Introduction

Zataria multiflora Boiss. (Lamiaceae) called Avishan Shirazi in Persian, is a perennial thyme-like plant that grows in southern and central parts of Iran, Afghanistan, and Pakistan (Fazeli et al. 2007). This plant species has several antibacterial, antioxidant, and anti-cancer properties associated with its phenolics and terpenoids such as 4-hydroxybenzoic acid, rosmarinic acids, catechin, and carvacrol (Fazeli et al. 2007). Given the restricted natural distribution of this plant, there is a need to find alternative ways to produce it in bulk and utilize its valuable secondary metabolites instead of harvesting it from natural habitats.

Plant secondary metabolites are important sources of industrial and medicinal compounds whose accumulation is a part of plant defense mechanism. Large-scale production of important secondary metabolites through cell and tissue cultures is an attractive alternative route to enhance the production of valuable phytochemicals (Haida et al. 2019). Furthermore, it is possible to induce plant defense machinery through biotic and abiotic elicitors, accompanied by the biosynthesis of phytochemicals in plant tissue culture (Balintova et al. 2019).

Chitosan, the de-acetylated form of chitin, is a biocompatible and non-toxic biopolymer derived from the cell wall of fungal species (Ahmad et al. 2019). This biopolymer can mimic the effects of pathogenic fungal species by inducing the production of secondary metabolites (Ahmad et al. 2019). It significantly increased the production of various phenylpropanoid compounds such as 4-hydroxybenzoic acid, p-coumaric acid, and ferulic acid in Cocos nucifera (Chakraborty et al. 2009) and echinacoside in Scrophularia striata (Kamalipourazad et al. 2016). The previous research has shown the role of chitosan in the induction of phenylpropanoid compounds in plant cells (Kamalipourazad et al. 2016). The present work investigated the effect of this elicitor on phenylpropanoids and terpenoids in Z. multiflora cell culture.

In addition, yeast extract is an important natural elicitor and a common nutrient available in large quantities at a moderate price. It contains various organic and inorganic derivatives such as vitamin B-complex, chitin, N-acetyl-glucosamine oligomers, β-glucan, glycopeptides, and ergosterol (Ahmad et al. 2019). It has been used effectively to stimulate the biosynthesis of phenylpropanoids and terpenoids in Ferulago angulata (Alavi Mehryan et al. 2020), Astragalus chrysochlorus, and Curcuma mangga (Nadeem et al. 2018).

The induction of secondary metabolites can be associated with the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and nitric oxide (NO) (Samari et al. 2020). These bioactive molecules trigger signal transduction pathways in plant cell culture. H2O2 signaling molecule drives the metabolic shift by altering genes expression and enzymes activities such as phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) in the phenylpropanoid pathway (Samari et al. 2020). It has also been found that NO is associated with an increase in cytosolic Ca2+, which is a defense role of this molecule (Arasimowicz and Floryszak-Wieczorek 2007).

In this work, we assumed that yeast extract stimulates the production of phenylpropanoids and terpenoids in Z. multiflora cell culture in response to the accumulation of H2O2 and NO molecules. In contrast, chitosan would only stimulate the production of phenylpropanoids due to H2O2-induced oxidative stress. To do this, a fast-growing Z. multiflora cell line was treated with chitosan and yeast extract. We then assessed the effects of these biotic elicitors on the levels of signaling molecules (NO and H2O2), the activity of PAL and TAL enzymes, and the biosynthesis of terpenoids and phenylpropanoids.

Materials and methods

Plant material

Z. multiflora seeds were collected from wild-growing plant populations in Kohpayeh region (altitude of 2000 m) located in Kerman province in the southeastern region of Iran. A voucher specimen (4477) was deposited at the Herbarium of Agricultural and Natural Resources Research Center, Kerman, Iran. The seeds were thoroughly washed in running tap water and surface sterilized according to the following steps: 70% (v/v) ethanol for 30 s, 5% (w/v) sodium hypochlorite with a few drops of Tween 20 for 5 min, then rinsed 3 times with sterile distilled water. The seeds germinated on MS medium (Murashige and Skoog 1962) containing 6.5 g L−1 (w/v) plant agar and 30 g L−1 (w/v) sucrose after incubation in a growth chamber at 25 ± 2 ℃ within a 16/8 h light/dark photoperiod.

