Within the plant kingdom, the polyketide pathway-derived naphthodianthrones are synthesized essentially by some Hypericum spp. Among prenylated polyketides identified in the representatives of Guttiferae (Clusiaceae), the acylphloroglucinols with prenylation patterns are commonly isolated from the genus Hypericum (Crockett and Robson, 2011; Porzel et al., 2014).

The accumulation of naphthodianthrones is restricted to the glandular structures, so-called dark nodules, which are found in approximately 2/3 of Hypericum sections (Ciccarelli et al., 2001; Crockett and Robson, 2011). New mass spectrometry imaging (MSI)-based techniques confirmed co-localization of hypericin, pseudohypericin and their protoforms protohypericin and protopseudohypericin in the dark nodules, as the sites of their accumulation within the leaf. The naphthodianthrones are co-localized in these structures even with other anthraquinones and bisanthraquinones. Similarly, hyperforin, together with its analogues adhyperforin or hyperfirin, were co-localized in the translucent pale cavities (Kusari et al., 2015; Kucharíková et al., 2016; Rizzo et al., 2019; Revuru et al., 2020). Since only members of the sections belonging to clade core Hypericum produce hypericins (Nürk et al., 2013), the application of naphthodianthrones as potential stress-induced markers in metabolic phenotyping is limited. Nevertheless, numerous studies have been focused on improvement of the Hypericum spp. chemical composition by stimulating biosynthesis of hypericins and acylphloroglucinol derivatives using different biotic/abiotic elicitors.

In Hypericum plants, the increased accumulation of hypericins and hyperforin usually signalizes stressful environmental conditions associated with higher altitudes, including the intensity and quality of light, extreme temperatures, or temperature fluctuations (Zobayed et al., 2005; Odabas et al., 2009; Germ et al., 2010; Brechner et al., 2011; Rahnavard et al., 2012). For example, each 70 to 100 µmol m^-2 s^-1 PAR (photosynthetically active radiation) resulted in a 1.5-fold increase of hypericin content in H. perforatum shoot cultures (Briskin and Gawienowski, 2001). In greenhouse-grown H. perforatum plants, increasing light intensities from 803.4 to 1618.6 µmol m^-2 s^-1 stimulated continuous rise of hyperforin, hypericin and pseudohypericin contents (Odabas et al., 2009). In relation to light quality, the red light and UV-B stimulated the production of hypericin, pseudohypericin and hyperforin in H. perforatum and H. retusum (Nishimura et al., 2007; Brechner et al., 2011; Namli et al., 2014).

Under higher temperature stress, the ambiguous plant responses were reported. In most of the studies aimed at H. perforatum, the naphthodianthrone and hyperforin content was found to be positively influenced by increasing temperature up to 30 or 35°C (Couceiro et al., 2006; Zobayed et al., 2006; Odabas et al., 2009). On the other side, Yao et al. (2019) and Couceiro et al. (2006) did not observe any stimulatory effect of increasing temperature on either hypericin or hyperforin accumulation in this species. However, accumulation of these compounds differs in plant organs and age of culture. The content of hypericins and hyperforin was the highest at 35°C in the shoots, while in the flowers, the maximum was reached at 20°C or 25°C (Zobayed et al., 2005).

In response to low temperatures, the accumulation of hypericins predominantly depends on a cold stimulus. While the exposure of H. perforatum plants to low but above zero temperature was accompanied by unchanged or significantly lower amount of hypericins, the subfreezing temperature (-4°C) resulted in a 1.6-fold increase of hypericin content (Petijová et al., 2014; Bruňáková et al., 2015). In post-cryogenic regenerants of H. perforatum, H. rumeliacum and H. tetrapterum, more than a 3-fold increase of hypericin accumulation was documented and a remarkable 38-fold increase of hyperforin content was observed in H. rumeliacum (Petijová et al., 2014; Bruňáková and Čellárová, 2017). The stimulatory effect of cryogenic treatment can be appended to its complex abiotic stress nature comprising the dehydration of cells, crystallization of free water, mechanical wounds caused by ice particles and cold itself. However, accumulation of hypericins and phloroglucinols was negatively correlated with a single stressor represented by the acute drought or osmotic stress (Gray et al., 2003; Zobayed et al., 2003).

