IntroductionMedicinal plants have been utilized throughout history for pharmaceuticals, spices, dyes, cosmetics, phytotherapy, and aromatherapy. Their significance continues to grow today, driven by scientific and technological advancements that enhance their practical applications1. Brassicaceae is one of the most important families with a large number of plant genera and species with medicinal properties. Isatis belonging to Brassicaceae is a genus of biennial herbaceous plants native to the Mediterranean, Eastern, and Central Asia, comprising around 79 species, with Isatis tinctoria being the most well-known2. In addition to Isatis tinctoria, which stands out for its dyeing and medicinal properties, there are also species within this genus, such as Isatis constricta, Isatis ermenekensis and Isatis floribunda that exhibit similar metabolite characteristics3,4,5.Isatis species are rich in indole alkaloids, especially indigotin (IND) and indirubin (INR). IND is a blue compound with a long history of use as a dye. The carcinogenic consequences of synthetic dyes generated from benzene, as well as their detrimental impacts on the environment have led to a recent surge in interest in natural colors. IND has a special place among natural dyes with its rare blue colour6. Indeed, several European nations made substantial contributions to projects involving the production of blue dye from Isatis and Polygonum species, as well as the extraction of IND and its application in the textile sector7. Besides being natural dyes, plants belonging to the genus Isatis have been used for medicinal purposes for a long time. The pharmacological properties of plants belonging to the genus Isatis were thought to be due to the indole alkaloids, especially INR8,9. It was determined that INR in the leaves, roots and seeds of the plant had a remarkable effect on controlling cell proliferation in cancer cells10,11. It was stated that the anti-cancer effect of INR may be related to the improvement of the body immune system12. It was reported that INR had anti-inflammatory effects as well as anticancer, antibacterial, toxin-removing and immune-enhancing properties13. Besides extracts from Isatis were found to be effective in the treatment of various diseases such as meningitis, encephalitis, hepatitis A, mumps, influenza, dermatitis and skin rash as well as antibacterial, anticancer and antimicrobial properties14,15,16,17.Traditional gathering of plants from native habitats in order to get medicinal and industrially important metabolites causes both the destruction of plants from their natural environment and the inability to obtain metabolites of standard quality and quantity due to geographical and ecological differences. Given the limitation in plant collection, in vitro methods are thought to be a more efficient and sustainable way to synthesize secondary metabolites. Applications of elicitors in plant tissue culture increase the production of secondary metabolites by inducing stress reactions and boosting the activity of enzymes involved in metabolic pathways18. However, it is of great importance that the elicitors to be used in in vitro cultures should be easily available and non-toxic as well as cost-effective for commercial applications. One of the elicitors that can be evaluated in this context is hydrogen peroxide (H2O2).H2O2 is a signaling molecule causing oxidative stress in plants rapidly promotes reactive oxygen species (ROS)19. H2O2 plays a role in modulating the expression of target genes related to defense systems and in other metabolic processes in plants20. When applied externally, H2O2 was found to increase the accumulation of several metabolites in different plants under both in vitro21,22,23,24,25 and in vivo26,27,28 conditions when used as an elicitor owing to its capacity to generate ROS and trigger secondary metabolic pathways.This study was conducted to comprehensively investigate, for the first time, the effects of H2O2 treatments on IND and INR production, antioxidant enzyme activities, intracellular H2O2 levels, and the expression levels of TSA and CYP79B2 genes involved in indole alkaloid biosynthesis in root cultures of four different Isatis species (I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria). Although the pharmacological potential of IND and INR is well-known, how these metabolites are modulated through biosynthetic pathways under H2O2 induced oxidative stress conditions and their relationship with antioxidant enzyme dynamics remain largely unclear. In this context, our study seeks to elucidate how H2O2, as a signaling molecule, influences secondary metabolite production at molecular and biochemical levels, shedding light on the complex interplay between oxidative stress response and alkaloid biosynthesis. By revealing how oxidative signaling directs metabolite production and gene expression, this work provides a novel perspective to the literature.Materials and methodsSterilization of seeds and culturing in nutrient mediaAfter being taken out of their capsules, the seeds of I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria provided by the Department of Field Crops, Isparta University of Applied Sciences were sterilized. The seeds were first submerged in 70% ethanol for one min and rinsed with sterile dH2O. The seeds were agitated for 10 min in a 15% sodium hypochlorite (NaOCl) supplemented with 1–2 drops of Tween 20 to accomplish surface sterilization. To get rid of any remaining sterilizing chemicals, the seeds were washed five sequential rinses with sterile dH2O. Murashige and Skoog (MS) medium29 supplemented with 3% sucrose and 0.6% agar was used to cultivate sterilized seeds. To encourage germination, cultures were maintained at 25 °C under a 16/8-h light (54 µmol/m²/s)/dark photoperiod30.Establishment and propagation of root culturesRoots of in vitro seedlings were used to establish root cultures. The root explants were transferred to MS medium added with 3% sucrose and 2 mg/L indole-3-butyric acid (IBA) and. The cultures were maintained in complete darkness at 24 ± 1 °C on an orbital shaker set at 90 rpm for 3 weeks. To increase root biomass, subculturing was performed at two-week intervals.H2O2 elicitationFor elicitation, 0.5 g of root material was inoculated into 30 mL of liquid MS medium including 3% sucrose and 2 mg/L IBA. Cultures were maintained under same conditions (24 °C, darkness, 90 rpm) for 15 d to allow acclimatization and growth. At the end of this period, H2O2 was applied to the cultures in the beginning of the stationary phase at final concentrations of 100, 200, 300, and 400 µM for 24 h. Control groups were cultured under the same conditions, with sterile dH2O replacing H2O2. After the elicitation period, root samples were harvested, thoroughly rinsed with sterile dH2O, blotted dry, and processed for further analysis, including metabolite quantification, gene expression, and enzyme activity assays.Quantification of IND and INR by HPLCIND and INR levels in root samples were quantified using high-performance liquid chromatography (HPLC). Root samples (0.5 g) were