Callus induction

Calli were prepared from 30 day-old seedlings leaf explants on modified B5 medium (Gamborg 1968) containing 1.86 mg L−1 (w/v) 1-naphthalene acetic acid (NAA), 0.2 mg L−1 (w/v) benzyl adenine (BA), 3% (w/v) sucrose, and 0.7% (w/v) agar. The pH was adjusted to 5.6. The calli emerged in the dark at 25 ℃ after 2 weeks and sub-cultured every 4 weeks.

Cell suspension culture

Soft and friable calli were selected for the establishment of cell suspension culture after 20 weeks. Approximately 3.0 g of the callus tissue was transferred to 25 mL of liquid B5 medium containing 1.86 mg L−1 (w/v) NAA, 0.2 mg L−1 (w/v) BA, 3% (w/v) sucrose in a 100 mL Erlenmeyer flask, and incubated on an orbital shaker at 120 rpm and 25 ℃ in the dark. According to the cell growth curve, Z. multiflora cells were sub-cultured every 7 days for 8 weeks.

Cell growth curve

The fresh cells (3 g) were transferred to 25 mL of the liquid medium in 100 mL Erlenmeyer flasks to determine the optimal time for treatment. The cell growth curve was drawn by measuring the dry weight (DW) of the cells for 14 days after sub-culture. The cells were separated from the liquid medium by a nylon mesh filtration under suction, rinsed 3 times with distilled water, and finally dried to reach a constant weight of 50 ℃. The experiment was conducted in triplicate.

Cell treatment

The cells were treated with yeast extract and low molecular weight chitosan (Chi, C3646), both purchased from Sigma-Aldrich (Darmstadt, Germany). Yeast extract was dissolved in distilled water, filter-sterilized (0.22 µm, Millipore), and added to the cell suspension cultures at the final concentrations of 0, 400, 800, and 1200 mg L−1 (w/v). Chitosan was dissolved in 1% (v/v) acetic acid at pH 5.6, autoclaved, and then added to the suspension-cultured cells in the logarithmic growth phase at final concentrations of 0, 10, 20, and 40 mg L−1 (w/v). Control groups were prepared by adding distilled water and 1% acetic acid (pH 5.6) to the suspension-cultured cells. Both elicitors were added to the cell cultures 7 days after sub-culture when cells were in the logarithmic growth phase. After 3 days, the cells were harvested by a nylon mesh on the Buchner funnel under reduced pressure, weighted, immediately frozen in liquid nitrogen, and stored at − 80 ℃ for further biochemical assays. All experiments were performed in triplicate, and data were expressed as a mean of three measurements with a standard error.

NO content

The fresh cells (0.2 g) were extracted in 100 mM potassium phosphate buffer (pH 7.0). The extract was centrifuged at 12,000 rpm and 4 ℃ for 15 min. The supernatant (250 μL) was incubated with 750 μL of potassium phosphate buffer and 500 μL of Griess reagent (1% sulfanilamide and 0.1% n-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid) for 10 min at room temperature (Green et al. 1982). The absorbance of the mixture was read at 540 nm and quantified by a calibration curve method using sodium nitrite as standard in µmol g−1 fresh weight (FW).

H2O2 content

The fresh cells (0.1 g) were homogenized in 3 mL of trichloroacetic acid (TCA; 0.1% w/v) on the ice bath. The reaction mixture was centrifuged (12,000 rpm; 15 min; 4 ℃), then 0.5 mL of supernatant was added to 0.5 mL of potassium phosphate buffer (10 mM; pH 7) and 1 mL of KI (1 M). The absorbance of the mixture was read at 390 nm by the spectrophotometer. H2O2 content was calculated using the calibration curve method and expressed as μmol H2O2 in g−1 fresh weight (Velikova et al. 2000).

