Harnessing the Potential of Harpin Proteins: Elicitation Strategies for Enhanced Secondary Metabolite Accumulation in Grapevine Callus Cultures

Abstract

Grapes and grape products are rich in secondary metabolites such as phenolic compounds and anthocyanins, which have antioxidant properties. These compounds possess health-promoting attributes, including cardioprotective, antimicrobial, and anticancer effects. In recent years, biotechnological methods have been employed to produce high quantities and purity of secondary metabolites under in vitro conditions, aiming to elucidate their complex functions and optimize production methods. However, the potential effects of harpin proteins on the accumulation of secondary compounds in callus cultures have not been investigated thus far. Harpin proteins, encoded by the hrp gene clusters in Gram-negative phytopathogens, are known to trigger defense responses in various plant species by promoting the accumulation of secondary compounds. These findings suggest that harpin proteins may have the potential to enhance secondary metabolite accumulation in callus cultures. This study therefore investigated the potential of applying different concentrations of harpin protein (0, 0.1, 1, 10, and 100 ppm) to increase secondary metabolite production in calluses derived from petioles of the “Horoz Karası” grape cultivar. Our findings revealed that 1 and 10 ppm harpin treatments resulted in the highest anthocyanin accumulations, with 17.21 and 16.57 CV/g, respectively, representing 1.95- and 1.87-fold increases compared to control treatments, respectively. Total phenolic content peaked at 0.39 mg GAE g⁻¹ FW with the 1 ppm harpin treatment, representing a 4.33-fold increase over the control. Total flavanol levels reached their highest levels at 0.027 mg CE g⁻¹ FW with 1 and 10 ppm harpin concentrations, resulting in a 2.25-fold increase compared to the control. The highest averages for total flavonol content were recorded at 0.024 and 0.021 mg RE g⁻¹ FW with 1 and 10 ppm harpin concentrations, respectively, representing 1.5- and 1.3-fold increases over the control. Principal component analysis (PCA) corroborated the results obtained from the heatmap analysis, indicating that harpin applications at 1 and 10 ppm were the most effective concentration range for maximizing secondary metabolite synthesis, while very low or high concentrations diminished these effects. These findings offered valuable insights for optimizing the production of high-value bioactive compounds, which can be utilized in various fields such as medicine, pharmaceuticals, food, and cosmetics. These results are expected to serve as a valuable reference for elucidating the mechanisms by which harpin proteins, rarely used in vitro, exert their effects on grapevine calluses, contributing to the literature in this domain.

1. Introduction

Plants constitute the primary source of carbohydrates, proteins, and fats essential for human sustenance. Secondary metabolites, classified based on their biosynthetic pathways and derived from intermediates of primary metabolic pathways, do not directly influence vital functions such as providing nourishment and energy. However, they determine quality criteria like color, taste, and aroma [1,2]. These metabolites protect plants against biotic and abiotic stresses, playing a crucial role in developing defense mechanisms against microorganisms and herbivores [3]. Phenolic compounds, a significant class of secondary metabolites, are aromatic ring compounds containing at least one hydroxyl group (OH) and exhibiting antioxidant properties. Studies have shown that these compounds inhibit the formation of low-density lipoproteins (LDL) [4], possess cardioprotective effects [5], exhibit strong antioxidant activities by scavenging free radicals that damage nucleic acids, somatic cells, and the immune system [6], and exhibit antimicrobial [7], and anticancer properties [8], making them crucial secondary metabolites for the healthcare sector. Additionally, they have potential applications in the food, cosmetics, and agricultural industries. Research has shown that grapes and grape products are rich in phenolic compounds [9]. Phenolic compounds found in grapes are classified as benzoic acids, hydroxycinnamic acids, stilbene derivatives (resveratrol), flavanols (catechin, epicatechin), flavonols (kaempferol, quercetin), and anthocyanins [10,11]. Among these compounds, anthocyanins are of great importance in secondary metabolite production. Anthocyanins, belonging to the flavonoid class of phenolic compounds, are natural red, blue, and purple color pigments. While there are 22 known different types of anthocyanins, the most important ones are delphinidin, malvidin, pelargonidin, peonidin, petunidin, and cyanidin. Anthocyanins have the potential to be used as natural food additives to enhance the appeal of food and beverages by providing natural coloration [12]. They also exhibit anticancer [13] and antioxidant [14] properties, making them valuable for the pharmaceutical industry. In grapes, anthocyanins are typically found in the skin and, in some varieties, in the berry.