air-dried, finely powdered, and macerated in 25 mL of 80% methanol at 50 °C for 15 h, followed by ultrasonic extraction at 50 °C for 25 min. The resulting filtrates were evaporated at 45 °C and reconstituted in HPLC-grade methanol.HPLC (Shimadzu, Japan) was used for the quantification of IND and INR according to the method of Zhang et al.31. Absorbance readings were done at 289 nm using diode-array detector (DAD). After going through a 0.45 μm filter, the extracts were examined on an Agilent TC-C18 column (5 μm, 250 mm x 4.60 mm). Chromatographic separation was achieved with an 80% methanol-water mobile phase. Flow rate and column temperature were adjusted as 1.2 mL/min and 60 °C, respectively. Standards of IND and INR supplying from Sigma were prepared with methanol at varying concentrations (µg/mL) and calibrated by HPLC. The retention times of IND and INR were determined as 4.35 and 5.72 min, respectively. The amounts of IND and INR were expressed as µg/g DW.Antioxidant enzyme activity assaysFor enzyme extraction, 1 g of root tissues were crushed in liquid nitrogen and homogenized in 4 mL potassium phosphate buffer (50 mM, pH 7.0) containing 1% polyvinylpyrrolidone (PVP) and 2 mM disodium ethylenediaminetetraacetic acid (Na2-EDTA). The homogenates were centrifuged at 12.000 rpm for 20 min at 4 °C, and the resulting supernatant was stored at − 80 °C until used for protein and enzyme assays. Protein determination was done by the Bradford method32 using bovine serum albumin (BSA) as a standard. Absorbance was measured at 595 nm, and protein concentrations were extrapolated from a standard curve.The activity of superoxide dismutase (SOD, EC 1.15.1.1) was measured based on the inhibition of nitroblue tetrazolium (NBT) photoreduction. The mixture comprised potassium phosphate buffer (50 mM, pH 7.3), methionine (13 mM), nitroblue tetrazolium (NBT, 75 µM), EDTA (0.1 mM), riboflavin (4 µM), and enzyme extract. Absorbance was recorded at 560 nm33. Since one SOD unit is defined as the amount of enzyme required to inhibit 50% of NBT photoreduction when riboflavin and light are present, SOD activity was calculated as unit/mg protein.The activity of catalase (CAT, EC 1.11.1.6) was assessed with monitoring the degradation of H2O2 at 240 nm over a 2 min interval34. The reaction mixture comprised potassium phosphate buffer (50 mM, pH 7.0) and H2O2 (10 mM). CAT activity was expressed as µmol/min mg protein using an extinction coefficient of 39.4/mM/cm.The activity of ascorbate peroxidase (APX, EC 1.11.1.11) was measured by observing the decline in absorbance at 290 nm for 2 min intervals. The mixture included potassium phosphate buffer (50 mM, pH 7.0), ascorbic acid (0.5 mM), EDTA (1 mM), and H2O2 (0.1 mM). APX activity was expressed as µmol/ min mg protein using an extinction coefficient of 2.8/mM/cm35.Intracellular H2O2 quantificationIntracellular H2O2 concentrations were determined by homogenizing root tissues (0.5 g) in 0.1% trichloroacetic acid (TCA). After centrifugation at 12.000 rpm, the supernatant was mixed with 10 mM potassium phosphate buffer (pH 7.0) and 1 M potassium iodide (KI). Absorbance was measured at 390 nm, and H2O2 concentration was calculated as µmol/g FW using a standard curve35.Gene expression analysisTotal RNA was isolated from root tissues harvested 24 h after H2O2 treatments. Harvested roots were rapidly frozen in liquid N₂ and maintained at -80 °C for subsequent analyses. Following the manufacturer’s instructions, 100 mg of root tissue was utilized for RNA extraction using PureZol reagent (Bio-Rad, USA). To remove residual genomic DNA contamination, RNA samples were treated with DNase I (QIAGEN, Germany). Following extraction, RNA samples were evaluated for integrity through 1% agarose gel electrophoresis, with subsequent quantification using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA).Complementary DNAs (cDNA) were synthesized with the RevertAid First Strand cDNA Synthesis Kit (Bio-Rad, USA) from the purified RNA. Reverse transcription (RT) reactions were conducted to generate cDNA templates for downstream gene expression analysis. This cDNA was used to evaluate the activities of and CYP79B2 (cytochrome P450) and TSA (tryptophan synthase alpha) genes related with the syntheses of INR and IND alkaloids in Isatis species (Fig. 1)36.Fig. 1Diagram of IND and INR biosynthesis pathway (Wang et al.36).Full size imageQuantitative real-time PCR (qPCR) was performed with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA) with 100 ng of cDNA per reaction. Gene-specific primers targeting CYP79B2 (GenBank: EU684741.1) and TSA (GenBank: AJ841705.1) were designed to quantify expression levels. Gene expression data were normalized using actin (GenBank Accession: AY870652.1) as the reference gene. The primers used in the RT-PCR reactions were as follows: TSA (forward primer, 5’-GTCCAGTTTCGCTGTTCACG-3’; reverse primer, 5’-CCACAAGTCCCTGTACACCA-3’), CYP79B2 (forward primer, 5’-CCGCCGATGAAATCAAACCC-3’; reverse primer, 5’-TTTGTTCACCATCTCCGCCA-3’) and actin (forward primer, 5’-ACTGGAATGGTGAAGGCTGG-3’; reverse primer, 5’-TCTGACCCATCCAAACCGTG-3’). Amplifications and fluorescence detection were performed on the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). Gene activities were calculated using the comparative 2−ΔΔCt method37.The experiment included two target genes, five H2O2 treatments (0, 100, 200, 300, and 400 µM), four Isatis species, and three biological replicates, resulting in a total of 120 plant samples. Each qPCR analysis was performed in duplicate to ensure reproducibility and accuracy.Statistical analysisThe experiments were conducted separately using a completely randomized experimental design with 3 replications and with 5 Erlenmeyer flasks per replication. Statistical analysis was conducted using SPSS 22.0 (IBM). One-way ANOVA and Tukey’s HSD post-hoc tests were applied to assess differences between treatment groups (p ≤ 0.05). Pearson correlation analyses were conducted in R (v4.3.1; R Core Team, 2023) using the corrplot package. Principal Component Analyses (PCA) were generated to visualize correlations and clustering patterns using ClustVis and JMP Pro 17 software.ResultsEffects of H2O2 treatments on IND and INR accumulation in roots of Isatis speciesThe effects of H2O2 applications at concentrations of 100, 200, 300, and 400 µM on the syntheses of IND and INR in roots of I. constricta, I. ermenekensis, floribunda, and I. tinctoria were evaluated in this study. Depending on the species and H2O2 concentrations, the results showed notable variations in alkaloid accumulation (p ≤ 0.05) (Fig. 2).Fig. 2Effects of H2O2 on IND and INR contents in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageIn I. constricta roots, the highest IND level (82.92 µg/g) was detected in the application of 100 µM H2O2 (Fig. 2A). Similarly, INR level peaked at the roots treated with 100 µM H2O2 (7.25 µg/g). The lowest values not only in IND but also in INR were detected at 300 µM and 400 µM H2O2 applications. In I. ermenekensis, quantitative analysis revealed significantly different IND accumulation levels across