MDA content

The level of cellular membrane lipids peroxidation was determined by measuring the MDA (a lipid peroxidation biomarker) content. Frozen samples (0.2 g) were homogenized in 0.1% TCA. The homogenate was centrifuged at 10,000 rpm for 15 min, and 500 µL of supernatant was mixed with 2 mL of 0.5% thiobarbituric acid (in 20% TCA). The mixture was incubated at 95 ℃ in the water bath for 30 min and quickly cooled on ice. The absorbance was read at 532 and 600 nm using the spectrophotometer. The MDA content was estimated using the extinction coefficient of 155 mM−1 cm−1 and expressed as µmol g−1 FW (Heath and Packer 1968).

PAL and TAL activity assays

PAL (EC 4.3.1.24) and TAL (EC 4.3.1.23) enzyme activities were evaluated based on the quantities of produced cinnamic acid and p-coumaric acid, respectively (Beaudoin-Eagan and Thorpe 1985). The fresh cells (0.2 g) were homogenized with 3 mL of buffer (50 mM Tris–HCl containing 15 mM β-mercaptoethanol at pH 8.2), and the homogenate was centrifuged at 15,000 rpm for 20 min. The extraction buffer (1 mL), 10 mM L-phenylalanine (0.5 mL), double distilled water (0.4 mL), and the enzyme extract (0.1 mL) were mixed and incubated at 37 ℃ for 1 h. The reaction was stopped by adding 0.1 mL of 6 M HCl. The cinnamic acid was extracted using ethyl acetate. The ethyl acetate phase was air-dried, then the residue was dissolved in 1 mL of NaOH (0.05 M) and the absorbance of the solution was read at 290 nm. TAL activity was conducted similarly except that L-tyrosine (0.1 M) was used as the enzyme–substrate, and the absorbance was measured at 320 nm. The enzyme activity was defined as μmol of cinnamic acid and p-coumaric acid per mg of protein for PAL and TAL in 1 min, respectively. Total protein was determined according to the Bradford assay using bovine serum albumin as the standard (Bradford 1976).

Total phenolic contents

To determine total phenolics, flavonoids, and anthocyanins, 0.2 g of the fresh cells were extracted in MeOH: HCl (99:1 v/v) at 4 ℃ in the dark for 24 h followed by centrifugation at 12,000 rpm for 15 min. The contents of anthocyanins were determined using a spectrophotometer at 550 nm, using the extinction coefficient of 33,000 cm−1 M−1 (Krizek et al. 1998). Total phenolic contents were measured at 280 nm using a spectrophotometer (Cintra6, GBC, Australia), then the content was estimated by a correlation curve using gallic acid as the standard (Pirie and Mullins 1976).

The acidic extraction was incubated in a water bath at 80 ℃ for 10 min to determine total flavonoids. The absorbance of the samples was read at 270, 300, and 330 nm by the spectrophotometer, and the contents of total flavonoids were determined using the extinction coefficient of 33,000 cm−1 M−1 (Krizek et al. 1998).

The total flavonols content was determined by mixing the methanolic extract of the cells with 3 mL of 5% sodium acetate and 1 mL of 2% AlCl3, followed by reading the mixtures’ absorbance at 445 nm after 2.5 h and estimating the content using rutin as standard (Akkol et al. 2008).

Profile of secondary metabolites

Freeze-dried cells (0.1 g) were homogenized in 2 mL of methanol using an ultrasonic bath (40 kHz, Falc Instruments, Treviglio, Italy) for 20 min. The homogenate was incubated overnight at room temperature on a shaker. The supernatant was separated by centrifugation at 12,000 rpm for 15 min. After evaporation, the residue was dissolved in 350 μL of methanol (HPLC grade) and filtered using a 0.22 µm pore size syringe filter before biochemical analysis using an HPLC system (Waters e2695, USA) equipped with a C18 column (Perfectsil Target ODS-3, 5 μm particle size, 250 mm length, and 4.6 mm width, MZ- Analysentechnik, Mainz, Germany) and a 2489 UV/Visible detector (Barreca et al. 2016). The mobile phase was the mixture of solvent A (water/acetic acid, 97/3, v/v) and solvent B (MeOH) under the following gradient conditions: 0–3 min, 0% B; 3–9 min, 3% B; 9–24 min, 12% B, 24–33 min, 20% B, 33–43 min, 30% B; 43–63 min, 50% B; 63–66 min, 50% B; 66–81 min, 60% B; 81–86 min, 0%, B and equilibrated 4 min for a total run time of 90 min. The flow rate, detection wavelength and sample injection volume were 1.0 mL min−1, 330 nm, and 20 μL, respectively. The column temperature was kept constant at 26 ℃. The identification of the secondary metabolites was carried out by comparing the absorption spectra and the retention times of each peak with those of pure (> 99%) standards. The quantitative estimation of the secondary metabolites was determined based on calibration curves of the reference standards (Barreca et al. 2016). All standards were purchased from Sigma-Aldrich (Seelze, Germany).

