Abstract
This study explored the impact of sodium chloride (NaCl) elicitation on the accumulation of primary and secondary metabolites and the oxidative stress responses of Inula crithmoides L. (golden samphire) in vitro shoot cultures. Elicitation involved applying different concentrations of NaCl (0, 50, 100, and 200 mM) for 4 weeks. This was followed by assessing its impact on plant growth, physiological parameters (pigments, hydrogen peroxide content, total soluble sugars and proteins, and proline), and secondary metabolism (phenylalanine ammonia-lyase activity, shikimic acid, phenolics, flavonoids, and hydroxycinnamic acids) in the shoots. The extracts were also analysed using high-performance liquid chromatography (HPLC). The NaCl elicitation did not affect shoot growth but increased physiological functions such as photosynthesis and oxidative stress management under moderate salinity levels. In addition, NaCl treatments increased the synthesis of soluble sugars and proteins, particularly proline, as well as bioactive phenolics such as gentisic acid, chlorogenic acid, 4-hydroxybenzoic acid, luteolin-7-O-glucoside, and naringenin-7-O-glucoside. The NaCl elicitation in golden samphire shoot cultures offers a significant method for enhancing the production of important nutritional and bioactive compounds. This underscores the species’ potential for cultivation in saline environments and provides valuable prospects for its utilization in the health and nutrition sectors.
Key message
NaCl elicitation significantly enhances the accumulation of metabolites, including sugars, proteins, and bioactive phenolic compounds in golden samphire, improving its nutritional and medicinal value.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Medicinal plants are invaluable sources of bioactive compounds utilized in various industries, such as pharmaceuticals, cosmetics, food additives, and veterinary products (Fierascu et al. 2021). However, relying solely on natural sources presents challenges such as environmental harm, loss of biodiversity, supply inconsistencies due to seasonal variations, and vulnerability to climate change. To tackle these issues, alternative sourcing methods must be explored to balance resource demand with environmental and ethical concerns for long-term sustainability (Caro et al. 2022).
Plant tissue culture provides a sustainable and controlled method for producing bioactive metabolites. Culturing plant cells, tissues, or organs in a controlled environment ensures year-round growth under optimal conditions and eliminates seasonal variations. Moreover, the synthesis of nutritional and bioactive metabolites may be enhanced through elicitation approaches that stimulate stress responses to activate specific metabolic pathways to produce desired compounds. Such methods may include biotic (microorganisms) and abiotic (temperature, light, chemicals such as jasmonic acid and sodium chloride-NaCl) elicitors (Chandran et al. 2020; Ozyigit et al. 2023). For instance, excessive sodium chloride (NaCl) stress can be detrimental to plants, hindering growth and potentially leading to plant death. However, when NaCl concentrations are moderate, they can promote growth, increase the accumulation of secondary metabolites, and enhance pro-health components, antioxidants, and nutritional quality (Acosta-Motos et al. 2017; Chiappero et al. 2021). NaCl elicitation has significantly increased the levels of total phenolics, flavonoids, anthocyanins, and phenolic acids in Melissa officinalis L. cultures (Hawrylak-Nowak et al. 2021). It also triggers stress markers (H2O2, catalase, and peroxidase), activates phenylpropanoid enzymes (phenylalanine ammonia-lyase and tyrosine ammonia-lyase), and enhances phenolic content in common bean sprouts (Ampofo and Ngadi 2021). Furthermore, it has been found to improve saponin production in Agave salmiana plants cultured in vitro (Puente-Garza et al. 2021).
Additionally, in recent years, salinity has emerged as a significant environmental challenge, affecting agricultural productivity and ecological balance (Egea et al. 2023), and posing a significant threat to plant crops, especially in regions with limited freshwater resources (Tessema et al. 2023). Addressing this issue is crucial for ensuring global food security and maintaining ecological stability in areas affected by salinity. Understanding plant stress response and resilience is key to adapting agricultural practices in these environments while preserving the desired nutritional and medicinal benefits (Chele et al. 2021; Mukhopadhyay et al. 2021).
Inula crithmoides L., commonly known as golden samphire, is a flowering plant of the Asteraceae family. It thrives in salt marshes, maritime cliffs, and rocky coastal areas along the Mediterranean coast, Britain, and Western Asia (Clapham, 1962; eHALOPH 2023). Notably, golden samphire is both edible and aromatic. Its young leaves or shoots are suitable for consumption whether raw, cooked, or pickled (Zurayk and Baalbaki 1996; D’Agostino et al., 2022). Furthermore, many Inula species, including I. crithmoides, possess significant therapeutic effects, such as antimicrobial (Deriu et al. 2008), anti-inflammatory (Hernández et al. 2007), antioxidant (Kogure et al. 2004), and antihepatotoxic activities (Saygi et al. 2003), attributed to pharmacologically active compounds like polyphenols, including numerous quinic acid derivatives such as chlorogenic acid (Trendafilova et al. 2020). Moreover, plant in vitro culture has proven to be a valuable platform for this species’ rapid and enhanced multiplication, providing a nursery for commercial cultivation systems (Rodrigues et al. 2023). Due to its therapeutic, nutritional, and aromatic properties, as well as its salt tolerance, golden samphire is considered a promising candidate for saline agriculture as a source of food and medicinal crops (Zurayk and Baalbaki 1996). Therefore, this study aimed to investigate the potential of using sodium chloride (NaCl) to enhance the accumulation of primary and secondary metabolites in Inula crithmoides L. shoots, as well as to evaluate its influence on their overall growth and oxidative stress responses aiming at increasing its nutritional and medicinal value.
Materials and methods
In vitro culture conditions and growth
Golden samphire plants were propagated in vitro using the micropropagation protocol outlined by Rodrigues et al. (2023). The shoots obtained were then cultivated in a basal MS medium supplemented with 0, 50, 100, and 200 mM NaCl. The medium, which contained 2% sucrose and 0.8% agar, was adjusted to a pH of 5.7–5.8 and autoclaved at 121 °C for 20 min. Cultures were maintained at a temperature of 25 ± 2 °C, with a 16/8 h light/dark photoperiod provided by LED light (2700 K) at an intensity of 3000 lx. The cultures were maintained for 6 weeks, and the number of shoots was counted. Additionally, the height of the tallest shoot and the length of the roots were measured.
