters.

Table 3.

Percentage of participation of factors and the best level of each factor on the parameter

Parameters Factors CdCl2 Pb(NO3)2 AgNO3 SA MJ
Root length (cm) PPF* 34.82 11.08 8.67 3.31 42.11
BLF** 0.25 12.5 0.25 0.5 0
Shoot length (cm) PPF 34.82 11.08 8.67 3.31 42.11
BLF 0.25 12.5 0.25 0.5 0.0
Leaf length (cm) PPF 13.44 11.95 18.71 8.06 47.81
BLF 0.25 50 0 0.25 0
Leaf area (mm2) PPF 23.21 3.97 24.69 17.10 31.02
BLF 0.25 12.5 0.25 0.25 0
Root fresh weight (g) PPF 3.36 3.98 11.01 6.34 75.30
BLF 0 12.5 0.25 0.125 0
Shoot fresh weight (g) PPF 25.23 28.70 14.47 23.35 8.23
BLF 1.0 12.5 0.5 0 0.5
Root dry weight (g) PPF 20.05 17.18 17.79 24.46 20.52
BLF 1.0 25 0.25 0.5 0
Shoot dry weight (g) PPF 8.01 1.36 17.28 43.68 29.27
BLF 0.25 12.5 0 0.5 0
Prolin (nMol/g FW) PPF 37.12 16.98 7.53 29.67 8.69
BLF 0 0 0 0.125 0
CAT (U/mgProtein) PPF 16.42 9.15 25.84 37.30 11.27
BLF 0 25 0 0.25 0
POD (U/mgProtein) PPF 6.78 19.45 15.50 9.16 49.10
BLF 1 12.5 1 0 0.5
APX (U/mgProtein) PPF 17.54 14.56 33.03 17.86 16.99
BLF 0.5 50 0 0.25 0.125
SOD (U/mgProtein) PPF 40.43 4.78 40.87 6.52 6.95
BLF 0.5 25 0 0.25 0.5
MDA (nMol/g FW) PPF 43.57 1.16 11.81 33.90 9.55
BLF 0.25 25 0 0.125 0
H2O2 (nMol/g FW) PPF 38.63 13.86 17.17 14.51 15.81
BLF 1 12.5 0.25 0.5 0.25
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*PPF = Percentage of participation of factors, **BLF = The best levels of factors

Morphometric features of St. John’s wort under cadmium chloride, lead nitrate, silver nitrate, salicylic acid, and methyl jasmonate by the Taguchi method

Among all the factors, methyl jasmonate had the greatest influence and participation on the root length of St. John’s wort, followed by cadmium chloride, lead nitrate, silver nitrate, and salicylic acid, respectively. Among all the levels of factors, level 1 methyl jasmonate, level 4 salicylic acid, and level 2 all three factors, silver nitrate, lead nitrate, and cadmium chloride, had the best performance and effect on root length of St. John’s wort (Table 3).

