Influence of Salinity on In Vitro Production of Terpene: A Review

Abstract

Terpenes are the largest group of plant secondary metabolites with many biological activities, such as anticancer, antimicrobial, anti-inflammatory, antifungal, and antiviral. They are natural plant products frequently used in many sectors, such as medicine, agriculture, and perfumery. Various biotechnological strategies have been developed to increase terpene production and variety in plants. Among these approaches, using stimulants that induce in vitro accumulation of plant secondary metabolites, such as elicitor, is one of the best alternatives. Successful effects of salt (NaCl), an abiotic elicitor, on terpene production in different plant species have been reported. This technique remains relevant as a promising approach to the yet unknown chemistry of many plant species. Therefore, this review aims to appraise the literature available for using NaCl stress as an elicitor in in vitro cultures to increase terpene compounds in plants.

1. Introduction

Plants are a valuable source of various secondary metabolites used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives [1]. Plant secondary metabolites (PSM) are low molecular weight organic compounds that do not directly affect plant growth and development but have essential roles as a defensive tool in interacting with the environment and adapting to environmental conditions. Under natural conditions, many PSMs accumulate in different parts of plants (vacuoles, specialized glands, trichomes, and sometimes only at certain developmental stages) to provide functional flexibility under the influence of environmental factors without affecting cellular and physiological developmental pathways [2]. Since these compounds have many properties, such as antioxidant, antimalarial, antifungal, antimicrobial, and antiviral, they are essential in protecting the plant’s defense system due to their toxicity. Thus, removing other microbes and herbivores protects them from all kinds of pathogens [3]. PSMs are diverse and numerous chemical compounds derived from primary metabolic pathways by the plant cell. There are over 100,000 known PSMs in the plant kingdom. These compounds are divided into three major classes according to their chemical structures: terpenes, nitrogen-containing compounds (e.g., alkaloids and glucosinolates), and phenolic compounds (e.g., phenylpropanoids and flavonoids) [4].

Terpenes are the largest and most structurally diverse group of natural products, with more than 80,000 characterized compounds [5, 6]. In addition to being a pigment, flavoring and solvent, terpenes have many functions, such as medical thermoprotectant and signal transduction processes. Although there are many types and varieties, it is impressive that different organisms use terpenes for common purposes. Many living organisms, such as microorganisms, fungi, and plants, are protected from abiotic and biotic stresses thanks to their synthesized terpenes [7]. Terpenes are aromatic metabolites found in plants and can improve plants’ adaptation to the environment. Some terpenes are of enormous value to humanity due to their application in medicine, industry, and agriculture. To date, 52 antimicrobial terpenes have been identified, including carvacrol, thymol, menthol, geraniol, carnosic acid, quercetin, and allicin [8, 9]. Other terpenes, such as beta-myrcene limonene, pinene, and caryophyllene, may be safe and cost-effective alternatives for treating malaria in the pharmaceutical industry because they have antiplasmodial potential [10, 11]. Recent studies suggest that another terpene, Tanshinone IIA, can prevent the occurrence of atherosclerosis and damage and hypertrophy of the heart [12]. Perillyl alcohol is a monocyclic monoterpene and is of great interest due to its potent antitumor activity.

On the other hand, geraniol has been found to have therapeutic effects on cancer diseases, such as lung, colon, prostate, pancreas, and liver. Artemisinin and its derivatives have been reported to affect tumors significantly and have specific inhibitory effects at low costs [13]. However, terpenes are challenging to produce in large quantities due to their complex chemical structure and low content [14]. Plant production in vitro conditions is preferred because it allows the production of plant-specific metabolites using elicitor and precursor compounds and even the increase in the number of metabolites and the synthesis of new metabolites. Elicitors can be defined as a substance that, when delivered to a living cell system in a small concentration, initiates or increases the biosynthesis of specific compounds. Different types of stress used as elicitors may promote or inhibit terpene production [15]. Abiotic elicitors, such as salinity, UV light, temperature, pH, and heavy metals, can stimulate the accumulation of terpenes [16].