The complex roles of secondary metabolites in plants have led to an increase in research efforts in this area. Studies in this field are crucial for both developing production methods and understanding the mechanisms of action of these compounds. The challenges faced in obtaining secondary metabolites from plants under natural conditions limit their production [12]. These challenges include the difficulty and expense of collecting plants from natural flora, the risk of extinction for some species due to excessive harvesting, the impact of climatic conditions on the quantity and quality of secondary metabolites, the synthesis of active compounds in plants during specific developmental stages or in very small quantities, failures in cultivating plants, and the need for extensive agricultural land and intensive labor to produce economically viable quantities of active compounds [10,11]. Due to these limitations, the restricted production cannot meet the increasing demand for natural products as consumer preferences change. In recent years, it has been determined that secondary metabolites can be obtained in high quantities and purity through in vitro techniques, and researchers have focused their efforts on this area. Producing secondary metabolites through biotechnological methods and optimizing culture conditions offers several advantages. Plant cell and tissue cultures eliminate environmental factors (climate, geographical challenges, seasonal limitations) encountered during plant cultivation, reduce land use, prevent the risk of extinction by avoiding harvesting from natural habitats, enable the production of economically valuable metabolites found in low quantities in plants, provide homogeneity, standard quality, and productivity in production, and assist in understanding the biosynthesis mechanisms of metabolites. Among biotechnological methods, the callus culture technique is a simple system that enables large-scale production of secondary metabolites and rapid, reliable multiplication of cells [15]. Due to these advantages, its commercial use has become more widespread in recent years [16].

Research has shown that applying biotic or abiotic elicitors to plants under in vitro conditions can lead to significant increases in the levels of secondary metabolites as a response to the plant’s stress mechanism [17,18]. For this purpose, various stress-inducing elicitors, such as proteins, glycoproteins, polysaccharides, jasmonic acid/methyl jasmonate, salicylic acid, ozone, ethylene, bacteria, fungi, light radiation, heavy metal ions (copper, mercury, cadmium, lead, etc.), or hydrogen peroxide, have been successfully applied individually or in combination [19,20]. In recent years, researchers have focused on natural elicitors such as seaweed, yeast extracts, Trichoderma harzianum fungus, or harpin proteins as sustainable approaches to promote plant growth and development [21,22]. Initially isolated from the apple fire blight pathogen Erwinia amylovora by Dr. Zhongmin Wei at Cornell University, harpins are acidic, heat-stable, glycine-rich proteins with a molecular weight of 44 kDa, encoded by the hrpN gene [23]. Harpin proteins produced by gram-negative bacteria are reported to increase tolerance to biotic and abiotic stress factors by activating the plant defense mechanism and contribute to improving quality attributes such as color and aroma, as well as yield [24]. However, the effectiveness of harpin proteins varies depending on plant species and intended use, and the effective concentration range also differs. Investigating the mechanisms of action of exogenously applied harpin proteins on secondary metabolites is of great importance for grapevines, a species known to naturally synthesize high levels of secondary metabolites with potential applications in various fields such as medicine, pharmaceuticals, food, and cosmetics.

While it is known that harpin proteins activate plant defense, little is understood about the changes they may induce in secondary metabolites during the activation of plant defense. Although there are a few studies evaluating the effects of harpin proteins on secondary metabolite production in plants comprehensive literature reviews [19,20,23], there are limited studies on their effects on biochemicals in grapevines callus environment. Therefore, this study aimed to investigate the potential of harpin protein applications at different concentrations (0, 0.1, 1, 10, and 100 ppm) to enhance secondary metabolite production in calluses derived from petioles of the “Horoz Karası” grape cultivar, a species known for naturally synthesizing high levels of secondary metabolites with potential applications in various fields. Given the limited understanding of the mechanisms by which harpin proteins influence secondary metabolite production in grapevines, this research holds significant importance by contributing valuable insights to the literature and potentially paving the way for optimizing the production of high-value bioactive compounds through biotechnological approaches.

2. Materials and Methods

2.1. Plant Material

This study, investigating the effects of harpin protein applications at different concentrations on secondary metabolite production in grapevine calluses, was conducted between 2023 and 2024 in the research greenhouse and tissue culture research laboratory of the Department of Horticulture, Faculty of Agriculture, Bozok University, Yozgat, Turkey. The plant material used in the research was petioles from the “Horoz Karası” grape cultivar. These plant materials were obtained by growing one-year-old cuttings from the Eastern Mediterranean Transition Zone Agricultural Research Institute during the dormant period. The “Horoz Karası” grape is a cultivar suitable for table and raisin purposes, with winged conical clusters weighing 700–800 g and blue-black berries containing 2–3 seeds. This cultivar ripens in mid-season and requires short-mixed pruning [25]. The research area where the plants were grown was a semi-circular, polycarbonate-constructed greenhouse with an approximate area of 200 m². This greenhouse was equipped with a 55% shade screen, fan heaters, a fan and pad system, and a ventilation system. Concrete-based rooting benches measuring 5 m in length, 1.20 m in width, 80 cm in height from the ground, and 20 cm in depth were used to place the pots.