treatments, with the minimum yield observed in 100 µM H2O2 treated roots and the maximum yield in the 300 µM H2O2 group, while 200 and 400 µM H2O2 treatments did not change IND accumulation compared to the control (Fig. 2B). In this species, 300 µM H2O2 application was found to increase the amount of INR as well as IND. H2O2 treatment also caused significant changes in alkaloid content in I. floribunda (Fig. 2C). The amount of IND, which was 30.10 µg/g in control roots, increased to 44.64 µg/g at 100 µM H2O2 and reached a maximum of 116.96 µg/g at 200 µM H2O2. As a result of the evaluations made in terms of INR accumulation, the amount of INR reached the highest level (7.83 µg/g) with 300 µM H2O2 application and decreased to the minimum level (1.06 µg/g) with 200 µM H2O2 application. In I. tinctoria, all H2O2 treatments significantly increased IND accumulation relative to untreated controls, though no significant differences were observed among the tested H2O2 concentrations (Fig. 2D). Among the H2O2 treatments, 300 µM H2O2 treatment increased INR accumulation comparison with untreated group, while the other H2O2 applications did not show a significant difference. These findings highlight the dose-dependent effects of H2O2 on secondary metabolite biosynthesis, demonstrating species-specific responses to oxidative stress in Isatis root cultures.Effects of H2O2 treatments on antioxidant enzyme activities in roots of Isatis speciesThe enzymatic activities of SOD, CAT and APX were quantitatively assessed in root cultures of four Isatis species after exposure to 0-400 µM H2O2 for 24 h. The results indicated substantial differences in enzyme activities depending on the species and H2O2 concentration (p ≤ 0.05).SOD activity, which catalyzes the breakdown of superoxide radicals to hydrogen peroxide and oxygen, showed significant differences according to treatments and species (Fig. 3). In I. constricta, SOD activity was the highest in the control roots as 20.90 U/mg protein while no significant differences were detected among the H2O2 treatments, with all treatments resulting in lower activity comparison with the control (Fig. 3A). The lowest SOD activity was detected in roots applied with 400 µM H2O2, with a value of 17.91 U/mg protein while no differences were observed among the other applications in I. ermenekensis (Fig. 3B). In I. floribunda, the greatest SOD activity was detected in the control roots, reaching 30.27 U/mg protein (Fig. 3C). Treatment with 100 µM and 200 µM H2O2 resulted in similar SOD activity levels. However, SOD activity declined significantly at higher H2O2 concentrations, with activity dropping to 18.17 U/mg protein at 300 µM, and further decreasing to 15.67 U/mg protein at 400 µM. Similar to I. floribunda, in I. tinctoria, the highest SOD activity (23.31 U/mg protein) was recorded in the control group. The lowest values were found at roots treated with 100 µM and 400 µM H2O2 as 14.39 U/mg protein and 16.91 U/mg protein, respectively (Fig. 3D).Fig. 3Effects of H2O2 on SOD activity in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageThe CAT activity responsible for the breakdown of H2O2 into oxygen and water has significantly varied among different Isatis species in response to H2O2 applications (Fig. 4). The highest CAT activity in I. constricta was detected at 200 µM H2O2, followed by 300 µM H2O2, while 100 µM and 400 µM were found to be the concentrations at which the minimum CAT activities were obtained (Fig. 4A). In I. ermenekensis, the greatest CAT activity was obtained from the roots treated with 200 µM H2O2 (Fig. 4B). CAT activity in the control group of I. floribunda was 5.99 µmol/min/mg protein, peaking at 11.84 µmol/min/mg protein at 300 µM H2O2. The control and H2O2 treatments did not differ significantly, with the exception of 300 µM H2O2, which produced the highest CAT activity (Fig. 4C). In I. tinctoria, CAT activity, which was 5.18 µmol/min/mg protein in the control, increased up to 15.51 µmol/min/mg protein with 200 µM H2O2 and reached the highest level (Fig. 4D). However, CAT activity decreased with increasing H2O2 concentration and remained at the lowest level at 400 µM H2O2.Fig. 4Effects of H2O2 on CAT activity in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageAPX activities of Isatis species varied significantly depending on the H2O2 treatments (Fig. 5). In I. constricta, it was determined that APX activity increased and reached the highest level (31.65 µmol/min/mg protein) with 100 µM H2O2 application compared to the control, but APX activity decreased as H2O2 concentration increased (Fig. 5A). APX activity of I. ermenekensis peaked at 200 µM H2O2 (24.49 µmol/min/mg protein) while the control and 400 µM H2O2 treatments exhibited lower values (Fig. 5B). The APX activity in root cultures of I. floribunda rose as the H2O2 concentrations increased, reaching its peak with the application of 400 µM H2O2 as 61.78 µmol/min/mg protein (Fig. 5C). In I. tinctoria, the highest APX activity (38.40 µmol/min/mg protein) was observed at 200 µM H2O2, while H2O2 applications at the concentrations of 300 and 400 µM resulted in the lowest values (Fig. 5D). Depending on the concentrations, H2O2 increased APX activity in roots, reflecting its role in oxidative stress defence.Fig. 5Effects of H2O2 on APX activity in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageEffects of H2O2 treatments on the intracellular H2O2 levels in roots of Isatis speciesAs shown in Fig. 6, intracellular H2O2 levels in root cultures of four Isatis species markedly changed depending on H2O2 concentrations and Isatis species (p ≤ 0.05). Intracellular H2O2 levels increased in a concentration-dependent manner and relative to controls, roots treated with H2O2 exhibited elevated intracellular H2O2 levels in all Isatis species. In I. constricta, the control group recorded the lowest level (0.20 µM/g), while 300 µM and 400 µM treatments significantly increased intracellular H2O2 level in roots (Fig. 6A). In I. ermenekensis roots, while the intracellular H2O2 content was measured as 0.41 µM/g in control group, the highest value (0.88 µM/g) was recorded in the roots applied with 400 µM concentration of H2O2 (Fig. 6B). I. floribunda exhibited a pattern similar to that of I. constricta. Intracellular H2O2 levels were highest at 300 µM and 400 µM H2O2 treatments, reaching 0.61 µM/g and 0.62 µM/g, respectively. In comparison, the control group exhibited significantly lower H2O2 level (0.14 µM/g) (Fig. 6C). In I. tinctoria, H2O2 levels in the control and 100 µM H2O2 treatment were the lowest. (Fig. 6D). However, a gradual increase was observed at higher concentrations, peaking at 0.85 µM/g at 400 µM H2O2.Fig. 6Effects of H2O2 on intracellular H2O2 content in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageEffects of H2O2 treatments on the gene expression levels in roots of Isatis speciesThe expression levels of TSA and CYP79B2 genes associated with IND and INR biosynthesis were also determined in roots of I. constricta, I. ermenekensis, I. floribunda and I. tinctoria, applied with H2O2. RT-PCR analyses revealed that