Salinity is one environmental factor limiting growth, development, and productivity among the abiotic stress variety. Under salinity conditions, terpenes protect cells from ion-induced oxidative damage [17]. It can also increase the tolerance of biological activity of some plant species, especially in vitro conditions. This trend is noteworthy because of the interesting biological properties of terpenes. This approach, which leads to the overproduction of terpenes, is highly desirable, especially in some medicinal plants. Plant production of terpenes against biotic and abiotic stress has been widely studied [18], and in some studies, it has been proven that salinity causes the accumulation of terpenes [16, 19, 20].

Recently, various strategies have been developed to synthesize terpenes, such as optimization of culture media, elicitation, use of precursors, bioreactor cultures, metabolic engineering, immobilization, and biotransformation methods [1]. This section focuses on the effect of salinity on terpene production in medicinal plants grown in vitro conditions.

2. Biosynthesis and classifications of plant terpenes

The name “terpene” is derived from the old French word “turpentine” and means “resin” [5]. Plants use two pathways to produce terpenes: the plastidial 2-C-methylD-erythritol-4-phosphate (MEP) pathway and the acetyl-CoA-linked cytosolic mevalonate (MVA) pathway. One of the basic terpene biosynthesis building blocks (C5 isoprene unit) is isopentyl pyrophosphate (IPP), and the other is allylic isomer dimethylallyl pyrophosphate (DMAPP). DMAPP and IPP building blocks combine to form mono-, di-, tri-, tetra-, and polyterpenes with higher molecular weights [21]. Terpenes are volatile unsaturated hydrocarbons with various structural properties [22]. They are commonly found in the leaves, flowers, stems and roots of higher plants, aromatic medicinal plants, citrus, conifers, and eucalyptus species.

The development of chromatographic and spectroscopic methodologies has accelerated the discovery of terpenes and terpenoids [18]. Terpenes and terpenoids are terms that are often used interchangeably. But terpenes undergo oxygenation, hydrogenation, or dehydrogenation to form terpenoids. Terpenes, such as pinene, myrcene, limonene, terpinene, and p-cymene, are compounds with simple hydrocarbon structures. However, terpenoids are a class of modified terpenes with different functional groups and oxidized methyl groups that have been moved or removed [23].

Terpene compounds are classified according to the number of isoprene units they contain. The chemical formula of terpenes is (C5)n. The n, in this formula, indicates the number of isoprenes present in the compound. The number of isoprene units forms the groups that provide the structural diversity of terpenes [14]. Accordingly, terpenes, as shown in Figure 1, are hemiterpenes (C5 isoprene), monoterpenes (C10 limonene, menthol, etc.), sesquiterpenes (C15 atractylone, caryophyllene, etc.), diterpenes (C20 taxol, etc.), sesterpenes (C25 disidiolide), triterpenes (C30 masticadienolic acid, ursolic acid, etc.), tetraterpene (C40, zeaxanthin, carotene, etc.) and polyterpenes (C˃40 resin, etc.)

Figure 1.

Classification of terpenes.

3. Production of terpenes via plant cell tissue and organ culture technics

Since terpenes are pharmaceutical compounds with important biological activities, studies on producing these compounds in plants have accelerated in the last 20 years. Plants generally produce low concentrations of terpene in their tissues, with terpene concentration less than 2–3% of a plant’s total dry weight. In particular, variation in terpene distribution caused by biotic, abiotic, and seasonal stimuli may vary depending on the plant’s chemotype, suggesting that different plants may respond to genetically different stimuli with various terpene syntheses. This situation may differ even between species and individuals [15].