Experiment number 8 is the optimum choice in sixteen Taguchi-designed experiments for the root length of St. John’s wort (Fig. 1a). Among all the factors, methyl jasmonate had the most significant influence and participation on the stem length of St. John’s wort. Consequently, salicylic acid, silver nitrate, cadmium chloride, and lead nitrate had similar effects on the stem length, respectively. Among the levels of factors, level 1 of methyl jasmonate, level 4 of salicylic acid, and level 2 of all three factors silver nitrate, lead nitrate, and cadmium chloride had the best performance and effect on stem length of St. John’s wort (Table 3). Experiment number 15 is the optimum choice of sixteen Taguchi-designed experiments for the stem length of St. John’s wort (Fig. 1b). Methyl jasmonate had the greatest effect and contribution among all the factors on the leaf length of St. John’s wort, as shown in Table 3. Likewise, silver nitrate, cadmium chloride, lead nitrate, and salicylic acid had similar effects on the leaf length, respectively. Among all the factors, level 1 of methyl jasmonate and silver nitrate, level 3 of salicylic acid, level 4 of lead nitrate, and level 2 of cadmium chloride had the best performance and effect on leaf length of St. John’s wort (Table 3). Experiment number 16 is the optimum choice of sixteen Taguchi-designed experiments for the leaf length feature of St. John’s wort (Fig. 1c). Among all factors, methyl jasmonate had the most significant effect and contribution on the leaf area feature of St. John’s wort. Following that, silver nitrate, cadmium chloride, salicylic acid, and lead nitrate had similar effects to the leaf area of St. John’s wort, respectively. Among the levels of factors, level 1 of methyl jasmonate, level 3 of salicylic acid, and level 2 of all three factors silver nitrate, lead nitrate, and cadmium chloride had the best performance and effect on the leaf area of St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method for the leaf area feature, experiment number 15 is the optimal choice for the leaf area of St. John’s wort (Fig. 1d). Among all the factors, methyl jasmonate had the most significant effect and participation on the root fresh weight of St. John’s wort. Silver nitrate, salicylic acid, lead nitrate, and cadmium chloride showed similar effects, respectively. Among the levels of factors, level 1 of methyl jasmonate and cadmium chloride, and level 2 of all three factors silver nitrate, salicylic acid, and lead nitrate had the optimum performance and effect on fresh weight of root (Table 3). According to Fig. 1e, the results of sixteen experiments designed by the Taguchi method experiment number 2 is the optimal choice for root fresh weight of St. John’s wort (Fig. 1e). Lead nitrate had the most significant effect and participation among all the factors on fresh weight of St. John’s wort stem. Also, cadmium chloride, salicylic acid, silver nitrate, and methyl jasmonate, respectively, affected on fresh weight of St. John’s wort stem. Among the levels of factors as well, level 4 of methyl jasmonate, level 1 of salicylic acid, level 3 of silver nitrate, level 2 of lead nitrate, and level 4 of cadmium chloride had the best performance and effect on fresh weight of stem (Table 3). According to the results of sixteen experiments designed by the Taguchi method for the fresh weight of stem at all levels followed experiment number 14 (Fig. 1f). Salicylic acid had the most significant effect and participation among all the factors on root dry weight. At the same time, methyl jasmonate, cadmium chloride, silver nitrate, and lead nitrate had a similar effect on the dry weight of St. John’s wort root, respectively. Among the levels, level 1 of methyl jasmonate, level 4 of salicylic acid and cadmium chloride, level 2 of silver nitrate, and level 3 of lead nitrate had the best performance and effect on root dry weight of St. John’s wort root (Table 3). The results of sixteen experiments designed by the Taguchi method for root dry weight, experiment number 15 is the optimal choice for dry weight of St. John’s wort root (Fig. 1g). Salicylic acid showed the greatest effect and participation among the factors on dry weight of St. John’s wort root. Likewise, methyl jasmonate, silver nitrate, cadmium chloride, and lead nitrate had similar effects, respectively. Among the factors, level 1 of methyl jasmonate and silver nitrate, level 4 of salicylic acid, and level 2 of lead nitrate, and cadmium chloride factors showed the best performance and effect on the dry weight of St. John’s wort stem (Table 3). The results of sixteen experiments designed by the Taguchi method revealed that experiment number 6 is the optimal choice for dry weight of St. John’s wort stem (Fig. 1h).

Fig. 1.

Fig. 1

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Sixteen experiments designed to morphometric features in St. John’s wort under cadmium chloride, silver nitrate, lead nitrate, methyl jasmonate, and salicylic acid treatment by the Taguchi method. a root length, b stem length, c leaf length, d leaf area, e fresh weight of root, f fresh weight of stem, g dry weight of root, and h dry weight of stem (p < 0/05)

Biochemical changes of St. John’s wort under cadmium chloride, lead nitrate, silver nitrate, salicylic acid, and methyl jasmonate by the Taguchi method

