Does phenotyping of Hypericum secondary metabolism reveal a tolerance to biotic/abiotic stressors?

In this review we summarize the current knowledge about the changes in Hypericum secondary metabolism induced by biotic/abiotic stressors. It is known that the extreme environmental conditions activate signaling pathways leading to triggering of enzymatic and non-enzymatic defense systems, which stimulate production of secondary metabolites with antioxidant and protective effects. Due to several groups of bioactive compounds including naphthodianthrones, acylphloroglucinols, flavonoids, and phenylpropanes, the world-wide Hypericum perforatum represents a high-value medicinal crop of Hypericum genus, which belongs to the most diverse genera within flowering plants. The summary of the up-to-date knowledge reveals a relationship between the level of defense-related phenolic compounds and interspecific differences in the stress tolerance. The chlorogenic acid, and flavonoids, namely the amentoflavone, quercetin or kaempferol glycosides have been reported as the most defense-related metabolites associated with plant tolerance against stressful environment including temperature, light, and drought, in association with the biotic stimuli resulting from plant-microbe interactions. As an example, the species-specific cold-induced phenolics profiles of 10 Hypericum representatives of different provenances cultured in vitro are illustrated in the case-study. Principal component analysis revealed a relationship between the level of defense-related phenolic compounds and interspecific differences in the stress tolerance indicating a link between the provenance of Hypericum species and inherent mechanisms of cold tolerance. The underlying metabolome alterations along with the changes in the activities of ROS-scavenging enzymes, and non-enzymatic physiological markers are discussed. Given these data it can be anticipated that some Hypericum species native to divergent habitats, with interesting high-value secondary metabolite composition and predicted high tolerance to biotic/abiotic stresses would attract the attention as valuable sources of bioactive compounds for many medicinal purposes.

Phytochemical profiling of several Hypericum species identified using genetic markers.

In the present study, we performed phytochemical profiling of several under-exploited Hypericum representatives taxonomically belonging to the sections Ascyreia, Androsaemum, Inodora, Hypericum, Coridium, Myriandra, and Adenosepalum. The authenticity of the starting plant material was confirmed using the nuclear ribosomal internal transcribed spacer as a molecular marker, DNA content and chromosome number. Phenolic constituents were analyzed using high-performance liquid chromatography to complement species-specific metabolic profiles. In several Hypericum representatives, the pharmacologically important compounds, including naphthodianthrones; phloroglucinol derivatives; chlorogenic acid; and some classes of flavonoids, particularly the flavonols rutin and hyperoside, flavanol catechin, and flavanones naringenin and naringin, were reported for the first time. Comparative multivariate analysis of chemometric data for seedlings cultured in vitro and acclimated to the outdoor conditions revealed a strong genetically predetermined interspecific variability in phenolic compound content. In addition to hypericins, which are the most abundant chemomarkers for the genus Hypericum, rarely employed phenolic metabolites, including phloroglucinol derivatives, chlorogenic acid, catechin, naringenin, naringin, and kaempferol-3-O-glucoside, were shown to be useful for discriminating between closely related species. Given the increasing interest in natural products of the genus Hypericum, knowledge of the spectrum of phenolic compounds in shoot cultures is a prerequisite for future biotechnological applications. In addition, phytochemical profiling should be considered as an additional part of the integrated plant authentication system, which predominantly relies upon genetic markers.

Targeted metabolomic profiling reveals interspecific variation in the genus Hypericum in response to biotic elicitors.

Shoot cultures of eight Hypericum species belonging to the sections Hypericum, Oligostema, Ascyreia and Webbia were evaluated for their phytochemical profiles by high-performance liquid chromatography. In total, 17 secondary metabolites assigned to the groups of anthraquinones, phloroglucinols, hydroxycinnamic acids and flavonoids were detected. Furthermore, the elicitation potential of 18 biotic factors derived from saccharides, endophytic fungi and Agrobacterium rhizogenes was examined and statistically analysed with the paired two-sample t-test and principal component analysis. The production of naphthodianthrones and emodin was predominantly stimulated by elicitors derived from Fusarium oxysporum and Trichoderma crassum, while Piriformospora indica promoted the phloroglucinols production. Among flavonoids, the aglycone amentoflavone was readily increased by several elicitors up to 15.7-fold in H. humifusum treated by potato-dextrose broth. However, the chlorogenic acid proved to be the most susceptible metabolite to elicitation, when 31.7-times increase was detected in H. maculatum shoots upon D-glucose treatment. In spite of several biotic factors have been tested, no metabolite was commonly induced in all Hypericum spp. as a response to elicitor treatments.