Physiological parameters
Photosynthetic pigment determination
Photosynthetic pigments (total chlorophylls and carotenoids) were extracted by macerating 25 mg of fresh plant material with 4 mL of pure methanol, in triplicate. The optical density was measured at 470 nm, 653 nm, 666 nm, and 750 nm (Lichtenthaler 1987).
Hydrogen peroxide (H2O2) content
The hydrogen peroxide content was determined using the protocol developed by Loreto and Velikova (2001), which was adapted for use with 96-well plates. Fresh plant material (100 mg) was homogenised with 1 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged for 15 min at 12,000 x g. Then, 50 µL of the supernatant was mixed with 50 µL of 10 mM potassium phosphate buffer and 100 µL of 1 M potassium iodide. After 30 min in the dark, the absorbance was measured at 390 nm. The results were calculated by comparing them with an H2O2 calibration curve and expressed as micromoles of equivalent H2O2 per gram of fresh weight (µmol/g FW).
Primary metabolism
Extraction
Fresh plant material (250 mg) was extracted three times with 500 µL of 80% ethanol (v/v) at 80 °C for 30 min (Martins et al. 2011).
Proline content
Extracts (500 µL) were mixed with 1% (w/v) ninhydrin reagent prepared in 60% (v/v) acetic acid, incubated for 1 h at 100 °C, and the absorbance was measured at 520 nm. Results were calculated using a proline calibration curve and expressed as micromoles of proline equivalents per gram of fresh weight (µmol/g FW).
Total soluble proteins
The extracts (5 µL) were mixed with 250 µL of Bradford reagent and incubated for 30 min at room temperature. The absorbance was measured at 595 nm, and the results were calculated using a bovine serum albumin (BSA) calibration curve. The results are expressed as milligrams of BSA equivalents per gram of fresh weight (mg/g FW).
Total soluble sugars
The extracts (100 µL) were mixed with 100 µL of distilled water, 200 µL of 9% (w/v) phenol, and 1 mL of 96% sulfuric acid (Abid et al. 2021). After incubating at room temperature for 30 min, the absorbance was measured at 490 nm. Glucose was used as a standard, and the results were expressed as milligrams of glucose equivalent per gram of fresh weight (mg/g FW).
Secondary metabolism
Shikimic acid content
Fresh plant material (100 mg) was macerated with 2 mL of 0.25 M HCl and then centrifuged at 20,000 g for 15 min at 4 °C. Fifty microliters of supernatant were mixed with 500 µL of 1% (w/v) periodic acid. After a 3-hour incubation at room temperature, 500 µL of 1 M NaOH and 300 µL of 0.1 M freshly prepared glycine were added, and the absorbance was measured at 380 nm (Alzandi and Naguib 2020). Results were calculated using a shikimic acid calibration curve and expressed as milligrams of shikimic acid equivalents per gram of fresh weight (mg/g FW).
Phenylalanine ammonia-lyase (PAL) activity
Fresh plant material (50 mg) was homogenized in 1 mL of 0.1 M sodium borate buffer (pH 8.8) and centrifuged at 20,000 x g for 20 min at 4 °C. In a 96-well plate, 10 µL of the supernatant was mixed with 50 µL of L-phenylalanine (10 mM) and 140 µL of 0.1 M sodium borate buffer (pH 8.8). The mixture was incubated at 37 °C for 1 h, and the absorbance was measured at 290 nm. Enzyme activity was determined by comparing it with a calibration curve for cinnamic acids and expressed as micromoles of cinnamic acid per hour per gram of fresh weight (µmol/h/g FW).
Total phenolics, flavonoids, and hydroxycinnamic acids
Fresh plant material was extracted following the reported method for the analysis of primary metabolites. The total contents of phenolics (TPC), flavonoids (TFC) and hydroxycinnamic acids (THA) of the extracts were determined in 96-well plates, as previously detailed in Rodrigues et al. (2015). The TPC was determined according to the method described by Velioglu et al. (1998) by mixing 5 µL of the extracts with 100 µL of the Folin-Ciocalteu 10-fold diluted solution and 100 µL of 75 g /L of sodium carbonate, incubated for 90 min and read at 725 nm. TFC were estimated using the aluminium chloride (AlCl3) colorimetric method described by Pirbalouti et al. (2013), modified to 96-well plates. For that, 50 µL of the extracts were mixed with 50 µL of a 2% AlCl3-ethanol solution, incubated for 10 min and read at 420 nm. The THA was estimated using the hydrochloric acid (HCl)-ethanol method (Mazza et al. 1999) adapted to 96-well plates by Rodrigues et al. (2015) by mixing 20 µL of the extracts with 20 µL of 95% ethanol containing 0.1% HCl, and with 2% HCl, and read at 320 nm. Results for TPC, TFC and THA were correspondingly expressed as equivalents of gallic acid (GAE), rutin (RE), and caffeic acid (CAE) in milligrams per gram of fresh weight (FW), using calibration curves with standard solutions at concentrations ranging from 0.002 to 2 mg/mL.