Statistical analysis

All experiments were conducted in a completely randomized design and assessed using ANOVA in SPSS software environment (version 19, Chicago, IL, USA). The experiments were performed in triplicates, and the results were expressed as mean ± standard error (SE). The statistical significance of data was evaluated using Duncan’s test at P ≤ 0.05. Hierarchical cluster analysis was performed by the algorithms embedded in the web-based MetaboAnalyst (https://www.metaboanalyst.ca).

Results

Effect of biotic elicitors on plant growth

Like other biological species, the suspension-cultured cells grew slowly during the first 3–4 days (lag phase), followed by exponential growth (with an approximately 3-folds increase) from days of 4 to 10, and the stationary growth afterward Fig. 1c in supplementary material. Z. multiflora cells showed a monotonic reduction in dry weight compared to the control after 3 days of treatment with increasing concentrations of chitosan and yeast extract. Accordingly, the highest reductions of 28.2 and 35% were observed at 40 mg L−1 of chitosan (Fig. 1a) and 1200 mg L−1 yeast extract (Fig. 2a), respectively.

Fig. 1
figure 1

The cell dry weight (a), nitric oxide (b), hydrogen peroxide (c), and MDA (d) contents of Z. multiflora cell culture treated with 0, 10, 20, and 40 mg L−1 chitosan. The data are means of three replicates. The different letters show statistically significant differences at P ≤ 0.05 based on Duncan’s test

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Fig. 2
figure 2

The cell dry weight (a), nitric oxide (b), hydrogen peroxide (c), and MDA (d) contents of Z. multiflora cell culture treated with 0, 400, 800, and 1200 mg L−1 yeast extract. The data are means of three replicates. The different letters show statistically significant differences at P ≤ 0.05 based on Duncan’s test

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The contents of NO, H2O2, and MDA

The NO content significantly decreased in the cells treated with 10, 20, and 40 mg L−1 of chitosan compared to the control (Fig. 1b), while the H2O2 content increased significantly (108–111%) at different treatments levels, respectively (Fig. 1c). The cells treated with 400, 800, and 1200 mg L−1 of yeast extract showed a monotonic increase in the NO content compared to the control (Fig. 2b). Consistently, the H2O2 level significantly increased (109–114%) compared to the control (Fig. 2c).

The accumulation of these peroxides in the cells was accompanied by lipid peroxidation and membrane damages. The MDA content is a well-known biomarker in this regard. It did not significantly change at the treatment concentrations < 40 mg L−1 of chitosan (Fig. 1d), while there was a significant increase (~ 14%) in the MDA content of the cells treated with three concentrations of yeast extract compared to the control (Fig. 2d).

PAL and TAL enzyme activities

PAL activity had no significant changes in the cells treated with concentrations < 40 mg L−1 of chitosan (Fig. 3a). There was an approximately 24% increase in PAL activity upon treatment with the highest concentration of chitosan. TAL activity increased monotonically with increasing treatment concentrations such that 40 mg L−1 of chitosan led to 48% higher activity than the control (Fig. 3b). PAL and TAL activities in the cells treated with yeast extract showed a similar response (Fig. 3c, d), so the treatment dosages < 800 mg L−1 did not bring any significant change compared to the control. Enzyme activities in the cells treated with 800 mg L−1 of yeast extract were approximately 3% lower than the control, while those treated with 1200 mg L−1 were approximately 20% higher than the control (Fig. 3c, d).

