Spearmint is a perennial herbaceous medicinal plant from the Lamiaceae family. The plants that belong to this family and genus are a rich source of secondary metabolites like terpenoids and polyphenols which have strong biological effects (Gulluce et al., 2007; Bimakr et al., 2011; Mahendran and Rahman, 2020). Therefore, such plants are often used as medicinal and aromatic plants or for culinary purposes.

Mint has been used by humans to disinfect homes after infectious disease or death for ages, and this way it came to represent cleanliness. Studies confirmed that the leaves of spearmint (Mentha spicata L. var. crispa) have anti-fungal, anti-microbial, anti-inflammatory, anti-tumor, and antioxidant activities (Mata et al., 2007; Sokovi et al., 2009; Lixandru et al., 2010; Guimarães et al., 2011; Chrysargyris et al., 2017; Salehi et al., 2018). Furthermore, its essential oil used to be an official drug in the Hungarian Pharmacopeia under the name Aetheroleum menthae crispae. In folk medicine, spearmint has been attributed a variety of therapeutic properties, including the prevention of chemotherapy-induced nausea and vomiting (CINV) and the treatment of respiratory and digestive troubles (Karousou et al., 2007; Cakilcioglu et al., 2011).

Spearmint essential oil is widely used in several products as an aromatic or fragrance agent such as medications and sweets, as well as chewing gum, toothpaste, and mouthwashes, and is an important compound of ecological pesticides and antimicrobial agents (Chrysargyris et al., 2017). Spearmint leaves and rarely essential oils are used to flavor teas, stews, and soups with the dried leaves used to preserve season meat such as lamb, fish, chicken, and vegetables such as rice (Mata et al., 2007; Igoumenidis et al., 2016; Salehi et al., 2018). Spearmint is also used as an ingredient in a variety of mixed drinks, including the mojito, and the mint julep.

The major constituent of spearmint essential oil is carvone, which provides spearmint its characteristic scent (Kokkini et al., 1995). In addition, spearmint essential oil also contains noteworthy concentrations of limonene and 1,8-cineole (Cirlini et al., 2016; Chrysargyris et al., 2017). Carvone is reported to inhibit bacterial growth and has fungicide and insect repellent properties (Oosterhaven et al., 1995).

Mint essential oil is among the 10 most commercialized essential oils. Moreover, spearmint is one of the most valuable flavors listed after vanilla and citrus (Chrysargyris et al., 2017). Therefore, spearmint is widely cultivated in all parts of the world and there is a growing demand for spearmint cultivation. The decrease in quality of arable land due to salinization, accumulation of heavy metals and organic pollutants, projected increase in temperature and erratic precipitation patterns represent a huge challenge to mankind. High soil salinity and high salt concentration in water available for irrigation effect and limit agricultural production in several areas worldwide including countries in the Mediterranean Region and the Sahara, e.g., Morocco and Algeria (Sarmoum et al., 2019), where mint production for pharmaceutical and food purposes is traditionally and economically important (El Hassani, 2020). Previous literature data indicated that wild mint is sensitive or moderately sensitive to salinity (Warrance et al., 2002). Peppermint is also highly sensitive to soil salinity, as it cannot maintain normal growth, biomass, and essential oil production in soils containing more than 75 mM NaCl (0.3%). Therefore, the breeding of salinity tolerant peppermint cultivars is important as well as understanding salinity stress induced cellular responses (Li et al., 2015). The effect of salt stress on photosynthesis and essential oil composition were studied in peppermint and other mint species (Aziz et al., 2008; Oueslati et al., 2010; Kasrati et al., 2014; Li et al., 2015; Yu et al., 2015). However, only one paper studied spearmint in the said respect (Chrysargyris et al., 2019) but it did not use a local, Moroccan cultivar. Data showed decreased growth, chlorophyll content, and altered essential oil profile under salt stress, but no 77 K fluorescence emission analyses and ultrastructural observations were carried out in these studies. Therefore, in this work, we wanted to investigate whether spearmint could be cultivated under salt stress conditions and how its photosynthetic tissues and especially the organization and structure of its photosynthetic apparatus were affected by salinity.