High-performance liquid chromatography (HPLC) analysis
The extracts at a concentration of 10 mg/mL in a mixture of 90% ultrapure water and 10% methanol were analysed by HPLC-DAD (Agilent 1260 Infinity II Series LC system, Germany), which consisted of the following modules: 1260 quaternary pump (G7111B), 1260 vial sampler (G7129A), and the 1260 diode array detector (G7115A). The data acquisition and instrumental control were performed using the OpenLab CDS software (version 2.6, Agilent Technologies). Analyses were performed using a Kinetex C18 column, 15 × 0.46 cm, with a 5 μm particle size (Phenomenex, USA). The mobile phase consists of a mixture of methanol (solvent A) and a 2.5% acetic acid aqueous solution with the following gradient: 0–5 min: 10% A, 5–10 min: 10–30% A, 10–40 min: 30–90% A, 40–45 min: 90% A, 45–55 min: 90 − 10% A, and 55–60 min: 10% A, using a flow rate of 0.5 mL/min. The injection volume was 20 µL at a draw speed of 200 µL/min. The detector was set at 255, 280, 320, and 350 nm. For identification purposes, the retention parameters of each assay were compared with the standard controls, and the peak purity was assessed using UV-visible spectral reference data. The levels of the various compounds were extrapolated from calibration standard curves. Commercial standards of gentisic, 4-hydroxybenzoic, chlorogenic, caffeic, vanillic, coumaric, cinnamic, and ferulic acids, luteolin-7-O-glucoside, naringenin-7-O-glucoside, rutin, naringenin, chrysin, and flavone were dissolved in ethanol (1000 mg/L) and then diluted with ultrapure water to the desired concentration.
Statistical analysis
The results were expressed as the mean ± standard deviation (SE), and each experiment was conducted at least three times. Significant differences were assessed using ANOVA followed by Duncan’s multiple range test (DMRT). P values less than 0.05 were considered significant. All statistical analyses were conducted using the XLSTAT statistical package in Microsoft Excel (version 2013, Microsoft Corporation).
Results
Growth and physiological parameters
Different concentrations of NaCl did not have a significant effect on the growth of aerial parts, as indicated by a similar number of shoots (1.67–2.29 cm) and height (8.79–10.67 cm) across all treatments (Figs. 1 and 2). However, the presence of NaCl in the medium significantly reduced the root length from 8.47 cm in the control plants (without NaCl) to 3.50 to 4.73 cm (50 to 200 mM NaCl) (Fig. 2). Furthermore, the overall moisture content of the plant has been observed to decrease from 94.1–94.5–91.4% when salinity exceeds 200 mM NaCl (data not shown). Interestingly, the total chlorophyll and carotenoid contents increased with the rising NaCl concentration in the media, following a dose-dependent trend. For instance, plants grown under controlled conditions (without salts) had 0.26 mg/g and 0.04 mg/g of total chlorophylls and carotenoids, respectively. However, with an increase in salinity to 50 and 100 mM NaCl, the levels correspondingly rose to 0.40–0.42 mg/g of FW and 0.6–0.7 mg/g of FW. When exposed to 200 mM, the golden samphire plants exhibited the highest levels of chlorophylls (0.72 mg/g FW) and carotenoids (0.10 mg/g FW) (Fig. 3A). Conversely, an opposite trend was observed for H2O2 levels, with its concentration decreasing as salinity increased. Plants grown without salts (control) had 5.71 µmol/g FW of H2O2, which decreased to 4.45–4.51 µmol/g FW when the golden samphire was grown at 50 and 100 mM, while the lowest levels were observed under the NaCl 200 mM condition (1.75 µmol/g FW) (Fig. 3B).
Effect of exposure to various concentrations of NaCl (50, 100, and 200 mM) on the shoot number, maximum shoot height, and root length of in vitro cultures of I. crithmoides. For each assay, the results were analysed using a one-way analysis of variance (ANOVA) and the bars on the graph bars labelled with different letters (a–d) are significantly different at p < 0.05 (Duncan’s test)
Effect of exposure to various concentrations of NaCl (50, 100, and 200 mM) on pigments (total chlorophylls and carotenoids) (A) in in vitro cultures of I. crithmoides. For each assay, the results were analysed by one-way analysis of variance (ANOVA) and the graph bars followed by different letters (a–d) are significantly different at p < 0.05 (Duncan’s test)
Primary metabolism
Higher contents of both total soluble proteins and sugars were observed in salt-free conditions (0.23 and 3.72 mg/g FW, respectively) and in plants growing in a saline medium. However, no significant differences were observed between the different concentrations of NaCl (proteins: 0.68–0.73 mg/g FW; sugars: 3.86–3.87 mg/g FW) (Fig. 4). In turn, there was a fivefold increase in proline content when plants were cultured under 200 mM NaCl conditions (8.49 µmol/g FW) compared to control plants (1.62 µmol/g FW) (Fig. 4).
Effect of exposure to various concentrations of NaCl (50, 100, and 200 mM) on primary metabolites (total soluble sugars, soluble proteins, and proline contents) in in vitro cultures of I. crithmoides. For each assay, the results were analysed using a one-way analysis of variance (ANOVA) and the bars on the graph bars labelled with different letters (a–c) are significantly different at p < 0.05 (Duncan’s test)
Secondary metabolism
The shikimic acid content showed a significant increase from plants grown without salt and 50 mM NaCl (0.11–0.14 mg/g of FW) to plants grown under 100 mM NaCl or more (0.22 mg/g of FW) (Fig. 5A). In turn, PAL activity increased 11-fold in plants exposed to 200 mM NaCl (15.9 µmol/h/g FW) compared to the control (1.40 µmol/h/g FW) (Fig. 5B), along with elevated levels of phenolics, flavonoids, and hydroxycinnamic acids (0.54, 0.95, and 4.16 mg/g of FW, respectively) (Fig. 5C).