the expression levels of both genes changed significantly depending on Isatis species and the H2O2 concentration (p ≤ 0.05) (Fig. 7).Fig. 7Effects of H2O2 on TSA and CYP79B2 gene expression in the roots of I. constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) (*Mean values with the same letter are not statistically significant (p ≤ 0.05)).Full size imageIn I. constricta, the lowest TSA gene activity was obtained in roots applied with 100 µM concentration of H2O2 as 0.57, while no other H2O2 treatments showed statistically significant differences relative to untreated controls. CYP79B2 expression level in I. constricta roots increased with rising H2O2 concentration and reached the highest level with 400 µM H2O2 treatment. (Fig. 7A). TSA expression in I. ermenekensis peaked at 1.22 at 200 µM H2O2 treatment (Fig. 7B). However, TSA level decreased as H2O2 level increased and fell to the lowest level at 400 µM. When the effects of H2O2 applied to I. ermenekensis roots on CYP79B2 gene activity were examined, it was found that 200 µM and 300 µM H2O2 applications increased the expression levels of CYP79B2 gene in comparison to the control. In I. floribunda, H2O2 treatments significantly increased TSA gene expression levels compared to un-treated group. The highest TSA activities were detected in 200 and 300 µM H2O2 treated roots with 4.59 and 4.00 values, respectively (Fig. 7C). In contrast to the differences seen in TSA gene, H2O2 treatments did not exhibit a notable difference in CYP79B2 gene activity in I. floribunda roots when compared with the control. In I. tinctoria, TSA expression increased from 1.43 in the control group to 3.96 at 100 µM and 8.87 at 300 µM, representing the highest value recorded (Fig. 7D). CYP79B2 gene activity decreased to the lowest level with 400 µM H2O2 application, while other H2O2 applications did not show a notable difference in CYP79B2 gene activity compared to the control.Correlation and principal component analysis (PCA)The results of the analyses of Pearson correlation matrix are given in Fig. 8. The analysis showed the correlations between alkaloids and antioxidant enzyme and gene activities differed according to the species. In I. constricta the amounts of IND and INR showed strong positive correlations with APX while formed negative correlations with the gene activities and intracellular H2O2 content (Fig. 8A). Correlation analyses in I. ermenekensis revealed that IND and INR accumulations in roots were supported by intracellular H2O2 content and CYP79B2 gene activity (Fig. 8B). In I. floribunda, IND accumulation showed positive correlations with APX, H2O2, and TSA, while CAT, H2O2, and CYP79B2 were found to support the accumulation of INR. In this species, SOD activity had a negative effect on both IND and INR accumulation (Fig. 8C). The levels of IND and INR in I. tinctoria was promoted by H2O2, and TSA, while a negative correlation was formed with the SOD and CYP79B2 gene activity (Fig. 8D). IND and INR accumulation in roots of Isatis species was affected by the antioxidant enzymes, H2O2 and the gene activities related to biosynthesis pathway of IND and INR depending on the species. Changes in the activities of antioxidant enzymes (APX and CAT) were observed only in I. constricta and I. floribunda. While a significant correlation was found between intracellular H2O2 levels and antioxidant responses in I. ermenekensis, I. floribunda and I. tinctoria, this correlation was not valid for I. constricta. A possible explanation for this may be that I. constricta exhibited a weaker or different physiological and molecular response to H2O2 treatments. In this species, oxidative stress mechanisms might not have been sufficiently activated in response to H2O2, which could have prevented the establishment of a clear correlation between H2O2 levels and specific biochemical responses.Fig. 8The correlation matrix of indole alkaloids (IND and INR), antioxidant enzymes (SAO, CAT and APX) and gene expression (TSA and CYP79B2) in Isatis constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D). The matrix illustrates the strength of the Pearson correlation, with the shape and color of each dot in the triangular matrix indicating the magnitude and direction (positive or negative) of the correlation among the parameters Dark blue dots represent strong positive correlations, while lighter colors indicate weaker relationships between the parameters.Full size imagePCA was employed to analyze the relationships among variables including IND, INR, SOD, CAT, APX, H2O2, TSA and CYP79B2 in I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria. In Fig. 9, two-dimensional scatter plots illustrating PCA for each Isatis species are presented. The analyses revealed that the principal components (PCs) had eigenvalues above 1.0, suggesting they carried significant information. In I. constricta, PC1 explained 54.65% of the variance -the highest among the four species- while PC2 and PC3 accounted for 21.91% and 16.62%, respectively, resulting in a total variance of 93.18% (Fig. 9A; Table 1). The high positive loadings of PC1 were IND and INR. The PC2 involved two variables including SOD and TSA with high negative loading. Figure 9B; Table 1 displayed the PCA results for I. ermenekensis. 82.05% of the variation was explained by the top two PCs. 47.52% of the variation was explained by PC1, while the TSA, APX, and CAT variables showed the highest factor loads. PC2 had strong positive loadings of IND, INR, and H2O2, accounting for 34.53% of the total variation. The first three PCs of I. floribunda accounted for 50.59%, 21.22%, and 14.04% of the total variance in the data, respectively, explaining 85.84% of the variation. PC1 exhibited high positive loadings for H2O2, TSA, and IND, while PC2 had two high positive loadings (CAT and INR) and two high negative loadings (APX and IND), according to Fig. 9C, a two-dimensional scatter plot showing the findings of PCA. The analyses in I. tinctoria were conducted using the first three PCs with eigenvalues greater than 1.0 (Table 1). With PC1, PC2, and PC3 explaining 41.93%, 29.92%, and 12.72% of the overall variation, respectively, the first three PCs collectively accounted for 84.57% of the data’s variance. PC1 exhibited large negative loadings for APX and CYP79B2, but PC2 had positive loadings for CAT, APX, CYP79B2, IND, and INR, as shown in Fig. 9D, a two-dimensional scatter plot showing the findings of PCA.Fig. 9PCA Biplot of H2O2 treatments of Isatis constricta (A), I. ermenekensis (B), I. floribunda (C) and I. tinctoria (D) in vitro roots obtained by principal component analysis.Full size imageTable 1 Factor loadings. Eigenvalues, and proportion of variation of the PCA of 8 variables in five H2O2 concentrations in roots of Isatis species.Full size tableDiscussionThe IND and INR alkaloids represent a significant class of secondary metabolites within the Isatis genus. These compounds are synthesized from tryptophan metabolism and represent a vital group of indigoid compounds within the plant kingdom38. The relatively low concentrations of these alkaloids in Isatis