Soil salinity is expected to affect the world more severely and extensively in the coming years. Increasing salinity in water resources is also associated with increasing salinity in the soil (McFarlane and Williamson, 2002). High soil salinity influences plant growth and productivity in a complex manner. It can trigger both hyperionic and hyperosmotic stress in plants (Hasegawa et al., 2000; Pagter et al., 2009; Isayenkov and Maathuis, 2019; Wani et al., 2020). Under salt stress conditions, photosynthetic parameters have been shown to be suppressed in a variety of plant species (James et al., 2002, 2006; Chaves et al., 2009; Bose et al., 2017). However, there are contradictions in the literature about the effect of salt stress on plastid structure. In many studies, the swelling of the thylakoid lumen and disorganization of the thylakoid system in chloroplasts was observed under salt stress (Salama et al., 1994; Yamane et al., 2008, 2012; Omoto et al., 2010; Evelin et al., 2013; Gao et al., 2015a; Goussi et al., 2018). Salt stress has an effect on chloroplast and thylakoid ion homeostasis (Bose et al., 2017), as well as on the lipid bilayer membrane, its composition and stability. Furthermore, by increasing lipid peroxidation, salt stress can cause membrane damage (Miller et al., 2010; Yamane et al., 2012; Suo et al., 2017). Increased number and size of plastoglobules have often been related to various stresses and were also observed under salt stress in susceptible species (Hernández et al., 1995; Solymosi and Bertrand, 2012; Acosta-Motos et al., 2017). It is assumed that plastoglobules are related to salt tolerance or may be related to degradation or aging processes during stress. Starch accumulation may be also affected by salinity stress (de Morais et al., 2019).

The above ultrastructural observations, especially those reporting thylakoid swelling, were reported in leaf samples prepared using conventional chemical fixation (using aqueous, ionic buffers), dehydration, embedding, and subsequent ultrathin sectioning, contrasting, and analysis by transmission electron microscopy (TEM). Small-angle neutron scattering (SANS) is another non-invasive method that also provides information about the ultrastructure of thylakoid membranes, i.e., their repeat distance, RD, and periodic order, averaged over the entire sample volume in the neutron beam in vitro (Nagy et al., 2013). During the sample preparation for electron microscopy, fixation or other artifacts related to osmotic shock or imbalance may occur.

Therefore, we wanted to develop model plant systems using living, intact, or almost intact plants in which the effects of salt stress on chloroplast structure could be compared critically using SANS and TEM. In this work, our objective was to check whether the effects of salt stress can be studied and compared in intact plants with these two methods yielding novel ultrastructural data. In addition, we aimed to investigate salinity stress in spearmint and compare the effects of ionic and osmotic components of salt stress on the essential oil composition, photosynthetic activity, and chloroplast structure in seedlings treated with various NaCl concentrations through the root or in a model system through the shoot.

Salinity affects an increasing amount of arable land, this way areas suitable for agricultural production are continuously diminishing. Therefore, we primarily wanted to investigate whether spearmint could be cultivated and propagated under low concentration salt stress conditions.

Salinity is complex stress, which under natural conditions is often combined with high temperature and drought stress resulting in increased salt concentrations in the soil water. According to recent estimations, salinity and sodicity affect one billion hectares worldwide, which represent 10% of the total arable lands on Earth. As a consequence, the annual economic losses associated with salinity stress can be as much as 27 billion USD (Shahid et al., 2018). Agriculture already faces challenges to produce enough staple food and feed crops to nourish the growing human population. Therefore, the increasing demand for the production of herbs and aromatic plants may not be met. In this respect, a better understanding of the physiological, cellular, and molecular mechanisms of spearmint under salinity stress may yield useful information for tolerance screening and may also provide a theoretical basis for further developments, potential future breeding, cultivation, and utilization of spearmint in saline areas.

When looking at literature data before starting these experiments in January 2019, we found several reports on salinity stress in peppermint (Li et al., 2015) or other mint species (Aziz et al., 2008; Oueslati et al., 2010; Yu et al., 2015), but almost no data were available about spearmint (Chrysargyris et al., 2019). In addition, literature data primarily focused on plant growth parameters (such as biomass), essential oil yield and composition, as well as the sometimes antioxidant response of the mint species under salt stress.

Therefore, one of the aims of this study was to better understand whether spearmint tolerates salinity stress and can be cultivated on saline soils. The results of the first experiment indicated that 50 mM NaCl stress inhibited adventitious root formation (Figure 1B), which under normal circumstances is an important means of vegetative reproduction, propagation, and regeneration in mint species (Li et al., 1999; Gomes et al., 2015; Salehi et al., 2018). Therefore, large-scale industrial cultivation of spearmint in soils with high salinity may be limited.