Effect of exposure to various concentrations of NaCl (50, 100, and 200 mM) on the secondary metabolism, including shikimic acid (A), PAL activity (B), and the total content of phenolics, flavonoids, and hydroxycinnamic acids (C), in in vitro cultures of I. crithmoides. For each assay, the results were analysed using a one-way analysis of variance (ANOVA) and the graph bars followed by different letters (a–c) are significantly different at p < 0.05 (Duncan’s test)
The HPLC-DAD analysis of phenolic compounds in golden samphire plants exposed different varying concentrations of NaCl (50, 100, and 200 compared comparison to the revealed distinct trends in the levels of these (Table 1 and Fig. S1). The phenolic compounds in golden samphire plants exhibit distinct variations in response to NaCl exposure, with some compounds increasing, others remaining stable, and some decreasing in concentration. For example, 4-hydroxybenzoic acid, the most abundant compound, exhibited an increasing concentration as the NaCl concentration increased. It reached its maximum at 200 mM NaCl with 0.29 mg/g of DW, compared to 0.08 mg/g of DW in the control. Chlorogenic acid, on the other hand, reached its highest concentration at 100 mM NaCl (0.31 mg/g DW), but decreased to 0.18 mg/g DW at 200 mM NaCl, showing a significant deviation from the control levels (< 0.01 mg/g DW). In contrast, naringenin-7-O-glucoside, luteolin-7-O-glucoside, and gentisic acid reached their peak at 50 mM NaCl (0.19, 0.07, and 0.06 mg/g DW, respectively), followed by a slight decrease at higher NaCl concentrations. Conversely, the rutin and chrysin content declined with higher NaCl concentration, reaching the lowest levels at 200 mM NaCl (0.02 and 0.01 mg/g DW, respectively). The concentration of naringenin decreased under salt stress compared to control levels (0.05 mg/g of DW), with no variation observed between the different NaCl concentrations (0.03 mg/g of DW). Flavone, vanillic, coumaric, cinnamic, caffeic, and ferulic acids exhibited modest fluctuations and generally maintained relatively low levels (0.01–0.02 mg/g DW).
Discussion
Growth and physiological parameters
Elevated soil salinity typically has a negative impact on plant growth by increasing osmotic stress on roots, inhibiting water absorption, disrupting nutrient uptake, and ultimately leading to reduced biomass and impaired overall plant health (Balasubramaniam et al. 2023). However, the golden samphire did not experience a significant impact on the growth of its aboveground parts up to 200 mM NaCl. Other halophyte species, such as Atriplex halimus, have shown an increase or unchanged leaf fresh weight at moderate salinities (0–200 mM NaCl) (Bendaly et al. 2016), as well as Arthrocnemum indicum, which exhibited unchanged shoot height up to 300 mM NaCl (Nisar et al. 2021). Furthermore, the salinity hindered the growth of golden samphire roots. In fact, roots are the primary organ involved in the uptake of water and nutrients before they are transported to the aboveground parts. Therefore, roots are the first organ to sense and respond to salt stress, enabling the plant to quickly adapt and remain functional by altering the root anatomy. This can include the formation of apoplastic barriers, which are modifications of the cell wall that help prevent the entry of toxic ions through the root apex, while also regulating the flow of Na+ to the shoots (Shahid et al. 2020). This strategy may explain the reduced root growth and the lack of impact on golden samphire shoots.
The average chlorophyll content of the golden samphire plants increased with increasing salinity, indicating that an increase in chlorophyll and carotenoid content appears to contribute to the salt resilience of golden samphire. An increase in photosynthesis can lead to higher growth rates and biomass, provided that the carbon source is not limited. This can explain the consistent plant growth observed in golden samphire in vitro cultures (Maschler et al. 2022). Moreover, similar to the current study, two halophyte species from the Juncaceae family (J. acutus and J. maritimus) have shown similar or increased pigment contents (chlorophylls and carotenoids) when grown in moderate salinities (100–200 mM NaCl) compared to plants irrigated with salt-free nutrient solution (Al Hassan et al. 2017). A similar trend was also observed in the halophyte grass Aeluropus littoralis under 600 mM NaCl (Hashemipetroudi et al. 2022), as well as in Limonium santapolense, L. virgatum, L. girardianum, and L. narbonense cultivated at moderate salinities (0–400 mM) (Hassan et al., 2017).
Salt stress usually induces the accumulation of Reactive Oxygen Species (ROS), such as hydrogen peroxide (H2O2), which can lead to increased oxidative damage to membrane lipids, proteins, and nucleic acids, causing irreversible metabolic dysfunction (Balasubramaniam et al. 2023). In this study, the concentration of H2O2 in golden samphire plants decreased as the salinity of the growing media increased, which is consistent with findings for several other halophytes, including Chrithmum maritimum (Hamdani et al. 2017), Atriplex halimus, Nitraria retusa (Boughalleb et al. 2010), Prosopis strombulifera (Reginato et al. 2019), and Suaeda salsa (Cai-Hong et al. 2005). It is known that the plant’s capacity to neutralize ROS is directly associated with the levels and performance of antioxidant enzymes, such as catalase, peroxiredoxin, glutathione peroxidase-like enzymes, and ascorbate peroxidase (Smirnoff and Arnaud 2019). When these mechanisms are present, it has been shown that NaCl does not significantly alter the levels of H2O2 due to the increased activity of APX (Tzortzakis et al. 2020). Moreover, the levels of osmoprotectants such as proline, and secondary metabolites like phenolic compounds may also contribute to decreasing the level of ROS (Kebert et al. 2022).
Overall, the NaCl treatment reveals the remarkable resilience of golden samphire, demonstrating its ability to sustain growth and physiological functions such as photosynthesis and management of oxidative stress under moderate salinity levels.
Primary metabolism
Primary metabolism refers to the fundamental biochemical processes that are essential for plant growth, development, and survival, such as sugars, proteins, and lipids (Reshi et al. 2023). Under saline conditions, plants often respond by accumulating organic solutes such as proteins, proline, soluble sugars, glycine betaine, and polyols. These small soluble compounds act as osmoprotectants, helping with intracellular osmotic adjustment and detoxification of ROS (Slama et al. 2015; dos Santos et al. 2022). For example, the accumulation of proline protects and stabilizes ROS-scavenging enzymes, activates alternative detoxification pathways, and enhances the activity of methylglyoxal detoxification enzymes, peroxidase, glutathione S-transferase, superoxide dismutase, and catalase. It also increases the glutathione redox state under salt stress (Shuyskaya et al. 2020). Interestingly, they accomplish these protective functions without negatively impacting cellular metabolism while safeguarding the integrity of the cell membrane (Slama et al. 2015). The levels of total proteins, soluble sugars, and notably proline have increased in golden samphire in vitro cultures when exposed to salinity. A trend already reported in Inula species grown in an environmental chamber (Pehlivan and Guler, 2019), as well as common to many other salt-tolerant plants subjected to a wide range of salinities (0–750 mM NaCl), including, for example, Frankenia pulverulenta, Atriplex prostrata (Bueno et al. 2020), Chrithmum maritimum (Hamdani et al. 2017), Aeluropus littoralis (Hashemipetroudi et al. 2022), or Lycium humile (Palchetti et al. 2021).