Fig. 3
figure 3

PAL (a, c) and TAL (b, d) activities in 7-days old Z. multiflora cell culture, respectively, treated with different concentrations of chitosan (0, 10, 20, and 40 mg L−1) and yeast extract (0, 400, 800, and 1200 mg L−1); the cells were collected 3 days after elicitation. Data represent the mean of three replicates ± SE. Significant differences at P ≤ 0.05 are indicated by different letters

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The contents of total phenolic, flavonoids, flavonols, and anthocyanins

Total phenolic contents in chitosan-treated cells did not change significantly at concentrations < 40 mg L−1 compared to the control (Fig. 4a). The highest contents were observed upon treatment with 40 mg L−1 of chitosan. Similarly, total flavonoids contents changed under chitosan treatment (Fig. 4a). However, total flavonols contents in the treated cells changed in a dose-dependent manner. Flavonols contents increased at the low level, significantly decreased at the moderate level, and showed no significant changes at the high level of chitosan treatment compared to the control. Total anthocyanins decreased at all treatment levels in chitosan-treated cells (Fig. 4a).

Fig. 4
figure 4

The contents of total phenolics, flavonoids, flavonol, and anthocyanins in Z. multiflora cell culture treated with 0, 10, 20, and 40 mg L−1 chitosan (a) as well as 0, 400, 800, and 1200 mg L−1 yeast extract. The 7 days-old sub-cultured cells were undergone treatment and collected three days after elicitation. Data are means of three replicates ± SE. Different letters show statistically significant differences at P ≤ 0.05 based on Duncan’s test

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Total phenolic contents decreased to a concentration of 800 mg L−1, followed by a 36% increase at a high treatment level (1200 mg L−1) compared to the control sample in the cells treated with yeast extract (Fig. 4b). Total flavonoids contents showed a similar trend (Fig. 4b) with the highest and lowest contents at the treatment levels of 1200 and 800 mg L−1 of yeast extract, respectively. The lowest flavonols contents were observed at 400 mg L−1 of yeast extract (Fig. 4b), and the lowest anthocyanins contents occurred at 800 mg L−1 (Fig. 4b). The increase in yeast extract concentration to 1200 mg L−1 returned flavonols and anthocyanins contents to those of their respective controls (Fig. 4b).

Secondary metabolites profiles

Rosmarinic acid, naringenin, rutin, protocatechuic acid, catechin, and cinnamic acid were detected as the major secondary metabolites of Z. multiflora before and after treatment with chitosan or yeast extract. These compounds showed a differential response to chitosan and yeast extract. Epicatechin, caffeic acid, 4-hydroxy benzoic acid, apigenin, and benzoic acid were induced after chitosan treatment. The induction of these compounds was accompanied by a decrease in carvacrol, thymol, salicylic acid, resorcinol, and vanillin in chitosan-treated cells compared to the control (Table 1). The biosynthesis of epicatechin, caffeic acid, 4-hydroxy benzoic acid, apigenin, benzoic acid, and syringic acid was induced upon treatment with 1200 mg L−1 of yeast extract. Meanwhile, there was a significant increase in the contents of gallic acid, vanillin, salicylic acid, catechin, carvacrol, thymol, p-coumaric acid, rutin, and quercetin, amounting to 6.3, 4.4, 2.7, 2.1, 1.8, 1.8, 1.6, 1.4, and 1.4 folds of their controls, respectively (Table 1).