Cadmium chloride had the most significant effect and participation on the proline content of St. John’s wort in all studied factors. Following that, salicylic acid, lead nitrate, methyl jasmonate, and silver nitrate showed similar effects, respectively. Among levels of factors, level 1 of all four factors cadmium chloride, lead nitrate, methyl jasmonate, and silver nitrate, and level 2 of salicylic acid had the best performance and effect on the proline content of St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method showed that experiment number 1 is the optimum choice for proline content in St. John’s wort (Fig. 2a). Salicylic acid revealed the most significant effect and participation among studied factors on the catalase enzyme activity in St. John’s wort. Also, silver nitrate, cadmium chloride, methyl jasmonate, and lead nitrate showed similar effects on the catalase enzyme activity in St. John’s wort, respectively. Among the levels of factors, level 1 of all three factors methyl jasmonate, silver nitrate, and cadmium chloride, and level 3 of both factors salicylic acid and lead nitrate had the best performance and effect on catalase enzyme activity in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method for catalase enzyme activity, experiment 1 is the optimum choice (Fig. 2b). Methyl jasmonate had the most significant effect and participation on the peroxidase enzyme activity in St. John’s wort. Also, lead nitrate, silver nitrate, salicylic acid, and cadmium chloride showed similar effects on the peroxidase enzyme activity in St. John’s wort, respectively. Among the levels of factors, level 1 of salicylic acid, level 4 of all three factors methyl jasmonate, silver nitrate, and cadmium chloride, and level 2 of lead nitrate had the best performance on the peroxidase enzyme activity in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method revealed that experiment number 14 is the optimum choice for peroxidase enzyme activity in St. John’s wort (Fig. 2c). Silver nitrate showed the most significant effect and participation on the ascorbate peroxidase enzyme activity. Also, salicylic acid, cadmium chloride, methyl jasmonate, and lead nitrate showed similar effects on the ascorbate peroxidase enzyme activity, respectively. Among levels of factors, level 1 of silver nitrate, level 2 of methyl jasmonate, level 3 of both factors salicylic acid and cadmium chloride, and level 4 of lead nitrate had the best effect on the ascorbate peroxidase enzyme activity in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method showed that experiment number 9 is the optimum choice for the activity of ascorbate peroxidase enzyme in St. John’s wort (Fig. 2d). Silver nitrate had the most significant effect and participation among the factors on the superoxide dismutase enzyme activity. Likewise, cadmium chloride, methyl jasmonate, salicylic acid, and lead nitrate showed similar effects on the superoxide dismutase enzyme activity in St. John’s wort, respectively. Among the levels of factors, level 1 silver nitrate, level 4 methyl jasmonate, and level 3 of all three factors cadmium chloride, salicylic acid, and lead nitrate had the best performance and effect on the superoxide dismutase enzyme activity in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method showed that experiment number 11 is the optimum choice for the superoxide dismutase enzyme activity in St. John’s wort (Fig. 2e). Cadmium chloride had the most significant effect and participation on malondialdehyde content in St. John’s wort. So, salicylic acid, silver nitrate, methyl jasmonate, and lead nitrate revealed similar effects on malondialdehyde content in St. John’s wort, respectively. Among the levels of factors, level 1 methyl jasmonate and silver nitrate, level 2 salicylic acid, and cadmium chloride, and level 3 lead nitrate had the best performance and effect on malondialdehyde content in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method showed that experiment number 6 is the optimum choice for malondialdehyde content in St. John’s wort (Fig. 2f). Cadmium chloride showed the most significant influence and participation among studied factors on hydrogen peroxide content in St. John’s wort. In addition, silver nitrate, methyl jasmonate, salicylic acid, and lead nitrate were affected by this feature, respectively. Among the factor levels, level 4 for both factors cadmium chloride and salicylic acid, level 2 for both factors lead nitrate and silver nitrate, and level 3 for methyl jasmonate had the best performance and effect on hydrogen peroxide content in St. John’s wort (Table 3). The results of sixteen experiments designed by the Taguchi method showed that experiment 6 is the optimum choice for hydrogen peroxide content in St. John’s wort (Fig. 2g).

Fig. 2.

Fig. 2

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Sixteen experiments designed in St. John’s wort under cadmium chloride, silver nitrate, lead nitrate, methyl jasmonate, and salicylic acid treatment by the Taguchi method. a proline content, b catalase enzyme activity, c peroxidase enzyme activity, d ascorbate peroxidase enzyme activity, e superoxide dismutase enzyme activity, f malondialdehyde content, g hydrogen peroxide content (p < 0/05)