Relatively low stimulation of naphthodianthrones biosynthesis reaching 1.3 to 4-fold increase was seen in Hypericum spp. shoot cultures subjected to various chemical elicitors. In H. perforatum and H. adenotrichum, the elevated production of hypericins was achieved by the addition of polyethylene glycol (PEG) or sucrose to culture media (Pavlík et al., 2007; Yamaner and Erdag, 2013), and more than 3-fold increase of hypericins was reported in nanoperlite-treated H. perforatum shoot cultures (Jafarirad et al., 2021). In reaction to phytohormones, the accumulation of naphthodianthrones depended on the type, combination, and concentration of phytohormones including the adenine-type (BAP, 6-benzylaminopurine; Kin, kinetin; ZT, zeatin), or phenylurea-type (TDZ; thidiazuron) cytokinins (Liu et al., 2007; Karakas et al., 2009; Coste et al., 2011). The hypericins content also increased when the stress-signaling molecules salicylic acid (SA), jasmonic acid (JA) and methyl-jasmonate (MeJA) were used for elicitation of Hypericum shoot cultures (Sirvent and Gibson, 2002; Liu et al., 2007; Pavlík et al., 2007; Coste et al., 2011; Gadzovska et al., 2013).

For stimulation of plant secondary metabolism by the elicitors of biotic origin, pathogenic or symbiotic microorganisms are usually inactivated, and a mixture of lipopolysaccharides, peptidoglycans and other cell wall components are used. The stimulatory effects of biotic elicitors depend on the type, intensity, and duration of stimuli. The accumulation of naphthodianthrones increased in Hypericum shoots treated with pectin or dextran (Gadzovska-Simic et al., 2014), mannan, pectin and β-1,3-glucan (Kirakosyan et al., 2000; Yamaner and Erdag, 2013). The elicitors derived from endophytic fungal mycelia of Thielavia subthermophila, Piriformospora indica, Fusarium oxysporum and Trichoderma crassum induced a 1.5-fold increase of hypericins in several representatives of the sections Hypericum and Oligostema regardless the elicitor type, while a 2.3-fold higher accumulation of phloroglucinols was observed in H. kouytchense after the treatment with P. indica (Bálintová et al., 2019). The content of phenolic compounds including hypericins and hyperforin also increased upon inoculation of Hypericum plants with pathogenic fungi, such as Colletotrichum gloeosporioides (Sirvent and Gibson, 2002), Diploceras hypericinum, Phytophthora capsici (Çirak et al., 2005), or Nomuraea rileyi (Meirelles et al., 2013). In contrast to unchanged hyperforin content, higher amounts of hypericins were detected in H. perforatum seedlings infected with a mix of arbuscular mycorrhizal fungi (Zubek et al., 2012; Lazzara et al., 2017). The content of naphthodianthrones in Hypericum plants was also stimulated by inoculation with bacteria, for example the bacterial strains isolated from rhizosphere of Nicotiana glauca wild population (Mañero et al., 2012). The inactivated Agrobacterium tumefaciens culture promoted production of hyperforin in H. perforatum shoots, although hypericin accumulation declined (Pavlík et al., 2007). In the transgenic plants of H. tomentosum regenerated form hairy root cultures induced by A. rhizogenes-mediated transformation, higher content of hypericins in comparison with control plants was observed (Henzelyová and Čellárová, 2018). Similarly, higher amount of hypericins along with a 7-fold increase of hyperforin was observed in transgenic lines of H. perforatum plants (Tusevski et al., 2017). Additionally, hypericins and hyperforin content increased upon feeding with herbivores represented by the generalist feeders Spilosoma virginica and S. congrua, or Spodoptera exigua (Sirvent et al., 2003).