2.2. Preparation of Growing Media and Planting of Cuttings

One-year-old cuttings of the “Horoz Karası” grape cultivar was prepared to be approximately 20 cm in length, each containing two buds. They were subjected to a rapid dipping treatment with 2000 ppm IBA (Indole Butyric Acid) and planted in 11 × 11 × 22 cm PE pots containing a sterile growing medium of 1:1 peat and perlite. Immediately after planting and continuing until sufficient shoot and leaf development was achieved, the cuttings were regularly irrigated with the nutrient solution recommended by Ollat et al. [26] (containing Ca(NO₃)₂·4H₂O (2.5 mM), KH₂PO₄ (1.0 mM), KNO₃ (2.5 mM), MgSO₄·7H₂O (1.0 mM), Na₂MoO₄ (0.013 µM), ZnSO₄·7H₂O (2.40 µM), CuSO₄ (0.5 µM), MnCl₂·4H₂O (9.2 µM), H₃BO₃ (46.4 µM), NaFe(III)-EDTA (45 µM), pH: 6.5). The irrigation amount was adjusted to maintain a 30% drainage rate. After an approximate 8-week growing period, the leaves, along with their petioles, were harvested from the cuttings.

2.3. Preparation of Explants and Callus Induction

Leaf samples taken from grapevine seedlings grown in the greenhouse were brought to the laboratory, washed under tap water, and then separated into leaf blades and petioles. The petioles were subjected to surface disinfection for 5 min in a 15% commercial sodium hypochlorite solution containing 0.05% (v/v) Tween-20, followed by three rinses with sterile distilled water. The nutrient medium was prepared by supplementing full-strength Gamborg’s B-5 basal medium (Sigma-Aldrich, St. Louis, MO, USA) with minimal organic components, 0.5 mg/L BAP, 0.5 mg/L IAA, 30 g/L sucrose, and 6 g/L agar [27]. pH of the medium was adjusted to 5.7 ± 0.1 before autoclaving at 120 °C for 15 min. The sterile nutrient solutions were poured into 9 cm diameter Petri dishes, with 25 mL per dish. Under sterile conditions, the disinfected petiole explants were cut into approximately 0.5–1 cm length and placed on the nutrient medium, with 15–20 explants per Petri dish. The cultures were incubated at 24 ± 1 °C in the dark for 21 days to facilitate callus development, and the resulting initial calluses were subcultured onto fresh medium with the same composition. The induced calluses were transferred to a new subculture medium after another 21 days, ensuring sufficient callus production for the subsequent harpin protein applications.

2.4. Harpin Protein Applications

The study utilized the commercial product “Messenger Gold” containing the active ingredient harpin protein, produced by the “Plant Health Care” company. The commercially obtained harpin is a second-generation harpin protein called Harpinαβ, consisting of a combination of active portions of harpin proteins isolated from various plant pathogenic bacteria such as Ralstonia solanacearum, Pseudomonas syringae, and Erwinia amylovora [24]. The solutions were prepared according to the procedure reported by Navarro-Acevedo [24]. The powder formulation was suspended in ddH₂O, and 0.05% (v/v) Tween-20 was added as a surfactant. Freshly prepared harpin protein solutions at concentrations of 0, 0.1, 1, 10, and 100 ppm were filtered through a 0.22 µm millipore membrane for in vitro experiments and added to the nutrient medium onto which the calluses from the third subculture would be transferred. For the 0 ppm (control) treatment, H₂O was used. The calluses transferred to the harpin-containing nutrient media were subcultured at 21-day intervals under the same culture conditions, and the experiment was terminated after approximately 4 weeks, at which point the calluses were collected and stored at −20 °C. Subsequently, to determine the effects of harpin protein applications on secondary metabolites, the anthocyanin, total phenolic compound, total flavanol, and total flavonol contents of the calluses were analyzed, and the most effective application concentration was identified based on the studied traits.