Discussion

In this study, according to the results of the proposed and optimal Taguchi experiment on morphometric and growth characteristics of St. John’s wort, application of cadmium and lead have shown reduced growth. The toxic effects of lead were more significant than cadmium as reported in different studies (Rad et al. 2014; Aghaei et al. 2019; Mok 1994; Shanker et al. 2005; Hassan et al. 2006; Fatemi et al. 2017; Ranjbar et al. 2020). Generally, cadmium and lead had adverse effects on yield and morphometric features of St. John’s wort. Salicylic acid and silver nitrate improved growth characteristics and compensated the harmful effects of cadmium and lead, which are growth reducers, also other studies showed the same results (Alirezaei et al. 2017; Mansour et al. 2020). Methyl jasmonate at higher levels harmed the growth features of St. John’s wort although methyl jasmonate had the greatest influence and participation on some morphometric features of St. John’s wort. Adding silver nitrate to the culture medium calluses increased the growth features of Lavandula Angustifolia Mill. The pieces of evidence that can interpret the effect of silver nitrate is the ionic effect mechanism. Silver particles release Ag+ gradually, can alter the structure of DNA during the substitution reaction, and change the expression of genes; subsequently through penetration into DNA, activated the transcription of enzymes is involved in the improvement of growth. Silver ions can bind DNA, so they cause transcription changes in some genes, and increase biomass by induction the transcription of genes involved (Alirezaei et al. 2017). Applying salicylic acid and methyl jasmonate in MS liquid culture medium (Murashig and Skog) had different effects on the stem growth of two different species of tea grass (Coste et al. 2011). External methyl jasmonate is commonly used in plant cell culture to activate secondary metabolism pathways. However, some studies showed that jasmonates have variety of biological activities, such as inhibiting the growth and germination of seeds, pollen grains, root growth, and photosynthetic organs (Rajabi et al. 2016). Also, methyl jasmonate improved vegetative and reproductive growth in pistachios (Pakkish and Asghari 2017). Salicylic acid is a natural phenolic compound and growth regulator that plays a critical role in plant growth and response to environmental stresses (Rajabi et al. 2016). Some indices such as plant height, number of leaves, leaf area, fresh and dry weight of shoots improved in Physalis peruviana using 2 mM salicylic acid ( Mansour et al., 2020). In this study, level 1 of methyl jasmonate had the most significant effect on growth while, the higher levels showed no effect on the growth indices. However, higher levels of salicylic acid had reduced the adverse effects of heavy metals on St. John’s wort.

In similar research methyl jasmonate at low concentrations improved growth, while at high concentrations reduced growth (Keramat and Daneshmand 2012). Cadmium is an environmental pollutant and has adverse effects on growth and yield of plants; nevertheless, salicylic acid pretreatment improved the growth and increased the tolerance of plants to cadmium stress (Gerami et al. 2018). Cadmium has been mentioned in various studies as a growth inhibitor (Gerami et al. 2018; Rad et al. 2014; Kolahi et al. 2018, 2020; Yousefi et al. 2018; Yazdi et al. 2019; Aghaei et al. 2019; Kazemi et al. 2020). Among the non-essential heavy metals, cadmium, due to its high solubility in water and rapid absorption by the roots, quickly enters the roots and shoots, subsequently affecting the division and growth of cells in the meristematic region, causing growth delay. The reducing root growth under cadmium is due to the reduced absorption of water and food; as a result, reduced respiration and metabolic processes. It seems the decrease of plant dry weight is due to impaired enzyme activity, the inhibition of photosynthesis, and impaired biosynthesis of chlorophyll pigments. This decrease may be due to the adverse effects of cadmium on energy production in mitochondria and chloroplasts (Aghaei et al. 2019). Cadmium reduces the activity of cytokinin hormone; it has significant effect on cell proliferation and plant growth (Mok 1994). Growth reduction due to cadmium may be due to loss of cell turgescence and reduction of mitosis or inhibition of cell elongation. For example, cadmium can affect the cell wall and especially the middle layer of the cell wall, which plays a role in cell elongation (Hassan et al. 2006). The decrease in calcium absorption due to the increase in the amount of cadmium in the environment, considering the physiological roles of calcium in plant cells, can be considered another reason for the decrease in plant growth and dry matter production (Rad et al. 2014). Lead is one of the toxic and unnecessary elements for plants that reduced root length, fresh and dry weight, stem length and height of plant; also salicylic acid played a role in reducing these damages (Mahdavian et al. 2016; Ranjbar et al. 2011; Padash et al. 2016; Fatemi et al. 2017). Lead poisoning primarily inhibits root growth, and the reduction in the development of the root system leads to limiting the growth of the aerial part. The decrease in the growth of the root and aerial parts under lead stress can be due to the accumulation of lead in the root and the ligninization of the cell walls under heavy metals (Fatemi et al. 2017).