The second experiment outlined the obvious, but still highly important role of roots in protecting plants against salinity and especially osmotic stress. In freshly cut, i.e., rootless shoots, the drying induced by osmotic stress (PEG treatment) and salt stress (150 mM NaCl) was faster, ultrastructural damage were more pronounced (compare Figures 7, 12), and the disorganization of the chlorophyll-protein complexes of the photosynthetic apparatus proceeded faster than in rooted plants (Figure 8). Indeed, our data outlined that rooted plants better tolerate osmotic stress alone than the combined specific ionic and osmotic stress induced by salt treatment.

Concerning the potential aromatic, culinary, and medicinal uses, in this work, we compared the effect of salt and osmotic stress on the essential oil composition (Tables 1, 2). Essential oils of Lamiaceae plants are produced by their capitate and peltate glandular hairs, which are carbon heterotrophic and depend on the metabolites produced by the adjoining photosynthetic cells for a continuous supply of carbon precursors (Aziz et al., 2008; Böszörményi et al., 2020). Stress-induced disruption of normal metabolism, e.g., photosynthesis and secondary metabolite production, often results in reduced growth of plants (Aziz et al., 2008; Kasrati et al., 2014), altered leaf or glandular hair differentiation, and could be expected to interfere with essential oil biosynthesis resulting in lower essential oil quantity and altered composition (Aziz et al., 2008; Kasrati et al., 2014; Sarmoum et al., 2019). Our data showed a typical spearmint essential oil profile characterized by the major presence of carvone (between 44 and 68%) as described by other studies (Kokkini et al., 1995; Cirlini et al., 2016; Chrysargyris et al., 2017, 2019). In the present study, limonene represented 11–17% of the essential oil, but mentone was not detected. Other important constituents of the samples were epi-bicyclosesquiphellandrene, germacrene D and β-caryophyllene (Tables 1, 2). Variation in the composition of essential oils can be attributed to different factors such as temperature, humidity, climate, soil type as well as the cultivar studied, and in this respect spearmint essential oil profiles greatly vary in the literature (Sarmoum et al., 2019; Zekri et al., 2019), and also showed some variation within our experiments. Therefore, we tried to standardize our treatments, and used the same clone and parallels measured at the same period of the year in each experiment. However, it has to be noted that we observed slight seasonal variations within the essential oil composition and the chlorophyll contents (SPAD index values were not shown). Therefore, we decided to include and compare only data obtained in identical parallel experiments recorded in the same period of the year. This way, the control values, as well as the effect of the applied treatments, can be directly compared within each experiment but may not be compared among the different experiments.

Surprisingly, our data showed that the applied treatments induced no important alterations in the essential oil composition in any of the experimental settings. This is somewhat surprising, as literature data reported both quantitative and qualitative alterations in the essential oil (Chrysargyris et al., 2019). Based on our data we may speculate that the essential oil biosynthesis pathway of the “Moroccan” cultivar and clone used in our study may be less influenced by salt stress than other cultivars. It is also important to mention that we determined the essential oil composition in fully differentiated leaf pairs which had already well-developed glandular hairs containing essential oil, which is in contrast to literature data that either used whole plants for similar measurements including leaf primordia with developing glandular hairs (Aziz et al., 2008; Kasrati et al., 2014) or even flowers (Zekri et al., 2019). This might explain why we have not observed important changes in the essential oil composition. In our opinion, the statistically significant, but very small differences observed in some minor components, not specifically reported in the literature data before, may rather be related to decreased essential oil content (Aziz et al., 2008; Kasrati et al., 2014; Sarmoum et al., 2019) observed under stress conditions. Due to this decrease, some minor components go beyond the limits of the detection. This resulted in alterations in the relative distribution of the remaining detected components, with the strongest apparent effect observed in components present in very low amounts in the original sample.

Another major aim of our study was to improve our understanding of the potential effects of salinity and osmotic stress on chloroplast structure and activity in the leaves of intact, i.e., rooted plants. In this respect, our data showed that osmotic stress-induced a significant decrease in the chlorophyll content (SPAD index—Figure 9), in the RWC (Figure 11) and a slight, but significant decrease in photosynthetic activity (Figure 10) of the leaves, while the native organization of the chlorophyll-protein complexes (Figure 8) and chloroplast ultrastructure was almost unaffected (Figure 12) by the osmotic component of the salt stress.