Due to these molecules’ complexity and extreme metabolic modifications, their chemical synthesis is inherently tricky, expensive, and relatively low-yield. By 2050, the need for food is expected to double its current level due to overpopulation, and therefore the use of PSMs will increase in the coming decades to meet this demand [24]. With the increase in the world population, modern technologies developed to meet the demand for PSMs and to overcome possible negative situations have begun to be used. Today, many biotechnological techniques such as plant cell and tissue cultures (shoot culture, callus culture, suspension culture, hairy root culture, plant cell immobilization, and bioreactors) and genetic engineering applications are widely used for terpene production [25]. Many other valuable pharmaceutical terpene compounds, such as ginsenosides in Panax ginseng [26, 27], terpenoid indole alkaloids (TIAs) in Catharanthus roseus [28, 29], and tanshinones in Salvia miltiorrhiza [30, 31], can be produced in high quantities through the shoot, callus, and cell suspension cultures technics. Some studies conducted to increase the production levels of some terpenes and produce them on an industrial scale have focused on bioreactor systems [32]. It has been reported that Rhizoma zedoariae cell suspension cultures provide cell proliferation and accumulation of β-element in the bioreactor [33]. Considering that secondary metabolite production may be higher in differentiated tissues, extensive research has also been conducted on hairy root cultures transformed with Agrobacterium sp. as an alternative research tool. Extensive research has also been carried out on hairy root cultures transformed with the Agrobacterium rhizogenes-mediated transformation method, which reduces the risk of somaclonal variation and provides rapid shoot regeneration, has yielded successful results for the production of terpenes. For example, although the concentration of triterpene detected in the leaves of Centella asiatica plants was >2 times higher than in the petiole, the amount of triterpene in the petiole-derived hairy root cultures was 1.4 times higher than in the leaf-derived hairy root cultures. In addition, it was determined that the amount of terpene obtained from leaf and petiole root cultures was higher than that of adventitious roots [34]. Advances in immobilization techniques contribute to a significant increase in the production of high value-added pharmaceutical compounds. Plumbago rosea, in which cell cultures were immobilized with 10 mM calcium alginate, plumbagin production was doubled compared to control cells [35].

Furthermore, understanding the function of genes involved in terpene production may lead to discovering new compounds or metabolic pathways that can reveal optimal properties. Accordingly, increased terpene emission was observed in Nicotiana tabacum plants’ leaves after adding monoterpene synthase genes [36]. In recent years, overproduction, co-overexpression, gene silencing, and genome editing techniques are among the current approaches used for synthesizing different terpene compounds through developments in metabolic engineering.

3.1 Using elicitors for terpene production

It has been shown that certain stress conditions can increase or inhibit terpene production and change the emission pattern and/or amount. Stress conditions generally affect both constitutive and induced terpene emission rates [15]. Treatment of in vitro cultures with biotic and abiotic elicitors has been seen as essential to increase the production of desired products [37]. This technique has become the subject of an increasing number of studies today. The elicitor triggers signal transduction and generates secondary signals, stimulating regulatory proteins (transcription factors) that coordinate the expression of biosynthetic genes [38]. Elicitors can be classified as biotic and abiotic elicitors depending on their source and effect on plants (Figure 2).

Figure 2.

Classification of elicitors used in secondary metabolite production.

Elicitor type, dose, and application time are the main factors in terpene production [39]. Apart from NaCl, an abiotic elicitor source, there is a lot of literature on methyl jasmonate (MeJA) and salicylic acid (SA). MeJA is a plant growth regulator belonging to the jasmonate family. Extensive research has been conducted on phytohormones, as they can influence many physiological and metabolic processes in plants. Given the great diversity of these compounds with specific biological functions, they are also referred to as biostimulants [24]. Biostimulants are essential as a signaling molecule that mediates intra- and inter-plant communication and modulates plant defense responses, including antioxidant systems. Farag et al. [37] studied the effects of six different biotic and abiotic elicitors, including MeJA, SA, ZnCl₂, glutathione, and β-glucan (BG; fungal stimulant) wounding, on terpene accumulation in soft coral Sarcophyton ehrenbergi. Based on the elicitation process, it has been discovered that the mere inclusion of 0.1 mM SA and 1.0 mM ZnCl₂ resulted in a remarkable increase in the levels of sarcophytolide I by 132 and 17 times, respectively, in just 48 hours.