Table 1 The changes in secondary metabolites contents in Zataria multiflora cell culture in response to two certain concentrations of chitosan and yeast extract. Data are means ± SE. Significant differences (P ≤ 0.05) are indicated by different letters
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Discussion

Biotic elicitors are versatile tools for triggering plant cells’ secondary metabolic pathways in a dose and time-dependent manner. Chitosan and yeast extract are two common biotic elicitors applied for the overproduction of valuable secondary metabolites in plant tissue and cell cultures (Ahmad et al. 2019; Kamalipourazad et al. 2016). In the present work, the growth of Z. multiflora cells decreased significantly after treatment with certain concentrations of yeast extract and chitosan (Figs. 1a and 2a). Reduced cell growth has also been reported in Salvia miltiorrhiza, Scrophularia striata, Vitis vinifera, Pueraria candollei and Linum usitatissimum treated with chitosan and yeast extract (Zhao et al. 2010; Kamalipourazad et al. 2016; Nadeem et al. 2018). This reduction in the biomass can be attributed to stress induction and peroxide accumulation in plant cells (Figs. 1b and 2c). In fact, these compounds have been shown to mimic the effects of pathogenic fungal species and stimulate plant defense mechanism (Ahmad et al. 2019). Stress induction is accompanied by deterioration of cell membranes due to lipid peroxidation (Figs. 1d and 2d). Our results on the effects of chitosan and yeast extract on lipid peroxidation and MDA production are in agreement with Sharifi-Rad et al. (2020) and Yu et al. (2001) studies on Momordica charantia and Taxus chinensis. Lipid peroxidation is caused by the elicitor-induced ROS generation that can shift normal metabolism towards defensive responses, such as the production of secondary metabolites under severe stress conditions (Figs. 3, and 4).

The NO content did not change significantly after chitosan treatment compared to the control (Fig. 1b). The immediate reaction of NO and O2 to form peroxynitrite may be the reason for a low level of NO in the treated samples (Arasimowicz and Floryszak-Wieczorek 2007). Phenolics have the ability to scavenge NO and peroxy nitrites (Arasimowicz and Floryszak-Wieczorek 2007), which can result in low levels of phenolics at moderate treatment dosages (Fig. 4). The cells under salinity stress also showed reduced NO contents due to suppressed expression of AtNOA1 gene and inhibited activation of NOS enzyme in Arabidopsis taliana (Qiao et al. 2014; Zhao et al. 2007). In general, NO is an enigmatic molecule which cannot be directly associated with stress conditions. According to previous reports, it appears that H2O2 plays a key role in the fate of chitosan-treated Z. multiflora towards the induction of cell death or defensive responses by the accumulation of phenylpropanoid compounds (Table 1). Previous studies have suggested that elicitors can initiate oxidative stress leading to the production of secondary metabolites through the generation of ROS as well as the induction of signal transduction pathways. ROS homeostasis is controlled through the activation of enzymatic and non-enzymatic antioxidants to prevent oxidative damage (Samari et al. 2020). ROS molecules at low concentrations act as signaling agents in the cellular responses to biotic and abiotic stresses, initiating the expression of specific genes and the production of secondary metabolites. However, high levels of ROS molecules are toxic, which can cause oxidative stress and damage to the cellular membranes (Kamalipourazad et al. 2016).

The high NO content in the cells treated with yeast extract can be associated with the presence of various components such as Ca2+ in yeast extract. This higher NO content in the treated cells can be attributed to the role of NO in the activation of the defensive mechanism in plant cells. In addition, supplementation of culture media with Ca2+ increases the activity of nitric oxide synthase (Rodriguez-Serrano et al. 2009). Increased cytosolic Ca2+ level is also correlated with the NO accumulation (Arasimowicz and Floryszak-Wieczorek 2007; Samari et al. 2022). However, cytosolic Ca2+ burst contributes to the production of terpenoids as potent defensive compounds (Rao et al. 2020). Consistently, the contents of NO signaling molecule and terpenoids (carvacrol and thymol) increased significantly in Z. multiflora cells treated with yeast extract (Fig. 2b, Table 1).