2.5. Analysis of Secondary Metabolites

2.5.1. Determination of Anthocyanin Content

Anthocyanin extraction and analysis were performed according to the spectrophotometric method reported by Zhang et al. [28]. For extraction, 0.15 g of fresh callus tissue was weighed and thoroughly ground with the aid of liquid nitrogen. Three mL of 50% acetic acid solution (Sigma-Aldrich, Merck CAS number 64-19-7, Darmstadt, Germany) was added, and the mixture was extracted at room temperature for 1 h, with vortexing for 10 s at 15 min intervals. One mL of the supernatant obtained after centrifugation of the extract at 9000 rpm for 5 min was collected, and 3 mL of McIlvaine buffer (14.7 g L⁻¹ di-Sodium hydrogen phosphate dodecahydrate (Sigma-Aldrich, CAS: 10039-32-4) and 16.7 g L⁻¹ anhydrous citric acid (Sigma-Aldrich, CAS: 77-92-9) was added. This ensured that the pH of the resulting solution remained consistent at 3.0, as the solution’s pH can affect the measurement of pigments in the extract. The absorbance of the obtained solution was measured at 535 nm using a UV–Vis spectrophotometer (Perkin Elmer Lambda 25, Shelton, CT, USA) with 50% acetic acid as a blank. The anthocyanin content is represented as the color value (CV), calculated using the following equation:

CV = 0.1 × A535 × DF (CV g⁻¹ FW)

Here, A535 represents the absorbance value measured at 535 nm wavelength, and DF is the dilution factor.

2.5.2. Determination of Total Phenolic Content

For the determination of total phenolic, total flavanols, and total flavonol contents, extraction procedures were carried out based on the method of Kiselev et al. [29]. Accordingly, 0.1 g of fresh callus tissue was thoroughly ground with the aid of liquid nitrogen. Five mL of 96% ethanol (Merck, CAS: 64-17-5) was added and homogenized for 3 min. After homogenization, the samples were incubated overnight at 45 °C and then centrifuged at 9000 rpm for 5 min. The supernatant was transferred to a new tube, and the ethanol was evaporated using a rotary evaporator (Hahn Shin, HS-2005V-N, Bucheon, Republic of Korea). After complete evaporation of ethanol, the remaining portion was dissolved in 1 mL of methanol (Merck, CAS: 67-56-1). The total phenolic content of the calluses was analyzed using the Folin-Ciocalteu colorimetric method [30]. Absorbances were measured using a UV–Vis spectrophotometer at a wavelength of 765 nm. The total phenolic compound content was determined as milligrams of gallic acid equivalents (GAE) per gram of fresh weight (mg GAE g⁻¹ FW) using a calibration curve prepared from a standard gallic acid solution.

2.5.3. Determination of Total Flavanol Content

The total flavanol content was estimated using the 4-(Dimethylamino) cinnamaldehyde (DMACA, Sigma-Aldrich, CAS: 6203-18-5) method [31]. One mL of the extract was transferred to test tubes, and 1 mL of a 0.1% DMACA solution dissolved in methanol containing 1 N hydrochloric acid (Sigma-Aldrich, CAS: 7647-01-0) was added. The mixture was vortexed and allowed to react at room temperature for 10 min. Subsequently, the absorbance at 640 nm was read in a UV–Vis spectrophotometer against a blank prepared without DMACA. The total flavanol concentration was estimated using a calibration curve constructed with catechin solutions. The results were expressed as catechin equivalents per fresh weight (mg CE g⁻¹ FW) for three replicates.

2.5.4. Determination of Total Flavonol Content

The total flavonol analysis was performed using the Neu solution according to the method employed by Kumaran and Joel Karunakaran [32]. Accordingly, a mixture of 1% 2-aminoethyl diphenylborinate solution (CAS: 524-95-8) and methanol was added to the extracts and thoroughly mixed, and the absorbance values were determined at 410 nm. The total flavonol contents were expressed as rutin equivalents per initial gram of fresh tissue (mg RE g⁻¹ FW) by comparing with the values obtained from the rutin standard.

2.6. Experimental Design and Statistical Analysis

The study was designed as a completely randomized design with four different harpin protein concentrations, three replicates, and ten Petri dishes per replicate. The numerical data obtained were subjected to one-way analysis of variance (one-way ANOVA) using the IBM SPSS vrs. 20.0 software package, and the Duncan’s multiple range test (p < 0.05) was used to determine the differences between means. Mean values and standard deviations (SD) were calculated and visualized for each treatment group. Additionally, the 95% confidence interval was calculated, and the estimated marginal means, lower, and upper limits were determined. Line graphs and principal component analysis (PCA) were generated using the Python programming language and the Matplotlib library for data visualization. Correlation analysis and heatmap were created using the SRPLOT software (https://bioinformatics.com.cn/srplot).