Again, in Mentha x piperita plants, where SA or MeJA was applied exogenously, a significant increase in menthol, pulegone, linalool, limonene, and menthone concentrations was achieved with the application of 2.0 mM MeJA [40]. Depending on the dose and time, significant increases in the accumulation of TIAs (eburenin, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine) occurred in hairy root cultures of Rhazya stricta, elicited by the application of different concentrations of MeJA [38]. The roots of Salvia miltiorrhiza, a plant rich in tanshinones, an essential active diterpene, are used medicinally for cardiovascular diseases and inflammation. In a study on Salvia miltiorrhiza, with 0.2% (v/v) bacterial inoculum as a biotic elicitor, the total tanshinone content of the roots increased more than 12-fold [41]. Li et al. [42] reported that selected genes in the tanshinone biosynthetic pathway of Ag (+), MeJA, and yeast extract were significantly upregulated in S. castanea f tomentosa Stib hairy root cultures. Yeast extracts increased the expression level of isopentenyl diphosphate isomerase 13.9-fold at 12 hours. It was determined that yeast extracts increased the expression level of isopentenyl diphosphate isomerase 13.9-fold in 12 h. In contrast, the contents of tanshinone IIA were increased by 1.8-fold and 1.99-fold compared to the control with Ag (+) and MeJA elicitation, respectively.

The addition of the biotic elicitor Anabaena sp. (265 cells/mL) to cell suspension cultures of Azadirachta indica triggered the synthesis of the triterpene azadirachtin (0.32 g/μL) [43]. UV-B caused a significant increase in lochnericine concentrations in hairy root cultures of C. roseus. It has also been shown that increasing the exposure time to UV-B up to 20 minutes causes significant increases in lochnericine, serpentine, and ajmalicine and a decrease in horhammericine [44].

3.2 Effect of salt as an elicitor

NaCl is one of the most important abiotic stress factors that cause different changes in plants’ morphological, physiological, and biochemical responses, limit their growth and development, and consequently negatively affect total crop production [45, 46]. In the early stages of salinity-induced stress, the ability of roots to absorb water is highly affected and reduced [47]. Higher NaCl concentrations pose a significant threat to the plant by inhibiting physiological processes through osmotic stress, nutrient imbalance, ionic toxicity, and oxidative stress [48]. Oxidative stress is a process in which reactive oxygen species occur as a result of excessive accumulation of sodium (Na⁺) and chloride (Cl⁻) in plant tissues [49]. After exposure to excess NaCl, plants first sense the potential source of stress and then activate a multifaceted response that includes a signaling network and the synthesis of several compounds that help reduce the effects of high salinity and maintain cellular homeostasis. At this point, secondary metabolites play critical roles in plant adaptation to NaCl stress [4]. Depending on the salinity levels that plants are exposed to in vitro, the type and amount of these metabolites they synthesize to survive may vary. Thus, plants can often produce species-specific secondary metabolites in shoots, roots, leaves, etc., at different stages of plant development [10]. Some studies have reported that terpenes exhibit antioxidant activities and thus their function in overcoming oxidative stress [4]. During NaCl stress, terpenes can reduce the consequences of oxidative stress either by reacting directly with intercellular oxidants or altering the signaling of reactive oxygen species. Various terpenes can minimize NaCl stress in different plant species by providing membrane stabilization and direct antioxidant effects. In addition to their antioxidant effects, isoprenes and monoterpenes can react rapidly with ozone and reduce the toxicity caused by NaCl stress. Amphipathic isoprene can prevent membrane and protein degradation by improving hydrophobic interactions between membrane proteins and lipids [20].