Salinity has a negative impact on chlorophyll biosynthesis (Abdelkader et al., 2007) and also destabilizes pigment-protein complexes (Jaleel et al., 2008). In control and mildly stressed leaves, chlorophyll fluorescence emission bands corresponding to PSII core antenna (band maximum position at 685 and 695 nm) as well as to PSI light-harvesting complex (emission maximum at around 735 nm) were observed (Figures 5, 8), similarly to green leaves in the literature (Briantais et al., 1986). However, long-term, high concentration salt stress in the rootless plants, as well as the osmotic stress, had induced a strong decrease in the RWC values and alterations in the native chlorophyll-protein complexes, especially in the fluorescence of the PSI complex. Similarly to literature data, these changes were accompanied by decreased chlorophyll content (Li et al., 2015) and SPAD values (Choi and Chiang, 2015), while control SPAD values were similar to or slightly lower than data in the literature in mint species (Ronga et al., 2018; Yan et al., 2020). It must be noted that these values strongly vary among species, cultivars, developmental stages, as well as environmental conditions, e.g., light intensity. Therefore, the comparison was mostly possible using the same genotype and experimental settings as well as leaves of the same developmental stages. In dark-adapted leaves, the Qy dark values corresponded to the maximum light energy conversion efficiency of PSII. Qy dark values lower than 0.6 indicated serious disturbances of PSII activity as described in the literature data for other mint species under severe salinity stress (Li et al., 2015; Khalvandi et al., 2021).

We collected several pieces of information about the strong deleterious effect of salinity on the photosynthetic activity and chloroplast structure of spearmint. Our comparative investigations using isosmotic PEG solutions clearly outlined that (i) the loss of granum regularity (Figures 12, 13), (ii) increased electron-density of the stroma, (iii) and the appearance of the crystalline inclusions resembling electron-transparent spotted bodies, (iv) as well as the stronger decrease in physiological parameters (Qy values, SPAD index, RWC, organization of the chlorophyll-protein complexes; Figures 8–11), can be attributed to the specific ionic components of salt stress. These data are important since often in other crops, e.g., in fenugreek, the swelling of the thylakoid lumen and other ultrastructural alterations are attributed to the osmotic component of salinity (Evelin et al., 2013), while we had proved that it was not the case for spearmint plants used in this experiment. Similar spotted bodies sometimes defined as stroma centers have been described in several species under disturbances in ion homeostasis of the plastids (Solymosi and Bertrand, 2011, 2012). The loss of granum regularity observed by TEM (Figure 12) was also confirmed using for the first time SANS to analyze spearmint granum structure in vivo (Figure 13). Neutron scattering techniques are widely used for the characterization of the structure and dynamics of condensed matter. A key advantage of neutrons is their electric neutrality, allowing them to penetrate deeply into most materials, without affecting the mesoscopic organization of soft or liquid phase samples, such as biological membranes and tissues. Our data about the effect of abiotic stresses on the ultrastructural organization of grana indicated a substantial degree of flexibility in the membrane ultrastructure. In addition, they paved the way for further experiments to study the in vivo effects of combined stressors or different stresses (light or heat stress) on the organization and short- or long-term dynamic structural alterations of grana in spearmint. Also, the findings of this study outlined the clear biological limitations of experiments carried out on excised leaf segments or injured, e.g., freshly cut, plant organs.