Relation between hypericin content and morphometric leaf parameters in Hypericum spp.: A case of cubic degree polynomial function.

Higher plants often accumulate secondary metabolites in multicellular structures or in secretory reservoirs. Biotechnological production of such compounds by cell cultures lacking proper morphological structures is difficult, therefore possibilities for an efficient increase of their formation by organ cultures are being searched. The genus Hypericum comprises many species that store photoactive and phototoxic naphthodianthrones in the dark nodules on their above-ground parts. To date, the relation between the content of hypericins and their proto-forms accumulated in the nodules, and morphological characters of the plant parts containing these structures has not been sufficiently explained. The content of hypericins and leaf morphology characters were measured in 12 selected diploid seed-derived Hypericum species cultured in vitro. The leaf volume and the volume of the nodules per leaf were calculated. Based on these data, a cubic degree polynomial regression model with high reliability was constructed. The model enables an estimate of the biosynthetic capacity of the cultures, and may be useful in designing the experiments aimed at elicitation of these unique secondary metabolites in shoot cultures of Hypericum spp. An analogous model may be developed for interpretation of experimental results for other plant species which accumulate metabolites in specialized morphological structures.

Modulation of anthraquinones and phloroglucinols biosynthesis in Hypericum spp. by cryogenic treatment.

Beside the high post-cryogenic recovery rate, a reinstated scale of secondary metabolites in recovered plant tissues represents another inevitable aspect of an effective cryopreservation protocol for medicinal plants. The current study was aimed at evaluation of the elicitation potential of cryogenic treatment on secondary metabolism of some Hypericum species. In agreement with our assumption, the cold stimuli applied during the pre-cryogenic phase increased the tolerance to low temperatures (-196°C) in H. perforatum, H. rumeliacum and H. tetrapterum reaching a maximum of 46% recovery rate in St. John's wort plants. The effect of cryogenic treatment-associated stressors on the spectrum of the profiling secondary metabolites, naphthodianthrones and phloroglucinols, was ambiguous. The content of hypericins in both pre-cultured H. tetrapterum donor plants and H. perforatum shoots regenerated from cryopreserved meristems increased more than 3-times. The highest 38-fold enhancement of phloroglucinols was observed in H. rumeliacum shoots recovered after cryostorage. Our findings indicate that modulated biosynthesis of secondary metabolites represented by naphtodianthrones and phloroglucinols can be considered as a part of overall plant adaptations to stress conditions associated with liquid nitrogen (LN) treatment.

Effect of cryoprotectant solution and of cooling rate on crystallization temperature in cryopreserved Hypericum perforatum cell suspension cultures.

BACKGROUND: The increasing demand for hypericins and hyperforins, the unique pharmaceuticals found in the Hypericum genus, requires the development of effective tools for long-term storage of cells and tissues with unique biochemical profiles. OBJECTIVE: To determine the temperature of crystallization (T(C)) and of ice formation of 14 cryoprotectant mixtures (CMs) for their use in cryoprotection of H. perforatum L. cell suspensions and to evaluate the impact of the lowest Tc on post-cryogenic recovery. MATERIALS AND METHODS: T(C) was determined by real-time microscopy of ice formation during slow cooling to -196° C and heating to 20° C. RESULTS: Exposure of cells to CMs CM2 (PVS3) containing sucrose and glycerol or CM12 and CM13 containing sucrose, glycerol, dimethylsulfoxide and ethylene glycol decreased T(C) below -60° C, prevented intracellular crystallization and considerably reduced both the size of crystals and the rate of extracellular ice propagation. CONCLUSION: The selected CMs proved suitable for cryopreservation of H. perforatum cell suspensions with the maximum of 58 % post-thaw recovery.

Conservation Strategies in the Genus Hypericum via Cryogenic Treatment.