In addition, the increased soluble sugar and protein content in plants, as a result of their adaptive responses to environmental stresses such as NaCl elicitation, can directly benefit human health when these plants are consumed. Soluble sugars such as glucose, fructose, and sucrose are essential energy sources in human nutrition and also play significant roles in carbohydrate metabolism. The increased presence of these sugars in plants not only reflects their stress adaptation mechanisms but also potentially enhances the nutritional value of the plants, providing a richer energy source for human consumption (Prinz 2019). Soluble proteins, including proline, play vital roles in supporting immune function and providing energy for collagen synthesis, essential for wound healing. Proline, in particular, supports gut health maintenance, contributes to antimicrobial peptides, and aids in skin barrier function, highlighting its critical role in innate immunity and metabolic regulation. These diverse roles underscore the importance of dietary proteins and amino acids in maintaining health and supporting physiological processes (Patriarca et al. 2021; Hepworth 2023).
Secondary metabolism
Secondary metabolism involves the synthesis of a diverse range of specialized compounds, such as polyphenols, that play a role in the defence of plants against environmental stress conditions (dos Santos et al. 2022). These compounds may also play a role in signalling pathways that regulate stress responses and cellular homeostasis, enhancing the adaptability of a plant and exhibiting potent antioxidant properties, essential for eliminating reactive oxygen species (ROS) such as H2O2 (Reshi et al. 2023). The shikimic acid pathway is essential for the biosynthesis of phenolic compounds via the phenylpropanoid pathway, with phenylalanine serving as the primary substrate for their synthesis. This process is facilitated by an essential enzyme called phenylalanine-ammonia-lyase (PAL), which is crucial for phenolic biosynthesis (Reshi et al. 2023). In this study, the golden samphire plants exhibited higher levels of shikimic acid above 100 mM NaCl. Additionally, they showed increased PAL activity and higher levels of phenolics, flavonols, and hydroxycinnamic acids at maximum salinity (200 mM NaCl). The same trend has been previously observed in I. chrithmoides cultivated in a greenhouse under different levels of NaCl (0–600 mM) (Chaura et al. 2015; Al Hassan et al. 2016), as well as in other halophyte species exposed to salt stress conditions (0–600 mM NaCl), such as Frankenia pulverulenta (Bueno et al. 2020), Juncus maritimus, J. acutus, and J. articulatus (Al Hassan et al. 2017b).
Furthermore, phenolic acids, such as 4-hydroxybenzoic and chlorogenic acids, were the compounds most affected by NaCl elicitation. These acids have been described as crucial signalling molecules in plants’ adaptive responses to abiotic stress, especially salinity. These compounds play a multifaceted role in detoxifying harmful substances, protecting plants against oxidative damage, and increasing antioxidant capacity. As a result, they mitigate the detrimental effects of ROS on plant metabolism by enhancing photosynthetic activity (Soviguidi et al., 2021). The increased production of secondary antioxidant metabolites in golden samphire under salinity treatments is likely to contribute to the observed rise in chlorophyll and carotenoid levels, as well as the significant reduction of H2O2. This suggests a crucial role for these compounds in reducing ROS, thereby improving photosynthetic efficiency.
Additionally, phenolic acids and flavonoids, which are key secondary metabolites that are particularly increased by NaCl elicitation in golden samphire shoots, are known for various medicinal benefits. For example, gentisic acid has been linked to several health-promoting effects, such as antioxidant, anti-inflammatory, hepato- and neuroprotective, and antimicrobial activities (Abedi et al. 2020). Chlorogenic acid, a compound commonly found in Inula species, has also attracted attention for its wide range of pharmacological effects, including neutralizing oxygen radicals, boosting superoxide dismutase concentrations, and moderating the synthesis and secretion of inflammatory mediators. Moreover, it has demonstrated antibacterial and antiviral properties, along with its potential role in managing blood sugar and lipid levels, which could be beneficial for treating and preventing chronic diseases such as cardiovascular diseases and diabetes (Miao and Xiang 2020). Similarly, 4-hydroxybenzoic acid exhibits antimicrobial, antialgal, antimutagenic, antiestrogenic, hypoglycemic, and anti-inflammatory properties. It also has anti-platelet aggregation, nematicide, antiviral, and antioxidant effects. It is also commonly used as a preservative in various products, including cosmetics, pharmaceuticals, food items, and beverages (Manuja et al. 2013). Additionally, the flavonoid derivatives luteolin-7-O-glucoside and naringenin-7-O-glucoside also offer a multitude of medicinal benefits, including anticancer, antioxidant, anti-inflammatory, antiviral, neuroprotective, and cardioprotective properties (Ullah et al. 2020). The heightened concentration of bioactive compounds such as gentisic acid, chlorogenic acid, 4-hydroxybenzoic acid, luteolin-7-O-glucoside, and naringenin-7-O-glucoside in NaCl-treated golden samphire shoots indicates that NaCl elicitation may substantially boost the medicinal properties of golden samphire, potentially elevating its efficacy as a natural remedy for various health conditions.