species render them highly valuable compounds5,30. In vitro techniques are thought to be the best way to obtain these alkaloids from roots, negating the need to extract them from the environment39. Additionally, the biosynthesis pathways of the targeted secondary metabolites can be modified, and the efficiency of secondary metabolite production can be improved by employing various techniques including precursor supplementation, growth regulator adjustment, and elicitor treatments18.Secondary metabolite production in plants represents a defensive response to environmental stress40. In response to the accumulation of reactive oxygen species (ROS) that occurs during stress in plant cells, plants enhance the synthesis of secondary metabolites, which serve as their primary defensive mechanism, with the objective of eliminating or reducing the effects of ROS41. As a result, the use of elicitors that induce stress in plant explants to boost secondary metabolite production has become a common and effective approach. However, it is very important for practical applications that these elicitors are easily applicable, cost-effective and environmentally benign. H2O2, one of the most important signaling molecule, stands out as an elicitor frequently used in plant tissue culture studies in recent years with its cost-effective and environmentally friendly properties42.Present study was carried out to determine the impact of H2O2 treatments on the accumulation of IND and INR in four different Isatis species and to uncover the molecular and biochemical mechanism responsible for the variations in metabolite accumulation induced by H2O2 treatments. In the study, it was determined that IND and INR amounts varied significantly depending on the species and the H2O2 applications, and H2O2 used at appropriate concentrations increased IND and INR accumulation in Isatis species compared to the control. Similarly, H2O2 applied to Castilleja tenuifora plants at different concentrations for 7 d decreased the total phenolic content in the aerial parts of plant at low concentrations (50 µM, 75 µM, and 10 µM) comparison with the un-treated group, but increased significantly with increasing concentrations to 125 µM and 150 µM. On the other hand, in roots, H2O2 applications stimulated total phenolic content at all concentrations43. Vazquez-Hernandez et al. was also found to increase the amount of steviol glycosides in Stevia rebaudiana leaves with H2O2 applications at appropriate concentrations28. H2O2 applied to Ficus deltoide plants at concentrations of 15 mM and 20 mM significantly enhanced total phenolic, carotene, and total flavonoid levels compared to other H2O2 concentrations (5 mM and 10 mM) and control44.When applied externally, the accumulation of H2O2 in cells directly activates the synthesis of secondary metabolites by causing oxidative stress in plants25. H2O2 not only a harmful ROS but also acts as a signaling molecule in stress-response networks45. The increased production of H2O2 in plant cells under stress conditions activates systemic acquired resistance and promotes the synthesis of secondary metabolites19,46. Furthermore, H2O2 is involved in regulating the expression of genes associated with plant defense systems and other metabolic processes20. Indeed, H2O2 treatments were found to promote the synthesis of many metabolites in different plants under in vitro conditions21,22,23,24,25. In accordance with the aforementioned studies, the present research demonstrated that H2O2 was efficacious in enhancing secondary metabolite production when utilized at suitable concentrations.In the study, it was also found that there were differences among the levels of exposure of species to oxidative stress. Compared to I. ermenekensis, I. floribunda and I. tinctoria, I. constricta was negatively affected by high concentrations of H2O2 such as 300 and 400 µM, and significant decreases in metabolite yield occurred at these concentrations. Indeed, high concentrations of H2O2 were found to exert a toxic effect on cells, inducing regulated cell death pathways47. This phenomenon can be attributed to the generation of hydroxyl radicals during the Haber-Weiss reaction via transition metals when H2O2 is unable to be detoxified at elevated concentrations48. Hydroxy radicals, one of the most reactive molecules known, cause protein degradation, formation of carbonyl derivatives, -SS-binding, lipid peroxidation and membrane damage49.Plants exposed to stressors show a range of physiological and biochemical changes linked to survival and adaptation strategies50. Additionally, SOD, CAT, and APX, antioxidant enzymes, play critical roles in maintaining cellular redox balance during H2O2 signaling. SOD transforms O2− into H2O2, playing a vital role in the cellular antioxidant defense system51. CAT is another key enzyme responsible for the breakdown of hydrogen peroxide (H2O2) into oxygen and water to prevent oxidative damage in cells52. APX is another antioxidant enzyme constituting a critical secondary antioxidant defense system, specifically scavenging H2O2 and reactive oxygen species (ROS) generated during chloroplast electron transport53. These enzymes mitigate potential oxidative damage, ensuring that H2O2 functions effectively as a signaling molecule41.Although there was no study investigating the effects of H2O2 applications on antioxidant enzyme activities in Isatis species, studies in different plant species showed that H2O2 changed the antioxidant enzyme activities54,55. In this study, the activities of SOD, CAT, and APX antioxidant enzymes were determined in root cultures of I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria treated with 100, 200, 300, and 400 µM H2O2. The findings revealed that the concentration of H2O2 had notable impacts on the activities of these enzymes. The study found that H2O2 applications, especially with increasing concentrations, reduced SOD activity, while CAT and APX activities significantly varied depending on the species and the H2O2 concentrations. CAT activity showed a significant increase compared to the control in I. constricta and I. ermenekensis at 200 μM, and in I. floribunda and I. ermenekensis at 300 µM H2O2 treatments. APX activity reached its highest levels at 100 µM in I. constricta, 200 µM in I. ermenekensis and I. tinctoria, and 400 µM in I. floribunda. The increased activity of these enzymes in the roots under H2O2 elicitation confirmed the presence of stress conditions. These findings elucidated the effects of varying H2O2 concentrations on SOD, CAT, and APX activities, demonstrating that plant species exhibit notable differences in their oxidative stress response. Similarly, previous studies showed that H2O2 applications caused significant changes in antioxidant enzyme activities depending on plant species and its concentrations12,55. Keshavarz et al. stated that the increase in antioxidant enzyme activities induced by H2O2 is due to the fact that it acts as a defence system for the removal of excessive ROS54. On the other hand, the decreases in antioxidant enzyme activities, especially at higher