Being an integral part of acquired systemic plant responses in Hypericum spp., the increased accumulation of naphthodianthrones and acylphloroglucinols was shown to be linked with various biotic and abiotic stress stimuli. However, applying phototoxic pigments hypericin and pseudohypericin to examine a stress tolerance is limited as the accumulation can only be stimulated to a certain physiological level. When compared with untreated Hypericum plants, the relatively low increase of hypericins content, usually not exceeding a 5-fold, could be attributed to morpho-anatomical structure of the tissues, in which these compounds accumulate. Based on the morphometric leaf parameters of 12 Hypericum species, a cubic degree polynomial regression function was proposed for estimation of the biosynthetic capacity of Hypericum shoot cultures (Kimáková et al., 2018). Furthermore, Saffariha et al. (2021) designed several models to predict changes of hypericin content in relation to ecological and phenological factors.

Within plant polyphenols, flavonoids are the best-known metabolites with antioxidant properties. Through the inhibition of ROS generation, scavenging of free radicals, absorption of UV-wavelengths, and protection of cell membranes, these compounds have a fundamental role in plant photoprotection. Moreover, flavonoids were found to act as signal molecules involved in plant developmental processes, including the morphogenetic responses induced by various biotic/abiotic stimuli (Ferdinando et al., 2012; Brunetti et al., 2013; Petrussa et al., 2013; Chandran et al., 2019; DeLuna et al., 2020). Based on distinct substitutions in the basic skeleton containing three phenolic rings, namely two 6-carbon rings A and B linked by the central 3-carbon C ring, the flavonoids can be subdivided into several major subgroups including flavonols, flavones, flavanones, flavanonols, flavanols, anthocyanins, isoflavonoids and chalcones (Panche et al., 2016). Flavonoids are often linked to mono- or oligosaccharides (glucose, galactose, rhamnose, xylose, arabinose) and can be mono-, di-, or tri-glycosylated (Nabavi et al., 2020). Due to a great antioxidant capacity and photoprotection, the flavonols represented by dihydroxy B-ring-substituted quercetin-3-O-glycosides along with monohydroxy B-ring-substituted kaempferol-3-O-glycosides, are the most studied flavonoids (Shah and Smith, 2020).

In relation to environmental conditions, the phytochemical profiling revealed substantial differences in the accumulation of polyphenolic compounds, especially flavonoids and hydroxycinnamates (Tattini et al., 2004). In common, the enhanced accumulation of flavonols and anthocyanins usually indicates a stress exposure (Jan et al., 2021b). Based on the spectrum and abundance of stress-induced metabolites, the flavonoid profile can be used to distinguish between the stress-exposed plants and their unstressed counterparts. For example, the quercetin-3-O-glucuronide/glucoside and kaempferol-3-O-galactoside/glucoside represent specific metabolites displaying significantly higher relative content in water-deficient plants of Vitis vinifera when compared with the unstressed control group (Gago et al., 2017). Furthermore, the species-specific responses to abiotic stress including differences in the content of quercetin and its glycosides enable to distinguish even between related species like Crataegus laevigata and Crataegus monogyna (Kirakosyan et al., 2004). The variation of biosynthetically related metabolites is usually further modified by other abiotic stimuli (de Abreu and Mazzafera, 2005) or concomitant biotic stresses (Schenke et al., 2019). As an example, the variation of quercetin and rutin in water stressed Hypericum brasiliense plants depended on the temperature regime (de Abreu and Mazzafera, 2005).