3. Results and Discussion

In recent years, the exploration of natural elicitors, such as harpin proteins, has gained significant attention for their potential to enhance secondary metabolite production in plants. Our study delves into this by investigating the impact of harpin proteins on the synthesis of key secondary metabolites under in vitro conditions. The findings present compelling evidence on the efficacy of harpin proteins in modulating metabolite accumulation. In our study, multivariate analysis of variance revealed that the model significantly and robustly explained the variance in the dependent variables (

Table 1. Multivariate and univariate analysis results for harpin applications on plant metabolites.

Multivariate Tests
		value	F	df1	df2	p
Harpin applications	Wilks’ Lambda	3.41 × 10−4	17.4	16	22	<0.001
Univariate Tests
	Dependent Variable	Sum of Squares	df	Mean Square	F	p
Harpin applications	Anthocyanin content	147.5588	4	36.88971	30.2	<0.001
	Total phenolic content	2.58 × 10−4	4	6.45 × 10−5	13.7	<0.001
	Total flavanol content	5.37 × 10−4	4	1.34 × 10−4	15.1	<0.001
	Total flavonol content	0.1684	4	0.04210	25.2	<0.001
Residuals	Anthocyanin content	12.2288	10	1.22288
	Total phenolic content	4.70 × 10−5	10	4.70 × 10−6
	Total flavanol content	8.89 × 10−5	10	8.89 × 10−6
	Total flavonol content	0.0167	10	0.00167

). The highest accumulation of anthocyanins, total phenolic compounds, flavanols, and flavonols was observed at harpin protein concentrations ranging from 1–10 ppm. The results demonstrated a profound effect of harpin protein applications at different concentrations on the accumulation of secondary metabolites in callus cultures. The optimal range of 1–10 ppm harpin is believed to trigger plant defense responses, stimulating biosynthesis and enhancing the accumulation of secondary metabolites. The application of harpin protein may have initiated a cascade of molecular events, including the induction of defense-related genes and modulation of metabolic enzymes, leading to increased production of these compounds. However, at higher concentrations, such as 100 ppm, the elicitation effect of harpin appeared to diminish or induce a different physiological response. The variations observed in the accumulation of different metabolite classes reflect the intricate interplay between elicitor signaling, metabolic regulation, and biosynthetic machinery. In the control samples without harpin application, the anthocyanin accumulation remained at the lowest level, averaging 8.84 CV/g, while the 1 and 10 ppm harpin applications resulted in the highest anthocyanin accumulations of 17.21 and 16.57 CV/g, respectively, causing 1.95- and 1.87-fold increases compared to the control treatments, respectively (Figure 1a). These findings indicate that harpin concentrations ranging from 1 to 10 ppm can significantly enhance anthocyanin synthesis. Higher production of secondary metabolites reflects greater antioxidant capacity in plant samples. It has been previously reported that the production and accumulation of anthocyanin pigments, a subgroup of flavonoids in plants, can vary under the influence of different elicitors [33]. In the present study, when the harpin concentration increased to 100 ppm, the anthocyanin content declined to 11.46 CV/g, suggesting that excessively high harpin concentrations may adversely affect anthocyanin accumulation. The lowest concentration (0.1 ppm) of harpin application yielded a moderate anthocyanin content of 13.13 CV/g. These results indicate that determining the effective concentration range of the harpin protein is crucial for optimizing anthocyanin accumulation. While harpin applications at levels between 1 and 10 ppm maximized anthocyanin accumulation, very low or very high concentrations diminished these effects.

Harpin proteins are known to trigger defense responses in plants by activating various biochemical pathways. Previous studies have demonstrated that harpin proteins induce hypersensitive response (HR) in plants and enhance disease resistance [34]. Similarly, Navarro-Acevedo [24] found that foliar application of harpin proteins to Arabidopsis plants promoted growth and increased drought tolerance under drought conditions. The effects of harpin proteins are mediated through the induction of ion fluxes in plant cell membranes, alkalization of the growth medium, depolarization of the cell membrane, and production of reactive oxygen species (ROS). Consequently, the synthesis of anthocyanins and other phenolic compounds increases [35]. In the present study, the increase in anthocyanin content of the calluses following harpin protein applications may be associated with ROS production and activation of cellular signaling pathways. The precursor molecule for anthocyanin biosynthesis is phenylalanine [36]. ROS may have accelerated the phenylpropanoid pathway by increasing the activity of Phenylalanine ammonia-lyase (PAL) enzymes, thereby promoting anthocyanin synthesis in the calluses. The activation of defense-related genes by harpin proteins likely leads to increased transcription and activity of key enzymes in the phenylpropanoid pathway, such as chalcone synthase and anthocyanidin synthase, which are critical for anthocyanin biosynthesis [21,22,23]. Although the mechanisms by which harpin proteins influence anthocyanin accumulation under in vitro conditions have not been previously investigated, our findings support previous reports on the positive effects of harpin proteins on coloration properties and anthocyanin accumulation in fruits grown under in vivo conditions. For instance, Crupi et al. [37] found that repeated applications of harpin proteins at a concentration of 400 g/Ha enhanced berry skin coloration, total soluble solids content, and maturity index values in the Crimson Seedless grape cultivar and effectively stimulated anthocyanin biosynthesis, increasing the levels of peonidin-3-O-glucoside, cyanidin-3-O-glucoside, and malvidin-3-O-glucoside by 2 to 10-fold. Similarly, Durmuş [38] reported that harpin proteins applied at a concentration of 120 ppm during different growth stages of the Red Globe grape cultivar improved color homogeneity, coloration rate, and other quality parameters of the berries. Likewise, Li et al. [39] found that pre-harvest applications of harpin protein at a concentration of 1.5 mg L⁻¹ were effective in enhancing flesh color in Lapins and Regina (Prunus avium) cherry cultivars.