H2O2 concentrations, had been attributed to the inactivation of antioxidant enzymes resulting from excessive ROS accumulation due to oxidative stress56.The effects of exogenously administered H2O2 on intracellular H2O2 levels in Isatis species was also evaluated in this study and it was observed that as the concentration of H2O2 applied to the roots increased, the intercellular H2O2 levels also rose accordingly. These results demonstrated that H2O2 had significant impacts on the quantity of intracellular H2O2 depending on the species and the concentrations. Similarly, H2O2 treated Nitraria tangutorum calluses had higher H2O2 content compared to the controls55. Catharanthus roseus calluses treated with 100 mM H2O257 and Fragaria vesca calluses treated with 50 µM H2O258 were found to contain higher levels of intracellular H2O2 compared to the controls. However, Yu et al. reported that a 200 mM H2O2 treatment on Vigna radiata reduced its intracellular H2O2 content. These differences highlight species-specific variations in responses to oxidative stress59.In the present study, the dose-dependent effects of H2O2 on transcriptional regulation of TSA and CYP79B2 genes, key enzymes in the IND and INR biosynthetic pathway, were quantitatively analyzed in root cultures of I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria using RT-PCR. Indole alkaloids, which perform critical defensive and physiological functions, are synthesized through complex enzymatic pathways. The TSA genes facilitate tryptophan production, a precursor for indole-ring-containing alkaloids60 while CYP genes participate in diverse hydroxylation and methylation reactions essential for secondary metabolite diversification61. In this study gene expression levels of TSA and CYP79B2 genes showed very notable changes according to H2O2 concentration and the species. Although varying by species, in some concentrations of H2O2 treatments, gene activities significantly increased compared to the control group. These findings are in agreement with the studies reported that H2O2 treatments affected gene expressions related to the secondary metabolite production through different ways. For instance, Vazquez-Hernandez et al. determined that H2O2 application at concentrations of 20 and 200 mM to Stevia rebaudiana leaves led to significant changes in SrUGT74G1, SrKA13H, SrUGT76G1, and SrUGT85C gene expressions related in steviol glycoside synthesis28. The researchers found that SrKA13H and SrUGT76G1 gene expressions did not change with H2O2 applications, but SrUGT74G1 gene expression increased with 20 mM H2O2 and decreased with 200 mM H2O2. SrUGT85C2 gene expression decreased at 20 mM H2O2 and increased at 200 mM H2O2. In aerial and roots of Castilleja tenuiflora, 150 µM H2O2 application caused significant changes in expressions of genes (Cte-TyrDC, Cte-GOT2, Cte-ADD, Cte-AO3, Cte-PAL1, Cte-CHS1, Cte-DXS1, and Cte-G10H) related to phenolic and terpene metabolism in the depending on plant tissue, application time and the genes43.H2O2 treatments led to an increase in TSA and CYP79B2 gene activities in I. consricta, but these increases negatively affected the accumulation of IND and INR. On the other hand, H2O2 encouraged the expression of the CYP79B2 gene in I. ermenekensis roots, giving rise to enhanced IND and INR accumulation. In I. floribunda, H2O2 boosted TSA gene activity, promoting IND accumulation, while CYP79B2 gene activity facilitated INR accumulation. In I. tinctoria, H2O2 had no significant effect on TSA expression, but it notably reduced CYP79B2 gene activity. Consequently, the syntheses of IND and INR increased in correlation with TSA gene activity, while CYP79B2 gene activity showed an inhibitory effect on alkaloid accumulation in I. tinctoria roots.The research results revealed that there was not always a positive correlation between gene activities and the metabolite accumulation in Isatis roots induced by H2O2, and that differences in species, H2O2 concentration, and genes affected the relationship between gene activities and the indole alkaloids including IND and INR in different ways. Similarly, H2O2 treatments of Stevia rebaudiana leaves greatly suppressed the expression of SrUGT76G1 and SrUGT85C2 genes involved in steviol glycoside biosynthesis, resulting in significant reductions in steviol glycoside amounts28. Similar results were found between the accumulation of verbascoside and aucubin and the expressions of genes Cte-TyrDC, Cte-GOT2, Cte-ADD, Cte-AO3, Cte-PAL1, Cte-CHS1, Cte-DXS1, and Cte-G10H) involved in the synthesis of these compounds in roots and aerial parts of Castilleja tenuifora plants treated with H₂O244. These findings demonstrate that transcriptional upregulation of biosynthetic pathway genes alone does not guarantee increased metabolite production and may vary across species.Principal Component Analysis (PCA) is a critical tool in scientific data analysis that enables the interpretation of complex datasets by revealing underlying patterns and variations62. PCA reduces the dimensionality of large, multivariate datasets, identifying the components that explain the largest variance and visualising them in 2D or 3D graphs. In this study, PCs with eigenvalues greater than one were selected according to the Kaiser criterion63. In this study, the top three variables most strongly associated with PC1 were H2O2, INR, and IND in I. constricta; TSA, CAT and APX in I. ermenekensis; H2O2, SOD, and IND in I. floribunda; and H2O2, IND and INR in I. tinctoria.ConclusionIn the present study, the impacts of different concentrations of H2O2 (100, 200 µM, 300 µM and 400 µM) on the accumulation of indole alkaloids (IND and INR), antioxidant enzyme activities (SOD, CAT and APX), gene expressions (TSA and CYP79B2) and intracellular H2O2 were investigated in I. constricta, I. ermenekensis, I. floribunda and I. tinctoria root cultures. The results showed that H2O2 significantly affected the biochemical and molecular responses of the plants. Treatments at certain concentrations significantly increased the production of secondary metabolites, gene expression levels and antioxidant defence mechanisms. By linking H2O2-mediated oxidative signaling to transcriptional activation of alkaloid biosynthetic genes, this work provides a framework for targeted metabolic engineering in Isatis species. This study establishes an important foundation for targeted metabolic engineering applications in Isatis species by directly linking H2O2 mediated oxidative signaling to the transcriptional activation of genes responsible for alkaloid biosynthesis. The findings reveal that H2O2 not only acts as a stress factor but also functions as a signaling molecule that regulates specific gene expression to direct secondary metabolite production. This makes it possible to target these signaling pathways through genetic or chemical approaches to enhance the production of specific metabolites. Therefore, the study provides a strategic framework for metabolic engineering by identifying molecular level intervention points to achieve directed improvements in metabolite levels and offers valuable data that will contribute to the development of potential applications in plant biotechnology and genetic engineering.Data availabilityThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.AbbreviationsAPX:Ascorbate peroxidaseCAT:CatalaseCYP79B2 :Cytochrome P450H2O2 :Hydrogen peroxideIND:IndigotinROS:Reactive oxygen speciesSOD:Superoxide dismutaseTSA :Tryptophan synthase alphaReferencesVarlı, M., Hancı, H. & Kalafat, G. Tıbbi ve aromatik Bitkilerin üretime Potansiyeli ve Biyoyararlılığı. Res. J. Biomed. Biotechnol. 