Similarly to literature data, we used hydroponic-like salinity treatments, and concentrations, as well as treatment lengths, similar to those used by other authors (Gao et al., 2015b; Goussi et al., 2018; Chrysargyris et al., 2019). We are aware of the limitations of hydroponic cultures as well as of data obtained with freshly cut shoots. Pot experiments have lower reproducibility due to local variations and inhomogeneities in soil composition and structure, unequal salt accumulation in the pots, and a constantly increasing concentration of salt in the pots during treatment which make it difficult to precisely control salt concentrations. Due to extensive vegetative propagation of mint species by rhizomes, it is also highly difficult to grow mint in soil culture with a well-controlled root-to-shoot ratio. In addition, due to the above factors, deciphering the osmotic and specific ionic components of salt stress is not possible in pot experiments, because osmolarity cannot be controlled in them precisely. We observed a strong decrease in the chlorophyll content (SPAD index) already at relatively low concentration (25 mM NaCl) but longer treatments (2 weeks) (Figure 3). Similar observations have been described in other works (Chrysargyris et al., 2019). When the RWC values of the leaves decreased during various salinity stress treatments to one-third of their original values, i.e., 35–37%—Figures 2, 11; Supplementary Tables 1, 4), the activity of the photosynthetic apparatus was strongly disturbed (Figures 4, 10; Supplementary Tables 2, 3), and the relative chlorophyll contents significantly decreased (Figures 3, 9; Supplementary Tables 2, 3). Similar observations were made in maize under PEG and salinity treatment, which resulted in a decrease in the RWC, chlorophyll content, and decreased photosynthetic values (F_v/F_m) (Gao et al., 2015b). Cellular integrity was lost, but still, grana could be recognized in the TEM images (Figures 6B–D, 12C–E), and the essential oil composition of the leaves was not affected (Tables 1, 2). Indeed, it is important to note that chloroplast structure showed the swelling of the lumen only in case of the still greenish, but strongly wilted leaves treated for 2 weeks using 50 mM NaCl (Figure 6), while leaves or leaf regions that were brownish and fully dry did not exhibit swelling. The swelling of the thylakoid lumen (Salama et al., 1994; Yamane et al., 2008, 2012; Omoto et al., 2010; Evelin et al., 2013; Gao et al., 2015a; Goussi et al., 2018) is thought to be associated with oxidative stress and membrane damage (lipid peroxidation and the formation of different reactive oxygen species) (Miller et al., 2010; Oueslati et al., 2010; Yamane et al., 2012; Suo et al., 2017) and chloride influx from the stroma into the lumen (Bose et al., 2017). The observed alterations in plastoglobuli (increased number and size, changed electron-density; Figures 6, 12) may also reflect changes and potential degradation in the membrane components under stress (Hernández et al., 1995; Solymosi and Bertrand, 2012; Acosta-Motos et al., 2017). The presence of an electron-dense luminal substance (Keresztes and Sárvári, 2001) in spearmint thylakoids may be worth further investigations in order to understand its role in preserving granum structure in almost fully dried and highly stressed leaves (Figures 6, 7, 12). Similarly, it might be interesting to analyze whether the increased electron-density of the stroma under salt stress (Figures 6, 7, 12) may be accompanied by the accumulation of antioxidants or polyphenolic compounds (Oueslati et al., 2010; Chrysargyris et al., 2017, 2019).

On the other hand, our studies also outlined that at low concentrations (50 mM NaCl and especially below) the native organization of the chlorophyll-protein complexes of the photosynthetic apparatus was well retained (Figure 5) and plastid ultrastructure was also well preserved even despite moderately decreased RWC, photosynthetic activity and relative Chl contents (Figures 2–4). This is in agreement with no important ultrastructural changes being observed in fenugreek at 50 mM NaCl treatment, while larger alterations including swelling appearing at 100 and 200 mM NaCl (Evelin et al., 2013). It is also important to note that relatively low concentration (50 mM), but longer (2-week-long) stress applied to freshly cut shoots induced a stronger decrease in the photosynthetic activity (compare Qy values in Figures 4, 10; Supplementary Tables 2, 3) than high concentration (150 mM), shorter (1-week-long) stress applied on rooted plants. This outlines the important role of roots in the salt tolerance mechanism.

Our data showed that the “Moroccan” spearmint cultivar used in this work could tolerate cultivation in areas exposed to moderate osmotic and salt stress for relatively short periods (1 and 2 weeks). Furthermore, these stressors did not affect the essential oil composition of the fully developed leaves. However, above 50 mM NaCl concentrations, adventitious root formation and, thus, vegetative reproduction and clonal propagation were inhibited, and the water homeostasis, photosynthetic activity, and chloroplast structure of the leaves were also severely damaged.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study was conceptualized and designed by KS. The indicated specific experiments and related data analyses were carried out by RÜ (SANS), FÁ, RO, and RH (SPAD, RWC, Qy values, and 77K fluorescence spectroscopy), KS (TEM and 77K fluorescence spectroscopy), BS-T, and AB (SPME and GC/MS). RO performed the RD calculations and analyses for TEM. Statistical analyses, Figures, and Tables were prepared by RO, FÁ, and KS. Data curation was made by RO, FÁ, RÜ, BS-T, and AB. The manuscript was mainly written by KS and RO but was complemented, edited, and approved by all authors.

This work was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (K.S.) and by the National Research, Development and Innovation Office (Grant OTKA FK 124748). RO and RH were financed by Stipendium Hungaricum Ph.D. Scholarship of the Tempus Public Foundation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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