In the genus Hypericum, flavonoids and hydroxycinnamic acids were shown to be the most prevalent phenolic compounds involved in antioxidant responses (Zdunić et al., 2017). The production of several flavonoid compounds and chlorogenic acid increased in H. perforatum adventitious roots cultured in media supplemented up to 70 g L^-1 of sucrose resulting in osmotic stress (Cui et al., 2010); the levels of quercetin aglycone and its glycoside rutin increased in shoots and roots of water-stressed H. brasiliense (de Abreu and Mazzafera, 2005); and the acute drought stress applied on H. perforatum shoots resulted in higher accumulation of quercetin glycosides in floral tissues (Gray et al., 2003). The content of several flavonoids including quercetin, hyperoside (quercetin-3-D-galactoside), rutin (quercetin-3-rutinoside), and quercitrin (quercetin-3-L-rhamnoside), along with kaempferol, amentoflavone, apigenin-7-glucoside, and chlorogenic acid also raised with the increasing light intensity and temperature; based on the relationship between the culture conditions (e.g. temperature, light intensity) and phenolics accumulation, a mathematical model was proposed for the prediction of phenolics content in H. perforatum plants (Odabas et al., 2010).

A visible tissue coloring caused by the enhanced accumulation of flavonoids is usually taken as general indicator of physiological status of a stressed plant (Lu et al., 2017). Sooriamuthu et al. (2013) observed a 3 to 5-fold elevation of flavonoids and anthocyanins in photoinduced reddish-colored plantlets after transferring the etiolated Hypericum hookerianum shoots to the light. In H. perforatum shoot cultures, the reddening of plant organs due to accumulation of anthocyanins signalized the increased osmotic strength of culture medium after addition of sucrose (Pavlík et al., 2007). In our previous research (Bruňáková et al., 2011; Bruňáková and Čellárová, 2016), a purple pigmentation of leaf tissues was also seen in the post-cryogenic regenerants of H. perforatum ( Figure 1 ).

The flavonoids and other phenolic compounds have been shown to strengthen the tolerance to a wide range of elicitors of biotic origin, including the pest and pathogen attacks. In our previous work, the changes in metabolic profiles of Hypericum spp. induced by glucose, starch, chitosan, and elicitors derived from H. perforatum-borne endophytic fungi were shown to be species-specific, depending on the type and intensity of stress stimuli (Bálintová et al., 2019). The content of rutin, hyperoside, isoquercetin (quercetin-3-glucoside) and quercitrin was elevated in all species regardless the elicitor, showing a maximum of 13-fold increase of isoquercetin in H. monogynum shoots treated with P. indica-derived elicitors in the presence of chitosan. On the other hand, the accumulation of amentoflavone and chlorogenic acid depended on the elicitation treatment and Hypericum species; the amentoflavone content raised maximally 15.7-times in H. humifusum shoots after culturing them on media supplemented with potato-dextrose broth (PDB), and a 13-fold increase was detected in H. tetrapterum after the treatment with elicitors derived from T. subthermophila, or A. rhizogenes. A maximum of 31.7-fold elevation of the chlorogenic acid was documented in H. maculatum shoots treated with D-glucose. Almost a 15-fold elevation of the chlorogenic acid was seen after the chitosan treatment in H. kouytchense shoots and a 11.5-fold increase of this metabolite was observed in H. maculatum treated by A. rhizogenes-derived elicitors (Bálintová et al., 2019).

The elevated accumulation of flavonols, hydroxycinnamic acids, xanthones, and other phenolic compounds stimulated by (poly)saccharides in in vitro cultures of Hypericum spp. proves the universal role of saccharides-based elicitation (Kirakosyan et al., 2000; Gadzovska-Simic et al., 2014; Gadzovska-Simic et al., 2015a). The elicitors derived from Aspergillus niger and F. oxysporum stimulated the biosynthesis of phenylpropanoids or flavonoid glycosides in several Hypericum spp. (Azeez and Ibrahim, 2013; Gadzovska-Simic et al., 2015b). Similarly, Franklin et al. (2009), and Tusevski et al. (2015) documented a significant elevation of xanthone biosynthesis in H. perforatum cell cultures stimulated by elicitors derived from A. rhizogenes and A. tumefaciens. An enhanced content of lignin and flavonoids was observed in H. perforatum cell wall biomass after A. tumefaciens elicitation (Singh et al., 2014). In common, the structural components of the cell wall of fungal or bacterial pathogens induce defense reactions in both, the intact plants or plant cell cultures via production of ROS, hypersensitive response, or production of SMs with antimicrobial effects (Angelova et al., 2006).