In the genus Hypericum, cryoconservation offers a strategy for maintenance of remarkable biodiversity, emerging from large inter- and intra-specific variability in morphological and phytochemical characteristics. Long-term cryostorage thus represents a proper tool for preservation of genetic resources of endangered and threatened Hypericum species or new somaclonal variants with unique properties. Many representatives of the genus are known as producers of pharmacologically important polyketides, namely naphthodianthrones and phloroglucinols. As a part of numerous in vitro collections, the nearly cosmopolitan Hypericum perforatum - Saint John's wort - has become a suitable model system for application of biotechnological approaches providing an attractive alternative to the traditional methods for secondary metabolite production. The necessary requirements for efficient cryopreservation include a high survival rate along with an unchanged biochemical profile of plants regenerated from cryopreserved cells. Understanding of the processes which are critical for recovery of H. perforatum cells after the cryogenic treatment enables establishment of cryopreservation protocols applicable to a broad number of Hypericum species. Among them, several endemic taxa attract a particular attention due to their unique characteristics or yet unrevealed spectrum of bioactive compounds. In this review, recent advances in the conventional two-step and vitrification-based cryopreservation techniques are presented in relation to the recovery rate and biosynthetic capacity of Hypericum spp. The pre-cryogenic treatments which were identified to be crucial for successful post-cryogenic recovery are discussed. Being a part of genetic predisposition, the freezing tolerance as a necessary precondition for successful post-cryogenic recovery is pointed out. Additionally, a beneficial influence of cold stress on modulating naphthodianthrone biosynthesis is outlined.

Shoot Tip Meristem Cryopreservation of Hypericum Species.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 µM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.

Gene expression profiling in Taxus baccata L. seedlings and cell cultures.

Limited native resources of paclitaxel from Taxus trees initiated the research to produce this compound by biotechnology. In vitro plant cell culture systems have been used for large-scale production of paclitaxel and related taxanes. In the past decade, several genes involved in the taxane biosynthetic pathway have already been sequenced and cloned. This protocol details how to derive cell cultures of Taxus baccata L. from young stems of mature trees and from all parts of in vitro- grown seedlings such as root segments, hypocotyls, and cotyledons. The time-course of expression of two genes - dbat and dbtnbt - coding for two enzymes of the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation has been investigated through a 64-day subculture interval of T. baccata cell cultures, during germination, and in early stages of seedling development. The expression level is measured by using quantitative real-time reverse transcriptase polymerase chain reaction. The intracellular content of baccatin III and paclitaxel is quantified by high-performance liquid chromatography HPLC.We have shown that although the increase in transcriptional activity of dbat and dbtnbt positively correlate with callus growth, the intracellular accumulation of paclitaxel varies during subculture with the maximum between the late linear and stationary phase. The expression of both genes peaks on day 8 of germination, followed by a decrease in the post-germination phase and during seedling growth. The increase of the steady-state mRNA level of both genes is followed by corresponding metabolite accumulation with a delay of approximately 14-28 d.

Expression of dbat and dbtnbt genes involved in paclitaxel biosynthesis during the growth cycle of Taxus baccata L. callus cultures.

The time-course of expression of dbat and dbtnbt genes involved in the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation were investigated through a 64-day subculture interval of VI/M1 and VI/M2 Taxus baccata callus cultures. HPLC proved traces of baccatin III and an intracellular content of paclitaxel up to 90 microg/g DW. The steady-state of the respective gene transcripts was measured by quantitative real-time RT-PCR. The expression profile of dbat and dbtnbt genes was slightly different and varied within the subculture. The highest level of dbat expression was detected 24 h after inoculation followed by a decrease in both cultures. In contrast with dbat no substantially high expression of the dbtnbt gene after inoculation was observed. The impact of the conditions during inoculation on gene expression is discussed. Although the increase in transcriptional activity of both genes positively correlated with callus growth, the intracellular accumulation of paclitaxel varied during subculture with the maximum in the stationary (VI/M1) or at the end of the linear (VI/M2) phase. The increase of the steady-state mRNA level of the dbtnbt gene was followed by paclitaxel accumulation with a delay of approx. 28 (VI/M1) and 14 days (VI/M2).