Although significant progress has been made on the accumulation mechanisms of terpene compounds in medical plants via NaCl elicitation [50], studies are still ongoing on terpene compounds whose chemistry and role are still unknown. To the present day, especially in recent years, research on accumulating these compounds has been presented in Table 1. Terpenes and terpenoids are the main components of volatile oils of medical and aromatic plants; it has a variety of chemical compositions ranging from monoterpenes, sesquiterpenes, triterpenes, alcohols, ethers, aldehydes, esters, and ketones [60, 61]. There are 49 different terpenes in the leaves and resins of the male and female mastic tree (Pistacia lentiscus) [62]. Tilkat et al. [16] investigated the effects of different NaCl concentrations applied to juvenile shoots of P. lentiscus in vitro on the accumulation of anticancer triterpenes, such as ursonic, moronic, oleanolic, masticadienolic, oleanolic, and ursolic acids. Accordingly, while 50 mM NaCl elicitation increased the amount of ursonic acid 2.16 times, 25 mM NaCl elicitation increased it 3.71 times. In addition, masticadienolic acid, which is not found in the control group, was induced in vitro leaves via elicitated 100 mM NaCl; and ursolic acid was induced in vitro leaves and stems by 25, 50, and 100 mM NaCl. It is known that C. roseus, which contains 200 different TIAs, is also a source of vinblastine and vincristine, which have high economic and medical importance. However, since it has been observed that these compounds accumulate in insufficient amounts in the natural growing environment of this plant, it has been tried to increase the production of these compounds with various strategies [63]. Studies have shown that TIA accumulation can be significantly altered through stress mechanisms such as NaCl stress [53, 64] or studies such as overexpression of these genes [65, 66]. In a survey conducted by Fatima et al. [53] using different levels of NaCl concentrations (0, 25, 50, 75, 100, and 125 mM) as an elicitor in various embryogenic tissues of C. roseus grown in vitro, the content of vinblastine and vincristine was increased. It was noted that the maximum accumulation of vinblastine and vincristine was obtained by elicitation with 25 mM NaCl. Thymol is a natural volatile monoterpene phenol and the main active ingredient of essential oil obtained from Thymus vulgaris, Ocimum gratissimum, Carum copticum, and Nigella sativa. Black seed (N. sativa L.) is a medicinal plant used to treat many diseases since ancient times. It has been reported that 250 mM NaCl elicitation in N. sativa calli leads to high thymol accumulation [19]. Diterpene steviol glycosides obtained from the leaves of the Stevia rebaudiana plant are calorie-free natural sweeteners. NaCl and Na₂CO₃ salts were investigated on steviol glycoside production in callus and suspension cultures. In calli, steviol glycosides increased from 0.27% (control) to 1.43% and 1.57%, respectively, with 0.10% NaCl and 0.025% Na₂CO₃. However, in suspension cultures, the same concentrations of NaCl and Na₂CO₃ increased the steviol glycoside content from 1.36 (control) at day 10 to 2.61% and 5.14%, respectively [52]. In addition to the studies on increasing the terpene compounds with a single type of elicitation in vitro, more than one type of elicitation has been tried in many plants, and successful results have been obtained. In this context, it has been reported that elicitors, such as NaCl, chitosan, MeJA, SA, and jasmonic acid (JA), are widely used in combination or separately for terpene production [25, 67, 68, 69]. Razavizadeh et al. (2020) investigated the potential effects of terpene accumulation by examining the combined effect of chitosan and NaCl on C. copticum shoot and callus cultures. They reported that combined elicitation led to an increased accumulation of thymol and p-cymene in both shoots and calli [25]. In another study on the production of vincristine and vinblastine from TIAs in C. roseus calluses of both osmotic and NaCl stresses, 75 mM NaCl elicitation significantly increased the content of both bioactive compounds [51]. In another combined elicitation study, the effect of NaCl and phenylalanine on rosmarinic acid production in vitro cultures of Mentha longifolia was investigated. Five different phenylalanine concentrations (0, 0.5, 5, 10, and 15 mg/l) and four different NaCl concentrations (0, 2000, 4000, and 6000 mg/l) were tested. Shoot tips were more efficient in rosmarinic acid production than callus cultures. In addition, low-concentration NaCl elicitation led to a high accumulation of rosmarinic acid [70].