The amount of proline and the activity of antioxidant enzymes increased in St. John’s wort under the stress of cadmium chloride, lead nitrate, and silver nitrate is consistent with the same research (Badpa et al. 2015; John et al. 2009; Yousefi et al. 2015). The salicylic acid and methyl jasmonate also improved the proline content. However, simultaneously salicylic acid and heavy metals reduced the antioxidant enzymes activity. One of the critical damages of heavy metals in a plant cell is the increase of reactive oxygen species (ROS). Plants counterattack with free radicals by using enzymatic and non-enzymatic antioxidant systems (Aghaei et al. 2019). Salicylic acid and methyl jasmonate decreased malondialdehyde content while improving both enzymatic and non-enzymatic antioxidant defense systems, thus play an important role in reducing free radicals, which have been reported in various studies ( Mansour et al., 2020; Keramat and Daneshmand 2012; Mahdavian 2017; Mohamadian et al., 2018). Salicylic acid reduced oxidative stress resulting from heavy metal toxicity, so increased plant tolerance to heavy metal stress (Gerami et al. 2018; Ranjbar et al., 2011; Padash et al. 2016). Low concentrations of silver nitrate and methyl jasmonate activated the antioxidant system of the plant by increasing ROS, while in high concentrations, they cause toxicity (Alirezaei et al. 2017; Keramat and Daneshmand 2012). Researches showed the application of salicylic acid and methyl jasmonate significantly increased the amount of polyphenolic and antioxidant compounds in artichokes (Coste et al. 2011). Salicylic acid inhibited the catalase and ascorbate peroxidase enzymes activity in leaves and medium cultures of salvia. Therefore, suggested the action mechanism of salicylic acid is likely to increase the amount of ROS by stopping the ability of the catalase enzyme to degrade H2O2 (Rajabi et al. 2016). In Physalis peruviana under 2 mM salicylic acid treatment, proline content improved compared to control or lower levels of salicylic acid; also it can decrease the content of malondialdehyde ( Mansour et al. 2020). Growth regulators such as salicylic acid inhibit the ROS; and ultimately reduce cell membrane damage so decrease malondialdehyde content ( Mansour et al., 2020). It has also been reported that salicylic acid treatment decreased malondialdehyde content in lemon balm and pepper (Mahdavian et al. 2016). Methyl jasmonate increased the enzymatic and non-enzymatic antioxidant defense system, in soybean (Glycine max L.); also it has the role in reducing free radicals (Keramat and Daneshmand 2012). Increasing methyl jasmonate in the plant under salinity stress, improves free proline content. Proline is not only an osmotic regulator but is also known as a powerful antioxidant and inhibitor of cell death. Therefore, proline can be considered the non-enzymatic antioxidant in plants which reduce the adverse effects of ROS. Methyl jasmonate helps plants to cope with stress through osmotic regulation and proline enhancement, also the synthesis of stress proteins (Mohammadian et al. 2018). It has been observed that silver in high concentrations causes oxidative stress and toxicity in the plant. Low concentrations of silver activate the plant’s antioxidant system by increasing the ROS. They increase antioxidant activity, but in high concentrations, they cause toxicity in the plants. One of the symptoms of toxicity in plants is the imbalance between the production of ROS and cellular antioxidant capacities (Gerami et al. 2018). Cadmium treatment reduces the activity of catalase and peroxidase enzymes; however, salicylic acid pretreatment increases the activity of these enzymes; in this experiment salicylic acid reduced oxidative stress due to cadmium toxicity and increased plant tolerance to cadmium stress. Under cadmium stress, the content of proline increased in peanuts and blue lentils (Dinakar et al. 2009; John et al. 2009). Increasing cadmium concentration causes the elevated proline content in the roots and shoots of Basil. Cadmium causes the release of free oxygen radicals such as superoxide (O2−), hydroxyl (OH−), nitric oxide (NO), and hydrogen peroxide (H2O2) in the plants like other heavy metals. Plants use enzymatic and non-enzymatic antioxidant systems to counterattack free radicals (Aghaei et al. 2019). Increasing the concentration of cadmium increased the superoxide dismutase enzyme activity in white berries (Zhang et al. 2010). A similar increase in superoxide dismutase enzyme activity been observed in bean Plants (Ahmadvand et al. 2013). The catalase enzyme activity is also increased in coordination with the superoxide dismutase enzyme activity in Lentil seedlings under cadmium stress (Rastgoo and Alemzadeh, 2011). In alfalfa, both of the above enzymes have increased (Mohammadi et al. 2011). Increased peroxidase enzyme activity have also been reported in Safflower plant (Badpa et al. 2015).