The ROS accumulation is probably the reason for the high activity of PAL and TAL enzymes at high concentrations of chitosan and yeast extract (Fig. 3). Similar results were also detected in the other plant cell cultures (Chen et al. 2021). These enzymes switch the function of normal cells from primary to secondary metabolism with the aim of increasing plant resistance to oxidative stress condition (Ahmad et al. 2019). The enhanced biosynthesis of phenolics through the phenylpropanoid pathway can be associated with the activation of defense mechanism in the elicited cells (Nadeem et al. 2018). Consistently, the contents of total phenolic and flavonoids increased significantly by approximately 25 and 55% in the cells treated with 40 mg L−1 of chitosan compared to the control, respectively (Fig. 4a). The contents of total phenolic and flavonoids in the cells treated with 1200 mg L−1 of yeast extract were 36 and 39% higher than the control cells (Fig. 4b). Similarly, yeast extract and chitosan significantly increased the total phenolic and flavonoids contents in the cell and tissue cultures of Astragalus chrysochlorus, Curcuma manga, Andrographis paniculata, and Scrophularia striata (Kamalipourazad et al. 2016; Nadeem et al. 2018). Therefore, this study indicates that the phenolic compounds accumulation could be regulated positively by the activation of key enzymes in the phenylpropanoid pathway under biotic elicitor treatment.

Oxidative stress induced the phenylpropanoids biosynthesis. The activation of this defensive pathway can lead to the induction of secondary metabolites accumulation after each treatment (Tashackori et al. 2021; Khodamoradi et al. 2022). End products (e.g., apigenin) and intermediates (e.g., caffeic acid) in the phenylpropanoid pathway were induced upon treatment with the highest concentrations of chitosan and yeast extract (Fig. 5). The results suggest that the biosynthesis of secondary metabolites is controlled by much more complicated means. A significant correlation was observed between peroxide accumulation and secondary metabolites biosynthesis. Also, there was a positive correlation between changes in the contents of caffeic acid and syringic acid, while the relationship between changes in cinnamic acid and p-coumaric acid levels was negative (Fig. 5). Correlations among metabolites after treatment with chitosan and yeast extract were assessed by Pearson correlation coefficient and visualized as a heat map (Fig. 5b, c). Interestingly, the terpenoids pathway significantly increased in Z. multiflora cells treated with yeast extract (Table 1). There were no significant changes in carvacrol and thymol contents after treatment with two concentrations of chitosan. Driving a plethora of results related to fluctuations in the abundance of intermediate metabolites in various common motifs of the metabolic network, Levine and Hwa (2007) found that steady-state fluctuations in different metabolic pathway nodes were not effectively correlated. In fact, the metabolic networks are highly interconnected and intermediate metabolites can be freely shared by different reactions. In other words, different pathways can be coupled together without creating complex relationships. In addition, it should be noted that some metabolites may only be induced during elicitation but converted to the related derivatives and therefore cannot be detected at the time of cell harvesting and measurement. This study is the first report on identifying medicinal metabolites of Z. multiflora cells suspension culture after biotic elicitation. It appears that these biotic elicitors can affect plant metabolism by modulating the metabolites biosynthesis, accumulation, vacuolar transit, turnover and degradation (Vasconsuelo and Boland 2007).

Fig. 5
figure 5

A schematic representation of the relationship between the secondary metabolite biosynthesis in Z. multiflora cell culture after oxidative stress (a), the heat map representation of the relationship between the biochemicals in the Z. multiflora cells treated with chitosan (b) and yeast extract (b) based on Pearson correlation coefficient. The colors in matrix boxes show the direction of correlation: Intense red denotes strong positive and blue denotes strong negative correlations

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Conclusion

Chitosan and yeast extract induced the phenylpropanoids biosynthesis in Z. multiflora cell culture in response to H2O2- induced oxidative stress. Depending on the treatment dosage, the cells showed different responses, so the highest concentrations of chitosan (40 mg L−1) and yeast extract (1200 mg L−1) resulted in the lowest cell dry weight and highest H2O2, phenolics, and PAL and TAL activities. However, the production of NO was only stimulated in the cells treated with yeast extract. The higher contents of epicatechin, caffeic acid, 4-hydroxy benzoic acid, apigenin, and benzoic acid were observed in the cells treated with chitosan; while both phenylpropanoids (epicatechin, caffeic acid, 4-hydroxy benzoic acid, apigenin, benzoic acid, gallic acid, vanillin, salicylic acid, and catechin) and terpenoids (carvacrol and thymol) increased in the cells treated with yeast extract.