Melatonin represents another stress-responsive metabolite involved in antioxidant defense in several Hypericum representatives, e.g., H. perforatum, or H. kouytchense (Murch and Saxena, 2006; Nguyen et al., 2018; Chung and Deng, 2020; Khan et al., 2020). According to Zhou et al. (2021), the overexpression of HpSNAT1 gene coding for serotonin N-acetyltransferase (SNAT), the key enzyme involved in melatonin biosynthesis resulted in more than 4-fold increase of melatonin content in response to high salt and drought treatment. In plants, melatonin was shown to contribute to the acquisition of tolerance against various abiotic/biotic stresses including cold (Han et al., 2017; Qari et al., 2022), or pathogen attacks (Lee et al., 2014).

To differentiate between stress tolerant and sensitive plants by metabolic phenotyping, the metabolite alterations induced by biotic/abiotic stimuli should be distinguished from a great naturally existing interspecific variability within this genus (Bálintová et al., 2019). Across the Hypericum spp., the spectrum and abundancy of SMs vary at the population, species and genotype levels (Çirak et al., 2008; Nogueira et al., 2008; Crockett and Robson, 2011; Çirak et al., 2014; Çirak et al., 2015; Çirak et al., 2016; Çirak and Radusiene, 2019; Carrubba et al., 2021), and usually changes during ontogenesis (de Abreu et al., 2004). Recent metabolomics studies reveal both the common and unique alterations in metabolic profiles of plants exposed to biotic/abiotic stresses related to polyphenolic compounds represented by chlorogenic acid, and flavonoids, namely the amentoflavone, quercetin or kaempferol glycosides.

In control plants, the PCA revealed a great interspecific variability of Hypericum phenolic composition related to anthraquinones, acylphloroglucinol derivatives, flavonoids and chlorogenic acid ( Figures 2A, B ). According to the determinant metabolites, Hypericum representatives were distributed to two relatively compact groups and a singleton. The HCA-based heatmap ( Figure 2C ) was used to visualize the differences in the number of phenolic compounds between control plants and plants exposed to the cold treatment.

According to Figure 2A , more than 50% of the variance was accounted for the first two components PC1 and PC2. The species found in the 2nd and 4th quadrants formed a relatively compact group corresponding to cluster I, representing the sections Hypericum (H. perforatum, H. maculatum, H. erectum), Adenosepalum (H. annulatum) and Oligostema (H. humifusum). The remaining representatives seen in the 1st quadrant formed the separate cluster II comprising the sections Webbia (H. canariense), Androsaemum (H. androsaemum), Bupleuroides (H. bupleuroides) and Ascyreia (H. kouytchense). Based on the relative metabolite content, H. kalmianum (Myriandra) was the most distantly related species, forming a singleton in the 3rd quadrant. The HCA dendrogram confirmed PCA clustering showing two separate clades of hierarchically nested species and H. kalmianum as a separate branch ( Figure 2C ). Using both the PCA and HCA hierarchical heatmap, the determinant metabolites were identified for each cluster ( Figures 2B, C ). Naphthodianthrones and their potential precursor emodin were identified as the main determinants for grouping the species in cluster I. The phloroglucinol derivatives were the major profiling compounds of species belonging to cluster II. The flavonoid quercetin, and its glycoside isoquercetin, along with catechin and astragalin (kaempferol-3-O-glucoside), were the most abundant metabolites in H. kalmianum.