The total phenolic content in the calluses remained at the lowest levels of 0.09 mg GAE g⁻¹ FW in the control samples without harpin application and 0.13 mg GAE g⁻¹ FW with the 0.1 ppm harpin application. In contrast, the 1 ppm harpin application resulted in a peak total phenolic level of 0.39 mg GAE g⁻¹ FW, representing a 4.33-fold increase compared to the control treatment. However, when the harpin concentration exceeded 1 ppm, the total phenolic content exhibited a significant decline, dropping to 0.21 mg GAE g⁻¹ FW with the 10ppm application and 0.20 mg GAE g⁻¹ FW with the 100 ppm application. Nevertheless, these averages were still 2.33 and 2.22 times higher, respectively, than the total phenolic content in the control treatment (Figure 1b). The findings of this study indicate that the harpin protein, when used at appropriate concentrations, can significantly enhance the accumulation of phenolic compounds in plant callus cultures. Particularly, the 1 ppm harpin concentration yielded the highest phenolic yield. However, higher concentrations diminished this effect, while lower concentrations did not induce a noticeable change, suggesting that harpin protein applications need to be carefully optimized. This effect is thought to be due to the harpin applications increasing the activity of the PAL enzyme, which plays a crucial role in the synthesis of phenolic compounds. These results are consistent with previous studies by Isah et al. [40] and Ullah et al. [41], which reported that various chemical and physical factors influence the accumulation of bioactive compounds in plant callus cultures. Additionally, our findings support the study by Fonseca et al. [42], which demonstrated the promoting effect of harpin proteins on the accumulation of plant phenolic compounds in vivo.

The applications of the harpin protein significantly influenced the total flavanol content of the calluses. The total flavanol levels reached the highest levels of 0.027 mg CE g⁻¹ FW at 1 and 10 ppm harpin concentrations, resulting in a 2.25-fold increase compared to the control. These averages were in the same statistical group as the 100 ppm application, which provided a flavanol content of 0.024 mg CE g⁻¹ FW. The lowest levels for total flavanol content were detected in the control group without harpin application at 0.012 mg CE g⁻¹ FW. The 0.1 ppm harpin concentration caused a moderate increase in total flavanol content, providing 0.018 mg CE g-1 FW, which was higher than the control treatment (Figure 1c). This increase in flavanol content is like the results reported for Taxus baccata cell suspensions treated with different elicitor applications [43]. These results can be attributed to higher trans-cinnamic acid production as a product of L-phenylalanine deamination by the phenylalanine ammonia-lyase enzyme and the increased binding of trans-cinnamic acid to different secondary metabolites under suitable concentrations of the elicitors used [44]. In this context, various factors have significant impacts on the trends of the elicitation process, including differences in the chemical properties of elicitors, their source and concentration, the duration of exposure to elicitation, the age and type of explant for culture, growth regulations and nutrient composition in the culture medium, and interactions with plant species and genotypes [45,46]. Considering that the production of plant secondary metabolites is strongly controlled by signaling events and environmental factors, further studies may be needed to gain a deeper understanding of the modulation effects at the callus level across different plant species. The significant increases in different phenolic contents could have been induced by the triggering effects of harpin on signaling pathways, which may have accelerated enzyme catalysis, leading to the formation of specific compounds, such as flavanols.