Conclusions
The investigation into NaCl elicitation’s impact on golden samphire demonstrated a significant enhancement in the accumulation of both primary and secondary metabolites within in vitro shoot cultures. This process notably augmented levels of soluble sugars, proteins (especially proline), and a range of bioactive phenolic compounds such as gentisic acid, chlorogenic acid, 4-hydroxybenzoic acid, luteolin-7-O-glucoside, and naringenin-7-O-glucoside, without adversely affecting plant growth. Moreover, it improved physiological responses such as photosynthesis and oxidative stress resilience at moderate salinity levels. These outcomes elucidate NaCl elicitation’s potential in elevating I. crithmoides shoots’ nutritional and medicinal properties, highlighting its adaptability for saline agriculture and its promising utility in the health and nutrition sectors.
Data availability
Data will be made available on request.
References
Abedi F, Razavi BM, Hosseinzadeh H (2020) A review on gentisic acid as a plant derived phenolic acid and metabolite of aspirin: Comprehensive pharmacology, toxicology, and some pharmaceutical aspects. Phytother Res 34:729–741. https://doi.org/10.1002/ptr.6573
Abid G, Ouertani RN, Muhovski Y, Jebara SH, Hidri Y, Ghouili E, Abdelkarim S, Chaieb O, Souissi F, Zribi F (2021) Variation in antioxidant metabolism of faba bean (Vicia faba) under drought stress induced by polyethylene glycol reveals biochemical markers associated with antioxidant defense. Plant Biosyst Int J Deal all Asp Plant Biol 155:797–806
Acosta-Motos JR, Ortuño MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ, Hernandez JA (2017) Plant responses to salt stress: adaptive mechanisms. Agronomy 7:18. https://doi.org/10.3390/agronomy7010018
Al Hassan M, Chaura J, López-Gresa MP, Borsai O, Daniso E, Donat-Torres MP, Mayoral O, Vicente O, Boscaiu M (2016) Native-invasive plants vs. halophytes in Mediterranean Salt marshes: stress tolerance mechanisms in two related species. Front Plant Sci 7:473. https://doi.org/10.3389/fpls.2016.00473
Al Hassan M, Chaura J, Donat-Torres MP, Boscaiu M, Vicente O (2017) Antioxidant responses under salinity and drought in three closely related wild monocots with different ecological optima. AoB PLANTS 9:plx009
Al Hassan M, Estrelles E, Soriano P, López-Gresa MP, Bellés JM, Boscaiu M, Vicente O (2017b) Unraveling Salt Tolerance mechanisms in Halophytes: a comparative study on four Mediterranean Limonium species with different Geographic distribution patterns. Front. Plant Sci 8:1438. https://doi.org/10.3389/fpls.2017.01438
Alzandi AA, Naguib DM (2020) Effect of hydropriming on Trigonella foenum callus growth, biochemical traits and phytochemical components under peg treatment. Plant Cell Tissue Organ Cult 141:179–190
Ampofo JO, Ngadi M (2021) Stimulation of the phenylpropanoid pathway and antioxidant capacities by biotic and abiotic elicitation strategies in common bean (Phaseolus vulgaris) sprouts. Process Biochem 100:98–106. https://doi.org/10.1016/j.procbio.2020.09.027
Balasubramaniam T, Shen G, Esmaeili N, Zhang H (2023) Plants’ response mechanisms to salinity stress. Plants 12:2253
Bendaly A, Messedi D, Smaoui A, Ksouri R, Bouchereau A, Abdelly C (2016) Physiological and leaf metabolome changes in the xerohalophyte species Atriplex halimus induced by salinity. Plant Physiol Biochem 103:208–218
Boughalleb F, Mhamdi M, Hailaoui H, Denden M (2010) Salinity effects on organic solutes and antioxidative enzymes in two halophytes, Nitraria retusa (Forssk) and Atriplex halimus (L). Res J Biol Sci 5:773–784
Bueno M, Lendínez ML, Calero J, del Pilar Cordovilla M (2020) Salinity responses of three halophytes from inland saltmarshes of Jaén (southern Spain). Flora 266:151589
Cai-Hong P, Su-Jun Z, Zhi-Zhong G, Bao-Shan W (2005) NaCl treatment markedly enhances H2O2-scavenging system in leaves of halophyte Suaeda salsa. Physiol Plant 125:490–499
Caro T, Rowe Z, Berger J, Wholey P, Dobson A (2022) An inconvenient misconception: climate change is not the principal driver of biodiversity loss. Conserv Lett 15. https://doi.org/10.1111/conl.12868
Chandran H, Meena M, Barupal T, Sharma K (2020) Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep (Amst) 26:e00450. https://doi.org/10.1016/j.btre.2020.e00450
Chaura J, al Hassan M, Daniso E, Vicente O, Boscaiu M (2015) Comparative analysis of the antioxidant response to salt stress in Inula crithmoides and Dittrichia viscosa. Bull Univ Agric Sci Vet Med Cluj-Napoca Hortic 72:11490
Chele KH, Tinte MM, Piater LA, Dubery IA, Tugizimana F (2021) Soil salinity, a Serious Environmental issue and plant responses: a Metabolomics Perspective. Metabolites 11:724
Chiappero J, del Cappellari R, Palermo L, Giordano TB, Khan W, Banchio N E (2021) Antioxidant status of medicinal and aromatic plants under the influence of growth-promoting rhizobacteria and osmotic stress. Ind Crops Prod 167:113541. https://doi.org/10.1016/j.indcrop.2021.113541
Clapham AR, Tutin TG, Warburg EF (1962) Flora of the British Isles. Cambridge University Press, New York, p 2
D’Agostino G, Badalamenti N, Franco P, Bruno M, Gallo G (2022) The chemical composition of the flowers essential oil of Inula crithmoides (Asteraceae) growing in aeolian islands, Sicily (Italy) and its biocide properties on microorganisms affecting historical art crafts. Nat Prod Res 36:2993–3001