As we have shown previously, H. kalmianum, H. perforatum and H. humifusum were characterized as FT species; for example, H. kalmianum plants tolerated ice crystallization up to -11°C (Petijová et al., 2014). In the context of metabolic adaptations related to the freezing tolerance strategy, a relatively higher basal level of the flavonols quercetin and its glycosides, as well as astragalin in H. kalmianum suggests an interconnection between the flavonoids accumulation and plant ability to tolerate ice crystallization. Additionally, the elevated basal content of naringenin and amentoflavone was observed in other FT representatives, e.g., H. perforatum and H. humifusum. On the other side, no substantially elevated basal level of flavonoids was seen in H. canariense or H. kouytchense, which are known as FS species avoiding freezing by supercooling (Petijová et al., 2014).

In Hypericum plants exposed to 4°C, the HCA dendrograms did not reveal any substantial metabolic alterations excepting the enhanced quercetin content in H. kalmianum ( Figure 2C ). It should be noticed that the subjection of plants to 4°C for 7 days did not represent any lethal stress for Hypericum plants involved in this study. The plants did not change their habitus and continued their growth. However, the exposure of FT Hypericum spp. to temperature of 4°C for several days induced the processes typical for the cold acclimation (Bruňáková et al., 2011; Petijová et al., 2014). Under cold acclimation, a major reprogramming of the transcriptome, proteome, and metabolome results in the accumulation of various cryoprotective substances like saccharides, saccharide alcohols, low-molecular weight nitrogenous compounds, amino acids including proline, cold-regulated (COR) proteins, antioxidant enzymes, polyamines, and polyphenolic metabolites, including several subgroups of flavonoids (Fürtauer et al., 2019; Xu and Fu, 2022).

The relationship between the enhanced accumulation of flavonoids and plant tolerance to the under-zero temperatures is known and well documented. In the model species Arabidopsis thaliana, the flavonols, particularly kaempferols and quercetin glycosides, along with the anthocyanins, and phenylpropanoids were shown to be involved in the freezing tolerance by increasing the antioxidant capacity of tissues exposed to low temperatures (Schulz et al., 2015; Schulz et al., 2016; Zhang et al., 2019; Schulz et al., 2021). Apart from Arabidopsis, higher flavonoid contents, including flavonoid aglycones and glycosides, was associated with freezing tolerance in the number of species, for example Primula malacoides (Isshiki et al., 2014) or Rhododendron cultivars (Swiderski et al., 2004).

Despite the causal relationship between the accumulation of flavonoids and freezing tolerance has not been revealed yet, flavonoids have been successfully applied for prediction of the freezing strategy. For example, in A. thaliana, the unique combinations of these compounds were proposed as metabolic markers of the freezing tolerance (Korn et al., 2008; Schulz et al., 2016). The metabolic interactions between saccharides and plant SMs suggest a possible relationship between accumulation of flavonoid glycosides and enhanced tolerance to freezing in cold-acclimated plants (Korn et al., 2008; Fürtauer et al., 2019). Among the mechanisms, by which plant polyphenols contribute to the protection of plants against a freezing damage, the flavonoids and phenolic acids are directly involved in scavenging free radicals, modulation of the phytohormone-mediated stress responses, stabilization of the cell membranes, or preventing the proteins aggregation (Sharma et al., 2012; Hasanuzzaman et al., 2020).

On the other side, the molecular, biochemical, and physiological changes induced by cold acclimation lead to suppression of some subgroups of SMs accumulation in FT plants exposed to low but above-zero temperatures (Fürtauer et al., 2019). In our previous research, the subjection of FT Hypericum spp. to 4°C resulted in the decrease of some SMs, e.g., carotenoids and naphthodianthrones (Petijová et al., 2014; Bruňáková et al., 2015). Similarly, a decrease of terpene indole alkaloids content was seen in Catharanthus roseus plants under acclimated conditions (Dutta et al., 2013).