Plant species	Elicitor type	Culture Type	Increased Terpene Compounds	References
Carum copticum	Chitosan + NaCl	Shoot and callus culture	Thymol and p-cymene	[25]
Catharanthus roseus	75 mM NaCl	Callus culture	Vincristine and vinblastine	[51]
Mentha longifolia	4000/ 6000 mg/l for callus, 2000 mg/l for the shoots	Shoot and callus culture	Rosmarinic acid	[51]
Stevia rebaudiana	NaCl and Na2CO3	Callus and suspension cultures	Steviol glycosides	[52]
Nigella sativa	250 mM NaCl	Callus culture	Thymol	[19]
Catharanthus roseus	25 mM NaCl	Shoot culture	Vinblastine and vincristine	[53]
Pistacia lentiscus	25 and 50 mM NaCl	Shoot culture	Ursonic acid	[16]
Pistacia lentiscus	100 mM NaCl	Shoot culture	Masticadienolic acid	[16]
Rauwolfia serpentina	100 mM NaCl	hairy root culture	ajmalicine and solasodine	[54]
Datura stramonium	1 and 2 g/l NaCl	Hairy root culture	Hyoscyamine	[55]
Panax ginseng	0.1% NaCl	Hairy root culture	Saponin	[56]
Withania somnifera	50 mM NaCl	Callus culture	Withanolide	[57]
Glycyrrhiza uralensis	0.3%, 0.6%, 1.2%, 1.8%, and 2.4%	Shoot culture	Saponin	[58]
Stevia rebaudiana	150 mM NaCl	Shoot culture	Steviol glycosides	[59]

Table 1.

Effect of salt stress on the accumulation of some terpene compounds in plants.

As discussed above, salt, an abiotic stress factor, actually increases the synthesis of terpenes in plants and is an essential factor for plants to combat the negativities they encounter under natural conditions (Figure 3). In addition, it is possible to increase the production of terpenes with economic value by plants through salt elicitation carried out in vitro. From this point of view, it can be seen that salt, which negatively affects agricultural production, is an essential factor that triggers different metabolic pathways in plants and contributes to the production of many other secondary metabolites, including terpenes, that are beneficial for humanity. Terpenes, in particular, and all secondary metabolites in general, which plants synthesize to make their own life easier and more efficient under salinity, form active compounds used in agricultural and industrial fields and necessary pharmaceutical raw materials for human beings.

Figure 3.

Terpenes production under NaCl stress [71, 72, 73].

4. Future prospects

Many studies have expressed and emphasized elicitation’s effect on gene expression in medicinal plants, including salt treatments. With plant tissue culture and genetic engineering applications, regulating the biosynthesis pathways of commercially valuable, potent, effective, and specific terpenes can be modified and improved. By revealing the effects of these applications holistically, it will be possible to produce terpenes, which are especially important in medical terms, on an industrial scale, and at an affordable cost.

Overall, since high salt stress affects the total amount and metabolite profile of secondary metabolites produced, it is clear that salinity regulation can be a promising way to obtain new compounds from plants to produce active PSMs such as raw pharmaceutical materials. In addition, considering the changing climatic conditions and soil profile, it is vital to understand the plant biosynthetic pathways that lead to the production of terpenes, which have a wide range of economic value through salt stress, and ultimately determine how to manipulate these pathways. Accordingly, applying transcriptome and metabolome data to examine gene-metabolite networks at both regulatory and catalytic levels for secondary metabolism in plants will also be an essential approach to reveal the PSM production profile. Additionally, modulation of plant metabolic pathways, leading to target metabolite production through the development of efficient modeling strategies and optimization of growth conditions may help improve the production of important bioactive molecules through bioreactors.