Deriu A, Zanetti S, Sechi LA, Marongiu B, Piras A, Porcedda S, Tuveri E (2008) Antimicrobial activity of Inula helenium L. essential oil against Gram-positive and Gram-negative bacteria and Candida Spp. Int J Antimicrob Agents 31:588–590
Diaz-Vivancos P, Sanchez-Blanco MJ, Hernandez JA (2017) Plant responses to salt stress: adaptive mechanisms. Agronomy 7:18. https://doi.org/10.3390/agronomy7010018
dos Santos TB, Ribas AF, de Souza SGH, Budzinski IGF, Domingues DS (2022) Physiological responses to drought, salinity, and heat stress in plants: a review. Stresses 2:113–135
Egea I, Estrada Y, Faura C, Egea-Fernández JM, Bolarin MC, Flores FB (2023) Salt-tolerant alternative crops as sources of quality food to mitigate the negative impact of salinity on agricultural production. Front Plant Sci 14:1092885
eHALOPH (2023) Salt-tolerant plants. https://ehaloph.uc.pt/showplant/11358 (accessed 2 August 2023)
Fierascu C, Fierascu I, Baroi AM, Ortan A (2021) Selected aspects related to Medicinal and Aromatic Plants as alternative sources of Bioactive compounds. Int J Mol Sci 22:1521. https://doi.org/10.3390/ijms22041521
Hamdani F, Derridj A, Rogers HJ (2017) Diverse salinity responses in Crithmum maritimum tissues at different salinities over time. J Soil Sci Plant Nutr 17:716–734
Hashemipetroudi SH, Ahmadian G, Fatemi F (2022) Ion content, antioxidant enzyme activity and transcriptional response under salt stress and recovery condition in the halophyte grass Aeluropus littoralis. BMC Res Notes 15:201
Hawrylak-Nowak B, Dresler S, Stasińska-Jakubas M, Wójciak M, Sowa I, Matraszek-Gawron R (2021) NaCl-Induced Elicitation alters physiology and increases Accumulation of Phenolic compounds in Melissa officinalis L. Int J Mol Sci 22:6844. https://doi.org/10.3390/ijms22136844
Hepworth MR (2023) Proline fuels ILC3s to maintain gut health. Nat Metab 5:1848–1849. https://doi.org/10.1038/s42255-023-00895-8
Hernández V, Recio MC, Máñez S, Giner RM, Ríos JL (2007) Effects of naturally occurring dihydroflavonols from Inula viscosa on inflammation and enzymes involved in the arachidonic acid metabolism. Life Sci 81:480–488
Kebert M, Vuksanović V, Stefels J, Bojović M, Horák R, Kostić S, Kovačević B, Orlović S, Neri L, Magli M, Rapparini F (2022) Species-level differences in Osmoprotectants and Antioxidants Contribute to stress tolerance of Quercus robur L., and Q. Cerris L. Seedlings under Water Deficit and High temperatures. Plants 11:1744. https://doi.org/10.3390/plants11131744
Kogure K, Yamauchi I, Tokumura A, Kondou K, Tanaka N, Takaishi Y, Fukuzawa K (2004) Novel antioxidants isolated from plants of the genera Ferula, Inula, Prangos, and Rheum collected in Uzbekistan. Phytomedicine 11:645–651
Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology; plant cell membranes, vol 148. Academic: Cambridge, MA, USA;, pp 350–382
Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127:1781–1787
Manuja R, Sachdeva S, Jain A, Chaudhary J (2013) A Comprehensive Review on Biological activities of p-hydroxy benzoic acid and its derivatives. Int J Pharm Sci Rev Res 22(2):109–115 ISSN 0976–044X
Martins N, Gonçalves S, Palma T, Romano A (2011) The influence of low pH on in vitro growth and biochemical parameters of Plantago almogravensis and P. algarbiensis. Plant Cell Tissue Organ Cult 107:113–121
Maschler J, Bialic-Murphy L, Wan J, Andresen LC, Zohner CM, Reich PB, Lüscher A, Schneider MK, Müller C, Moser G, Dukes JS, Schmidt IK, Bilton MC, Zhu K, Crowther TW (2022) Links across ecological scales: Plant biomass responses to elevated CO2. Glob. Chang Biol 28:6115–6134
Mazza G, Fukumoto L, Delaquis P, Girard B, Ewert B (1999) Anthocyanins, phenolics, and color of cabernet franc, merlot, and pinot noir wines from British Columbia. J Agric Food Chem 47:4009–4017
Miao M, Xiang L (2020) Pharmacological action and potential targets of chlorogenic acid. Adv Pharmacol (San Diego Calif) 87:71–88. https://doi.org/10.1016/bs.apha.2019.12.002
Mukhopadhyay R, Sarkar B, Jat HS, Sharma PC, Bolan NS (2021) Soil salinity under climate change: challenges for sustainable agriculture and food security. J Environ Manage 280:111736
Nisar F, Gul B, Aziz I, Hameed A, Egan T (2021) Increasing salinity leads to differential growth and H2O2 homeostasis in plants produced from heteromorphic seeds of the succulent halophyte Arthrocnemum Indicum. Plant Physiol Biochem 166:225–234
Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, Yalcin B, Erdogan A, Yalcin IE, Cabi E, Kaya Y (2023) Production of secondary metabolites using tissue culture-based biotechnological applications. Front Plant Sci 14. https://doi.org/10.3389/fpls.2023.1132555
Palchetti MV, Reginato M, Llanes A, Hornbacher J, Papenbrock J, Barboza GE, Luna V, Cantero JJ (2021) New insights into the salt tolerance of the extreme halophytic species Lycium Humile (Lycieae, Solanaceae). Plant Physiol Biochem 163:166–177
Patriarca EJ, Cermola F, D’Aniello C, Fico A, Guardiola O, De Cesare D, Minchiotti G (2021) The multifaceted roles of Proline in Cell Behavior. Front Cell Dev Biol 9:728576. https://doi.org/10.3389/fcell.2021.728576
Pehlivan N, Saruhan Guler N (2019) Salt stress triggered changes in osmoregulation and antioxidants in herbaceous perennial Inula plants (Asteraceae). Alinteri J Agric Sci 34:39–46