The PCA and HCA revealed a naturally occurring interspecific variability in the basal level of non-enzymatic AOXs (proline and carotenoids), and enzymatic activities (CAT, SOD and APX). As shown by the PCA, more than 60% of the variability could be explained by PC1 and PC2 ( Figure 3A ). Within the control group of plants without the cold exposure, H. kalmianum formed a clearly separated singleton in the 1st quadrant based on the contribution of the profiling metabolites ( Figures 3A, B ). The other species were mostly situated in the 2nd quandrant ( Figure 3A ). According to the HCA dendrogram and HCA-based heatmap, the control plants of H. kalmianum represented a separate branch due to substantially higher basal level of APX activity. The cold-treated plants formed a common and relatively compact cluster spreading among all four quadrants ( Figure 3A ). The HCA heatmap revealed a substantial elevation of AOX enzymatic activities, along with the increased content of proline and carotenoids, which followed exposure of Hypericum plants to 4°C ( Figure 3C ).

In response to acclimation temperature of 4°C, the metabolic and antioxidant phenotyping revealed substantial alterations in basal level of phenolic compounds, along with other AOXs non-enzymatic components and enzymatic activities among Hypericum species differing in their geographical distribution. In relation to phenolic composition, the flavonols quercetin and quercetin glycosides, as well as astragalin, and other flavonoid metabolites like naringenin and amentoflavone could be considered as metabolites contributing to the freezing tolerance in the genus Hypericum. Higher basal level of proline, carotenoids and enzymatic activities of SOD, CAT and APX might reflect the genetically predetermined mechanisms of a stress response in tolerant species or signalize the oxidative burst in sensitive Hypericum representatives.

The tolerance to cold stress is a highly complex trait influenced by multiple factors; the reconfiguration of primary and secondary metabolic pathways along with activation of antioxidant enzymatic defense usually reflect the genetically predetermined capacity of the species adaptation to the local stress conditions. Based on electrolyte leakage (LT50 values), the freezing adaptation strategy was shown to be consistent with the natural habitat of a species. Within the genus Hypericum, the extent of the freezing tolerance was shown to concur with the post-cryogenic regeneration reflecting the geographical distribution of a species (Petijová et al., 2014; Bruňáková et al., 2015). While the (sub)tropical H. canariense, endemic of Canary Island and Madeira (Robson, 1996), was referred as cold-sensitive species avoiding ice formation by supercooling, other studied Hypericum species originating from the temperate zones, or growing at higher altitudes of the subtropical areas were shown to be relatively tolerant to freezing (Petijová et al., 2014). For example, the nearly cosmopolitan H. perforatum, and H. kalmianum restricted to cold area of USA and Canada (adjacent to Ontario Lake and Ottawa River) were characterized as the FT species (Petijová et al., 2014; Bruňáková et al., 2015). However, the prediction of freezing resistance strategy based on metabolic and antioxidant profiling is usually complicated by a large intraspecific variability related to the origin of the plant accession. The conflicting evidence of tolerance/avoidance mechanism was reported for A. thaliana accessions originated from different habitats (Zhen and Ungerer, 2008; Hoermiller et al., 2018). As shown by Nägele and Heyer (2013), the cold-induced intensive accumulation of saccharides and amino acids was higher in FT accessions of A. thaliana when compared with their FS counterparts. Significant alterations in AOX metabolism were also observed between FT and FS cultivars of Hordeum vulgare (Dai et al., 2009).

In conclusion, we assume that the combination of metabolic and enzymatic AOXs, along with other physiological markers including the thermal properties of plant tissues (LT50), and knowledge of the plant origin could be used for prediction of a mechanism, by which Hypericum spp. adapt to a cold stress. Albeit some classes of phenolic compounds contribute to the enhanced stress tolerance, more data are needed to identify the unique combinations of metabolites that would indirectly allow to estimate the extent of freezing tolerance of a particular species in the genus Hypericum.