The total flavonol content was significantly influenced by the harpin concentrations, with the lowest value of 0.012 mg RE g⁻¹ FW obtained from the 100 ppm harpin concentration. The highest averages of 0.024 and 0.021 mg RE g⁻¹ FW were recorded at 1 and 10 ppm harpin concentrations, respectively, providing 1.5- and 1.3-fold increases compared to the control. The 0.1 ppm harpin application, with 0.017 mg RE g⁻¹ FW, was in the same statistical group as the control (0.016 mg RE g⁻¹ FW) (Figure 1d). The increase in total flavonol content with different elicitors (methyl jasmonate and salicylic acid) was previously reported in in vitro callus cultures of A. purpurata by Mounika and Giri [47]. Plants utilize different defense response pathways in response to environmental stresses, such as elicitor exposure at the cellular level [48]. These cellular responses lead to increased production and accumulation of various secondary metabolites, including total phenolics and their derivatives [45,48,49]. These results are supported by the findings of previous studies conducted under in vivo conditions with harpin proteins. Rodrigo-Garcia et al. [49]. reported positive effects of foliar-applied harpin proteins at concentrations of 45, 60, and 120 mg L⁻¹ on the polyphenol content in lettuce. Considering all the findings together, harpin protein concentrations in the range of 1–10 ppm maximized the accumulation of secondary metabolites. In contrast, when the harpin protein was applied at a low concentration, its effect was diminished, while concentrations exceeding 10 ppm resulted in a significant decrease in bioactive metabolite accumulation. Consistent with these results, various previous reports have shown that different components added to the callus growth medium can influence certain physiological and biochemical parameters of the calluses in varying ways, depending on the applied concentration [50,51]. These findings highlight the potential of harpin proteins in promoting the production of bioactive compounds in the fields of plant biotechnology and plant biochemistry, providing an important foundation for future studies. These results offer valuable insights for optimizing the production of high-value bioactive compounds that can be utilized in various fields—such as medicine, pharmaceuticals, food, and cosmetics (Figure 2a)—supporting previous studies in this area. A correlation analysis was conducted to determine the direction and strength of the relationship among the studied secondary metabolites (Figure 2b). The elicitation effect of harpin proteins may also involve the modulation of hormone signaling pathways, particularly jasmonic acid and ethylene, which are known to play crucial roles in regulating plant defense responses and secondary metabolite production.

The results of the correlation analysis indicate significant relationships among the examined secondary metabolites. The positive correlations observed between anthocyanin, total phenolic, total flavanol, and total flavonol components provided insights into their accumulation patterns in the callus cultures. In the correlation plot, the numbers in each cell, ranging from −1 to 1, represented the Pearson correlation coefficients between two features. Strong positive correlations were found between anthocyanin and total phenolic (0.79), anthocyanin and total flavanol (0.85), and anthocyanin and total flavonol (0.82). These strong correlations suggest that the accumulation of anthocyanins is closely associated with the overall phenolic compound content. It is important to note that this relationship is expected, as anthocyanins are a subclass of phenolic compounds. While these correlations initially suggested the possibility of overlapping metabolic pathways, we acknowledge that our study design does not provide direct evidence for this hypothesis. The observed changes in secondary metabolite levels could indeed be due to the activation of additional metabolic reactions rather than effects on main metabolic chains. Future studies incorporating enzyme inhibition experiments, such as targeting common enzymes in these pathways, would be necessary to substantiate any claims about shared biosynthetic routes. Similarly, a strong positive correlation (0.80) was observed between total phenolic and total flavanol contents. This relationship is logical, as flavanols are a subclass of phenolic compounds, and their increased presence would naturally contribute to higher total phenolic content. A moderate positive correlation (0.68) was found between total phenolic and total flavonol content, which is also consistent with flavonols being a subclass of phenolic compounds. The weak positive correlation (0.47) detected between total flavanol and flavonol content suggests that while both are subclasses of flavonoids, their accumulation may be influenced by different factors or regulated by distinct mechanisms within the callus cultures. The differential accumulation of flavanols and flavonols in response to harpin treatment suggests that this elicitor may selectively activate specific branches of the flavonoid biosynthetic pathway, possibly through the regulation of transcription factors such as MYB, bHLH, or WD40 proteins. However, we recognize that our current data do not allow us to definitively determine the specific mechanisms underlying these increases. The observed changes could be due to various factors, including the activation of multiple independent pathways, rather than shared biosynthetic routes as we initially hypothesized. Further investigation using more targeted analytical techniques, such as enzyme activity assays, gene expression analyses, or metabolic flux studies, would be necessary to elucidate the precise metabolic changes induced by harpin protein treatment. Furthermore, the role of ROS as secondary messengers in harpin-induced metabolite accumulation warrants investigation, as ROS have been implicated in the activation of defense-related genes and the stimulation of secondary metabolite biosynthesis in other elicitor systems. Our findings demonstrate an overall positive relationship between the analyzed components, indicating that an increase in one component is generally associated with increases in the others. These results suggest that biotechnological approaches and natural elicitors used to enhance secondary metabolite production in callus cultures may have a broad impact on various phenolic compounds. However, we recognize that our current data do not allow us to definitively determine the specific mechanisms underlying these increases. The observed changes could be due to various factors, including the activation of multiple independent pathways, rather than shared biosynthetic routes as we initially hypothesized.