Pirbalouti GA, Firoznezhad M, Craker L, Akbarzadeh M (2013) Essential oil compositions, antibacterial and antioxidant activities of various populations of Artemisia chamaemelifolia at two phenological stages. Rev Bras Farmacogn 23:861–869. https://doi.org/10.1590/S0102-695X2013000600002
Prinz P (2019) The role of dietary sugars in health: molecular composition or just calories? Eur J Clin Nutr 73:1216–1223. https://doi.org/10.1038/s41430-019-0407-z
Puente-Garza CA, Espinosa-Leal CA, García-Lara S (2021) Effects of saline elicitors on saponin production in Agave salmiana plants grown in vitro. Plant Physiol Biochem 162:476–482. https://doi.org/10.1016/j.plaphy.2021.03.017
Reginato MA, Turcios AE, Luna V, Papenbrock J (2019) Differential effects of NaCl and Na2SO4 on the halophyte Prosopis strombulifera are explained by different responses of photosynthesis and metabolism. Plant Physiol Biochem 141:306–314
Reshi ZA, Ahmad W, Lukatkin AS, Javed SB (2023) From nature to lab: a review of secondary metabolite biosynthetic pathways, environmental influences, and in vitro approaches. Metabolites 13:895
Rodrigues MJ, Soszynski A, Martins A, Rauter AP, Neng NR, Nogueira JMF, Varela J, Barreira L, Custódio L (2015) Unravelling the antioxidant potential and the phenolic composition of different anatomical organs of the marine halophyte Limonium algarvense. Ind Crop Prod 77:315–322
Rodrigues MJ, Castañeda-Loaiza V, Fernandes E, Custódio L (2023) A first approach for the micropropagation of the edible and medicinal halophyte Inula crithmoides L. Plants 12:2366
Saygi Ş, Konuklugil B, Kutsal O, Uzbay İT, Deniz G, Gören Z (2003) Assessment of therapeutic effect of Inula Heterolepsis Boiss in alcoholic rats. Phyther Res 17:683–687
Shahid MA, Sarkhosh A, Khan N, Balal RM, Ali S, Rossi L, Gómez C, Mattson N, Nasim W, Garcia-Sanchez F (2020) Insights into the physiological and biochemical impacts of salt stress on plant growth and development. Agronomy 2020(10):938
Shuyskaya EV, Rakhmankulova ZF, Toderich KN (2020) Role of proline and potassium in adaptation to salinity in different types of halophytes. In: Grigore MN (ed) Handbook of Halophytes. Springer, Cham
Slama I, Abdelly C, Bouchereau A, Flowers T, Savouré A (2015) Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Ann Bot 115:433–447
Smirnoff N, Arnaud D (2019) Hydrogen peroxide metabolism and functions in plants. New Phytol 221:1197–1214. https://doi.org/10.1111/nph.15488
Tessema N, Yadeta D, Kebede A, Ayele GT (2023) Soil and Irrigation Water Salinity, and its consequences for Agriculture in Ethiopia: a systematic review. Agriculture 13:109
Trendafilova A, Ivanova V, Rangelov M, Todorova M, Ozek G, Yur S, Ozek T, Aneva I, Veleva R, Moskova-Doumanova V, Doumanov J, Topouzova‐Hristova T (2020) Caffeoylquinic acids, cytotoxic, antioxidant, acetylcholinesterase and tyrosinase enzyme inhibitory activities of six Inula species from Bulgaria. Chem Biodivers 17:e2000051. https://doi.org/10.1002/cbdv.202000051
Tzortzakis N, Nicola S, Savvas D, Voogt W (2020) Soilless cultivation through an intensive crop production scheme. Management strategies challenges and future directions. Frontiers Media SA
Ullah A, Munir S, Badshah SL, Khan N, Ghani L, Poulson BG, Emwas AH, Jaremko M (2020) Important flavonoids and their role as a therapeutic Agent. Molecules 25(22):5243. https://doi.org/10.3390/molecules25225243
Velioglu YS, Mazza G, Gao L, Oomah BD (1998) Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J Agric Food Chem 46:4113–4117
Zurayk RA, Baalbaki R (1996) Inula crithmoides: a candidate plant for saline agriculture. Arid L Res Manag 10:213–223
Funding
Open access funding provided by FCT|FCCN (b-on). This study received Portuguese national funds from FCT—Foundation for Science and Technology through projects UIDB/04326/2020 (DOI:https://doi.org/10.54499/UIDB/04326/2020), UIDP/04326/2020 (DOI:https://doi.org/10.54499/UIDP/04326/2020), LA/P/0101/2020 (DOI:https://doi.org/10.54499/LA/P/0101/2020), PIDDAC - UIDB/00100/2020 and UIDP/00100/2020, and LA/P/0056/2020 (IMS associated laboratory). Maria João Rodrigues was supported by the FCT program contract (UIDP/04326/2020), and Luísa Custódio by the FCT Scientific Employment Stimulus (CEECIND/00425/2017).
Open access funding provided by FCT|FCCN (b-on).
Author information
Authors and Affiliations
Contributions
Luísa Custódio: Conceptualization, formal analysis, investigation, resources, writing–review and editing, project administration, funding acquisition. Nuno Neng: methodology, formal analysis, writing– review and editing. Maria João Rodrigues: Conceptualization, methodology, formal analysis, investigation, writing– original draft preparation, review and editing. All authors have read and agreed to the published version of the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Additional information
Communicated by Ali R. Alan.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 changes were made. 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/4.0/.
About this article
Cite this article
Rodrigues, M.J., Neng, N. & Custódio, L. NaCl elicitation enhances metabolite accumulation and stress resilience in Inula crithmoides L. shoot cultures: implications for its nutritional and medicinal value. Plant Cell Tiss Organ Cult 157, 17 (2024). https://doi.org/10.1007/s11240-024-02750-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11240-024-02750-4