1(1), 24–32 (2020).Google Scholar Davis, P. H. Cruciferae. In Flora of Turkey and the East Aegean Island (Edinburgh University Press, 1965).Cessur, A., Albayrak, İ., Demirci, T. & Baydar, N. G. Silver and Salicylic acid-chitosan nanoparticles alter Indole alkaloid production and gene expression in root and shoot cultures of Isatis tinctoria and Isatis ermenekensis. Plant. Physiol. Biochem. 202, 107977 (2023).Article CAS PubMed Google Scholar Karakoca, K., Ozusaglam, M. A., Cakmak, Y. S. & Erkul, S. K. Antioxidative, antimicrobial and cytotoxic properties of Isatis floribunda Boiss. ex Bornm. extracts. EXCLI J. 12, 150 (2013).PubMed PubMed Central Google Scholar Yıldız, N. & Karakaş, Ö. Qualitative and quantitative determination of tryptanthrin, indirubin, indican and Isatin Indole alkaloids during vegetative and flowering stages in the roots and leaves of Isatis constricta PH Davis. J. Field Crops Cent. Res. Inst. 28(2), 59–66 (2019).Google Scholar Kizil, S. Research on frequency of appropriate planting of some woad (Isatis tinctoria L., Isatis constricta Davis) species and establishment of dyeing properties thereof. Doctoral dissertation, Ankara University (2000).Cordis Sustainable production of plant-derived indigo. https://cordis.europa.eu/project/id/QLK5-CT-2000-30962/fr.Hamburger, M. Isatis tinctoria–From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical. Phytochem. Rev. 1, 333–344 (2002).Article CAS Google Scholar Lin, C. W. et al. Anti-SARS coronavirus 3 C-like protease effects of Isatis Indigotica root and plant-derived phenolic compounds. Antiviral Res. 68(1), 36–42 (2005).Article CAS PubMed PubMed Central Google Scholar Meijer, L., Shearer, J., Bettayeb, K. & Ferandin, Y. Diversity of the intracellular mechanisms underlying the anti-tumor properties of indirubins. Int. Congr. Ser. 1304, 60–74 (2007).Article CAS Google Scholar Nam, S. et al. Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells. Proc. Natl. Acad. Sci. U. S. A. 102(17), 5998–6003 (2005).Article ADS CAS PubMed PubMed Central Google Scholar Du, G. H., Song, X. Y., Kong, L. L. & Chen, N. H. Indirubin. In Natural Small Molecule Drugs from Plants 529–532 (Springer, 2018).Speranza, J. et al. Isatis tinctoria L. (Woad): A review of its botany, ethnobotanical uses, phytochemistry, biological activities, and biotechnological studies. Plants 9(3), 298 (2020).Bown, D. Encyclopaedia of herbs and their uses dorling Kindersley (1995).Han, J., Jiang, X. & Zhang, L. Optimisation of extraction conditions for polysaccharides from the roots of Isatis tinctoria L. by response surface methodology and their in vitro free radicals scavenging activities and effects on IL-4 and IFN-γ mRNA expression in chicken lymphocytes. Carbohydr. Polym. 86(3), 1320–1326 (2011).Article CAS Google Scholar Miceli, N. et al. Chemical characterization and biological activities of phenolic-rich fraction from cauline leaves of Isatis tinctoria L. (Brassicaceae) growing in sicily, Italy. Chem. Biodivers. 14(8), e1700073 (2017).Ullah, I. et al. Antibacterial and antifungal activity of Isatis tinctoria L. (Brassicaceae) using the micro-plate method. Pak. J. Bot. 49(5), 1949–1957 (2017).CAS Google Scholar Fazili, M. A., Bashir, I., Ahmad, M., Yaqoob, U. & Geelani, S. N. In vitro strategies for the enhancement of secondary metabolite production in plants: A review. Bull. Natl. Res. Cent. 46(1), 35 (2022).Article PubMed PubMed Central Google Scholar Chen, Z., Silva, H. & Klessig, D. F. Active oxygen species in the induction of plant systemic acquired resistance by Salicylic acid. Science 262(5141), 1883–1886 (1993).Article ADS CAS PubMed Google Scholar Quan, L. J., Zhang, B., Shi, W. W. & Li, H. Y. Hydrogen peroxide in plants: A versatile molecule of the reactive oxygen species network. J. Integr. Plant. Biol. 50(1), 2–18 (2008).Article CAS PubMed Google Scholar Alvarado-Orea, I. V. et al. Photoperiod and elicitors increase steviol glycosides, phenolics, and flavonoid contents in root cultures of Stevia rebaudiana. In Vitro Cell. Dev. Biol. Plant. 56, 298–306 (2020).Article CAS Google Scholar Barba-Espín, G. et al. H2O2-elicitation of black Carrot hairy roots induces a controlled oxidative burst leading to increased anthocyanin production. Plants 10(12), 2753 (2021).Article PubMed PubMed Central Google Scholar Du, L. et al. Elicitation of Lonicera japonica thunb suspension cell for enhancement of secondary metabolites and antioxidant activity. Ind. Crops Prod. 156, 112877 (2020).Article CAS Google Scholar Goncharuk, E. A. et al. Effects of hydrogen peroxide on in vitro cultures of tea (Camellia sinensis L.) grown in the dark and in the light: Morphology, content of malondialdehyde, and accumulation of various polyphenols. Molecules 27(19), 6674 (2022).Article CAS PubMed PubMed Central Google Scholar Huerta-Heredia, A. A. et al. Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors. Eng. Life Sci. 9(3), 211–218 (2009).Article CAS Google Scholar Garcia-Mier, L. et al. Elicitor mixtures significantly increase bioactive compounds, antioxidant activity, and quality parameters in sweet bell pepper. J. Chem. 2015, 269296 (2015).Mejía-Teniente, L. et al. Oxidative and molecular responses in Capsicum annuum L. after hydrogen peroxide, Salicylic acid and Chitosan foliar applications. Int. J. Mol. Sci. 14(5), 10178–10196 (2013).Article PubMed PubMed Central Google Scholar Vazquez-Hernandez, C. et al. Controlled elicitation increases steviol glycosides (SGs) content and gene expression associated to biosynthesis of SGs in Stevia rebaudiana B. cv. Morita II Ind. Crops Prod. 139, 111479 (2019).Article CAS Google Scholar Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).Article CAS Google Scholar Gai, Q. Y. et al. Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. for the efficient production of flavonoids and evaluation of antioxidant activities. PLoS One 10(3), e0119022 (2015).Zhang, Q., Hong, B., Zheng, L., Wang, X. & Cai, D. Matrix solid-phase dispersion extraction followed by HPLC-diode array detection method for the determination of major constituents in a traditional Chinese medicine Folium isatidis (Da-qing-ye). J. Sep. Sci. 35(18), 2453–2459 (2012).Article CAS PubMed Google Scholar Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72(1–2), 248–254 (1976).Article CAS PubMed Google Scholar Gong, H., Zhu, X., Chen, K., Wang, S. & Zhang, C. Silicon alleviates oxidative damage of wheat plants in pots under drought. Plant. Sci. 169(2), 313–321 (2005).Article CAS Google Scholar Cakmak, I., Strbac, D. & Marschner, H. Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J. Exp. Bot. 44(1), 127–132 (1993).Article CAS Google Scholar Velikova, V., Yordanov, I. & Edreva, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant. Sci. 151 (1), 59–66 (2000).Article CAS Google Scholar Wang, W. et al. Accumulation mechanism of Indigo and indirubin in Polygonum tinctorium revealed by metabolite and transcriptome analysis. Ind. Crops Prod. 141, 111783 (2019).Article CAS Google Scholar Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25(4), 402–408 (2001).Article CAS PubMed Google Scholar Williams, D. & De Luca, V. Plant cytochrome P450s directing monoterpene Indole alkaloid (MIA) and benzylisoquinoline alkaloid (BIA) biosynthesis. Phytochem. Rev. 22(2), 309–338 (2023).Article CAS Google Scholar Albayrak, İ., Demirci, T. & Baydar, N. G. Enhancement of in vitro production of tropane alkaloids and phenolic compounds in Hyoscyamus niger by culture types and elicitor treatments. Plant. Cell. Tiss Organ. Cult. 156(3), 72 (2024).Article CAS Google Scholar Kumari, S. et al. Plant hormones and secondary metabolites under environmental stresses: Enlightening defense molecules. Plant. Physiol. Biochem. 206, 108238 (2024).Article CAS PubMed Google Scholar Hasanuzzaman, M. et al. Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants 9 (8), 681 (2020).Article CAS PubMed PubMed Central Google Scholar Narayani, M. & Srivastava, S. Elicitation: A stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Phytochem. Rev. 16, 1227–1252 (2017).Article CAS Google Scholar Rubio-Rodríguez, E. et al. Mixed elicitation with Salicylic acid and hydrogen peroxide modulates the phenolic and iridoid pathways in Castilleja tenuiflora plants. Planta 258(1), 20 (2023).Article PubMed Google Scholar Jamaludin, R. et al. Influence of exogenous hydrogen peroxide on plant physiology, leaf anatomy and Rubisco gene expression of the Ficus deltoidea Jack var. Deltoidea Agron. 10(4), 497 (2020).Article CAS Google Scholar Chamnongpol, S. et al. Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc. Natl. Acad. Sci. U. S. A. 95(10), 5818–5823 (1998).Alvarez, M. E. et al. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92(6), 773–784 (1998).Article CAS PubMed Google Scholar Gechev, T. S. & Hille, J. Hydrogen peroxide as a signal controlling plant programmed cell death. J. Cell. Biol. 168(1), 17–20 (2005).Article CAS PubMed PubMed Central Google Scholar Asada, K. Ascorbate peroxidase—a hydrogen peroxide-scavenging enzyme in plants. Physiol. Plant. 85(2), 235–241 (1992).Article CAS Google Scholar Shen, B., Jensen, R. G. & Bohnert, H. J. Mannitol protects against oxidation by hydroxyl radicals. Plant. Physiol. 115 (2), 527–532 (1997).Article CAS PubMed PubMed Central Google Scholar Zafar, S., Khan, M. K., Aslam, N. & Hasnain, Z. Impact of different stresses on morphology, physiology, and biochemistry of plants. In Molecular Dynamics of Plant Stress Management (eds Shahid, M. & Gaur, R.) 67–91 (Springer, 2024).Slesak, I., Libik, M., Karpinska, B., Karpinski, S. & Miszalski, Z. The role of hydrogen peroxide in regulation of plant metabolism and cellular signalling in response to environmental stresses. Acta Biochim. Pol. 54(1), 39–50 (2007).Article CAS PubMed Google Scholar Ahmad, P., Nabi, G., Jeleel, C. A. & Umar, S. Free radical production, oxidative damage and antioxidant defense mechanisms in plants under abiotic stress. In Oxidative Stress: Role of Antioxidants in Plants (eds Ahmad, P. & Umar, S.) 19–53 (Studium Press, 2011).Chang, C. C. C. et al. Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis. Plant. J. 38(3), 499–511 (2004).Article CAS PubMed Google Scholar Keshavarz, R. et al. Role of hydrogen peroxide on viability, morphology and antioxidant enzyme activity in callus cells of Catharanthus roseus L. Asia Pac. J. Mol. Biol. Biotechnol. 23, 235–242 (2015).Google Scholar Yang, Y. et al. Exogenous H2O2 increased catalase and peroxidase activities and proline content in Nitraria tangutorum callus. Biol. Plant. 56(2), 330–336 (2012).Article CAS Google Scholar Özden, M., Demirel, U. & Kahraman, A. Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. Sci. Hortic. 119(2), 163–168 (2009).Article Google Scholar Tang, Z., Yang, L., Zu, Y. & Guo, X. Variations of vinblastine accumulation and redox state affected by exogenous H2O2 in Catharanthus roseus (L). Plant. Growth Regul. 57, 15–20 (2009).Article CAS Google Scholar Tian, F., Liu, Y., Hu, K. & Zhao, B. The depolymerization mechanism of Chitosan by hydrogen peroxide. J. Mater. Sci. 38, 4709–4712 (2003).Article ADS CAS Google Scholar Yu, C. W., Murphy, T. M. & Lin, C. H. Hydrogen peroxide induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Funct. Plant. Biol. 30, 955–963 (2003).Article CAS PubMed Google Scholar Salvini, M. et al. Alpha-tryptophan synthase of Isatis tinctoria: Gene cloning and expression. Plant. Physiol. Biochem. 46(7), 715–723 (2008).Article CAS PubMed Google Scholar Chakraborty, P., Biswas, A., Dey, S., Bhattacharjee, T. & Chakrabarty, S. Cytochrome P450 gene families: Role in plant secondary metabolites production and plant defense. J. Xenobiotics 13(3), 402–423 (2023).Article CAS Google Scholar Marini, F. & Binder, H. pcaExplorer: An R/Bioconductor package for interacting with RNA-seq principal components. BMC Bioinform. 20, 1–8 (2019).Article Google Scholar Kaiser, H. F. The varimax criterion for analytic rotation in factor analysis. Psychometrika 23, 187–200 (1958).Article Google Scholar Download referencesAcknowledgementsThe authors are thankful to TUBITAK (Scientific and Technological Research Council of Turkey) for the financial support for this research Project (Project number: KBAG-123Z490).Author informationAuthors and AffiliationsDepartment of Agricultural Biotechnology, Faculty of Agriculture, Isparta University of Applied Sciences, 32270, Isparta, Turkeyİlknur Albayrak, Alper Cessur & Nilgün Göktürk BaydarDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Suleyman Demirel University, 32260, Isparta, TurkeyTunahan DemirciDepartment of Field Crops, Faculty of Agriculture, Isparta University of Applied Sciences, 32270, Isparta, TurkeyÜmmü TuğluAuthorsİlknur AlbayrakView author publicationsSearch author on:PubMed Google ScholarAlper CessurView author publicationsSearch author on:PubMed Google ScholarTunahan DemirciView author publicationsSearch author on:PubMed Google ScholarÜmmü TuğluView author publicationsSearch author on:PubMed Google ScholarNilgün Göktürk BaydarView author publicationsSearch author on:PubMed Google ScholarContributionsİ.A.: Writing – original draft, Conceptualization. A.C.: Visualization, Writing - review & editing, Data curation, Conceptualization, Formal analysis. T.D.: Writing – review & editing, Validation, Project administration, Methodology, Funding acquisition. Ü.T.: Writing – review & editing. N.G.B.: Writing – review & editing, Formal analysis, Methodology, Conceptualization, Visualization.Corresponding authorCorrespondence to Alper Cessur.Ethics declarationsCompeting interestsThe authors declare no competing interests.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.Reprints and permissionsAbout this article