The heatmap analysis, performed for data visualization, illustrates the responses of grapevine calluses to different harpin concentrations and how these responses vary between different groups (Figure 2c). Along the y-axis, five different concentration levels of harpin are present: 0 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm. Along the x-axis, the anthocyanin, total phenolic, flavanol, and flavonol components are arranged. The colors in the heatmap represent the intensity levels of each component.

The color scale ranges from −2 (green) to +2 (orange). Green represents low concentration levels, while orange represents high concentration levels. Yellow tones indicate intermediate levels. The heatmap essentially divided the harpin treatments into two main clusters. The first cluster includes the control group (0 ppm) and the lowest and highest harpin concentrations (0.1 and 100 ppm, respectively), generally showing lower secondary metabolite levels. Within the first main cluster, the 100 ppm harpin treatment, which had a more significant effect on total phenolic and flavanol accumulation, showed a separate branching, while the 0.1 ppm harpin concentration and the control treatment, which had a lower impact on the accumulation of these compounds, formed the second sub-cluster. In the heatmap, it can be observed that at 0 ppm concentration (control group), the levels of the examined compounds are generally low (green). The second main cluster consists of the 1 and 10 ppm harpin concentrations, and the effect of these treatments on the accumulation of secondary compounds is evident from the color distinction. In the heatmap, the 1 ppm treatment, showing higher secondary metabolite levels, is dominated by orange colors. The 1 ppm concentration was the most effective treatment, particularly in increasing total phenolic compound levels. These findings are consistent with previous reports stating that different components added to the callus growth medium can induce differences in the biochemical parameters of the calluses, depending on the applied concentration [52]. To visualize the relationship between harpin concentrations and secondary compounds, a principal component analysis (PCA) was performed, and a biplot was created (Figure 2d). The first two components explain a large portion of the total data variance (approximately 94%), indicating that the PCA analysis preserves a significant amount of information from the original data set and summarizes the structure of the data set in an understandable way. PC1 (Principal Component 1) accounts for 80.44% of the total variance. This suggests that a significant portion of the data varies along the PC1 axis, and this component is the main source of variation in the data. PC2 (Principal Component 2) accounts for 13.47% of the total variance. The orange dots represent different harpin concentrations (0 ppm, 0.1 ppm, 1 ppm, 10 ppm, 100 ppm). The red arrows indicate the direction and magnitude of the secondary metabolites (anthocyanin, total phenolic, flavanol, and flavonol) on the principal components. The anthocyanin and total phenolic vectors are represented by long arrows pointing towards the PC1 axis, indicating a strong relationship between anthocyanin and total phenolic values with PC1. The total flavanol and flavonol vectors have significant loadings on both axes, suggesting that these variables play a role in both principal components and are secondary sources of variation in the data. The biplot confirms the findings obtained from the correlation analysis and heatmap. According to the biplot, the y-axis separates the harpin treatments into two groups, with the control treatment, 0.1 ppm, and 100 ppm concentrations in the first group and the 1 ppm and 10 ppm treatments in the second group. The scatter plot shows that the 1 ppm concentration is the best-represented in terms of all secondary metabolite values, followed by the 10ppm treatment. Our findings are consistent with previous studies reporting that different elicitors can induce changes in the content of secondary metabolites [47].

4. Conclusions

In this study, the effectiveness of harpin proteins in promoting the production of secondary metabolites was investigated using calluses derived from grapevine petiole explants, which are known to naturally synthesize high levels of secondary compounds. Our results indicated that harpin applications within the concentration range of 1–10 ppm was effective in promoting the accumulation of secondary compounds in callus cultures. These findings will make a significant contribution to future research aimed at increasing secondary metabolite production using biotechnological methods and natural elicitors. Furthermore, this study will serve as a valuable reference for elucidating the mechanisms of action of harpin proteins, which are rarely used under in vitro conditions, on grapevine calluses. In this context, the utilization of harpin proteins holds significant potential for the development of new and effective strategies in the fields of plant biotechnology and metabolic engineering. Our results pave the way for sustainable and efficient enhancement of anthocyanin production for agricultural and industrial applications. Future studies could explore the synergistic effects of combining harpin proteins with other elicitors or optimizing the timing and duration of harpin treatments. Additionally, scaling up harpin-elicited callus cultures to bioreactor systems would enable large-scale production of valuable secondary metabolites.