Lead has increased the activity of ascorbate peroxidase, superoxide dismutase, and peroxidase enzymes (Padash et al. 2016). The high concentration of lead nitrate showed a significant increase in peroxidase and catalase enzymes activity in leaves and roots of Brassica napus L. Catalase and oxidase enzymes play a critical role in responding to non-biological stresses such as lead. According to the results of their research, the treatment of lead nitrate and salicylic acid decreased the catalase and peroxidase enzymes activity of leaves and roots compared to the treatments of lead alone (Ranjbar et al. 2011). The level of activity of antioxidant enzymes depends on the duration and type of stress, plant species, and plant parts (Ranjbar et al. 2020). One of the effects of lead toxicity is the induction of oxidative stress in the plant due to the production of ROS and thus disrupting the oxidation and reduction status. Although some ROSs may act as important signaling molecules that alter gene expression; so regulate the activity of specific defense proteins; but all ROSs can be very harmful in high concentrations. Although the processes of producing ROS are slow under normal conditions, lead accelerates these processes (Chehregani Rad et al. 2017).

Conclusion

Investigating the effect of lead and cadmium on St. John’s wort showed that this plant has an excellent potential to respond to heavy metal stresses. Although the toxic effects of lead are more significant than cadmium in St. John’s wort. The growth characteristics of St. John’s wort decreased under the stress of heavy metals, and the antioxidant pathways of the plant showed significant changes under the influence of heavy metals. In general, the results of this study showed that the simultaneous application of salicylic acid and silver nitrate with the heavy metals cadmium chloride and lead nitrate could reduce the toxic effects of these metals on the morphometric properties of St. John’s wort. Methyl jasmonate, according to the proposed experiments of Taguchi design, at the low levels, has a positive role in these characteristics while at the higher levels, inhibited growth characteristics of St. John’s wort. Also, according to the results, only salicylic acid was able to reduce the effects of heavy metals on biochemical properties; while silver nitrate acted like heavy metals, especially at higher levels. One of the ways to reduce the harmful effects of heavy metal stress is to use elicitors and plant growth regulators. In this research, salicylic acid and methyl jasmonate were used under cadmium and lead stress on St. John’s wort; although salicylic acid has greater role in reducing the adverse effects of heavy metals; it was able to create better induction on growth and metabolism of St. John’s wort, at all levels. Applied elicitor and plant growth regulators is strategy to reduce the harmful effects of heavy metal stress. Cultivation of the medicinal plant St. John’s wort in environments contaminated with heavy metals should managed according to the reduction of growth characteristics. The use of an effective elicitor suggested for compensating the toxic effect of heavy metals in St. John’s wort for improving growth and reducing the toxic effects of heavy metals. These elicitors generally change the adverse effects of heavy metals by strengthening the pathways of the antioxidant system and some antioxidant enzymes in St. John’s wort. The research assumptions were validated, which suggests that the Taguchi method could be considered in the optimum culture of medicinal plants under different treatments such as heavy metals and elicitors.

Acknowledgments

The authors sincerely thank the Vice Chancellor for Research of Tabriz University and Shahid Chamran University of Ahvaz for their financial support of this research.

Declarations

Conflict of interest

The authors report no declarations of interest and that they are responsible for the content and writing of the article.

Footnotes

Publisher’s Note

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Contributor Information

Elham Mohajel Kazemi, Email: e.mohajel.k@gmail.com.

Maryam Kolahi, Email: m.kolahi@scu.ac.ir.

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