Genotype-dependent response of St. Johǹ`s wort (Hypericum perforatum L.) shoot tips to cryogenic treatment: Effect of pre-culture conditions on post-thaw recovery

Abstract Cryopreservation of medicinal plants is an important tool for restoration of their potential to synthesize valuable secondary metabolites. Establishment of an efficient protocol requires detailed understanding of species/genotype-specific responses to changing external variables. The aim of this work was to study the genotype-dependent response of Hypericum perforatum L. shoot tips to different pre-cryogenic conditions. Shoot apices isolated from plants growing on the hormone-free medium and from clusters induced by addition of benzylaminopurine, were subjected to pre-culture using two agents (0.3M sucrose and 0.076µM abscisic acid) prior to cryopreservation. Although the genotypes represented the seed progeny of one mother plant, the results showed their different capability to withstand cryogenic treatment. The differences were apparent especially in case of plants cultivated on the hormone-free medium. The ability to recover depended also on the pre-culture agent, duration of the pre-culture period and under certain conditions also on interaction between benzylaminopurine and abscisic acid in the medium. The results are illustrated by anatomical alterations of the meristematic zones and adjacent leaf primordia. Long term exposure of cultures to benzylaminopurine resulted in the delay of abscisic acid effect during pre-culture step and thus impaired regeneration capability of shoot apices after cryopreservation

Occurrence and Distribution of Phytochemicals in the Leaves of 17 In vitro Cultured Hypericum spp. Adapted to Outdoor Conditions

A plethora of plants belonging to the genus Hypericum have been investigated so far owing to the biological efficacies of pharmacologically important secondary metabolites produced by several Hypericum species. However, there is currently a dearth of information about the localization (accumulation) of these compounds in the plants in situ. In particular, the biosynthetic and ecological consequence of acclimatization of in vitro cultured Hypericum spp. to outdoor conditions is not fully known. Herein, we report an application of matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) to reveal the distribution of major naphthodianthrones hypericin, pseudohypericin, protohypericin and their proposed precursor emodin as well as emodin anthrone, along with the phloroglucinol derivative hyperforin, the flavonoids quercetin, quercitrin, rutin and hyperoside (and/or isoquercitrin), and chlorogenic acid in Hypericum leaves. Plants encompassing seventeen Hypericum species classified into eleven sections, which were first cultured in vitro and later acclimatized to outdoor conditions, were studied. We focused both on the secretory (dark and translucent glands, other types of glands, and glandular-like structures) as well as the non-secretory leaf tissues. We comparatively analyzed and interpreted the occurrence and accumulation of our target compounds in different leaf tissues of the seventeen species to get an intra-sectional as well as inter-sectional perspective. The naphthodianthrones, along with emodin, were present in all species containing the dark glands. In selected species, hypericin and pseudohypericin accumulated not only in the dark glands, but also in translucent glands and non-secretory leaf tissues. Although hyperforin was localized mainly in translucent glands, it was present sporadically in the dark glands in selected species. The flavonoids quercetin, quercitrin and hyperoside (and/or isoquercitrin) were distributed throughout the leaves. Rutin was present only within sections Hypericum, Adenosepalum, Ascyreia and Psorophytum. Our study provides insights into the prospects and challenges of using in vitro cultured Hypericum plants, further adapted to field conditions, for commercial purposes

MALDI-HRMS Imaging Maps the Localization of Skyrin, the Precursor of Hypericin, and Pathway Intermediates in Leaves of Hypericum Species

Hypericum perforatum and related species (Hypericaceae) are a reservoir of pharmacologically important secondary metabolites, including the well-known naphthodianthrone hypericin. However, the exact biosynthetic steps in the hypericin biosynthetic pathway, vis-à-vis the essential precursors and their localization in plants, remain unestablished. Recently, we proposed a novel biosynthetic pathway of hypericin, not through emodin and emodin anthrone, but skyrin. However, the localization of skyrin and its precursors in Hypericum plants, as well as the correlation between their spatial distribution with the hypericin pathway intermediates and the produced naphthodianthrones, are not known. Herein, we report the spatial distribution of skyrin and its precursors in leaves of five in vitro cultivated Hypericum plant species concomitant to hypericin, its analogs, as well as its previously proposed precursors emodin and emodin anthrone, using MALDI-HRMS imaging. Firstly, we employed HPLC-HRMS to confirm the presence of skyrin in all analyzed species, namely H. humifusum, H. bupleuroides, H. annulatum, H. tetrapterum, and H. rumeliacum. Thereafter, MALDI-HRMS imaging of the skyrin-containing leaves revealed a species-specific distribution and localization pattern of skyrin. Skyrin is localized in the dark glands in H. humifusum and H. tetrapterum leaves together with hypericin but remains scattered throughout the leaves in H. annulatum, H. bupleuroides, and H. rumeliacum. The distribution and localization of related compounds were also mapped and are discussed concomitant to the incidence of skyrin. Taken together, our study establishes and correlates for the first time, the high spatial distribution of skyrin and its precursors, as well as of hypericin, its analogs, and previously proposed precursors emodin and emodin anthrone in the leaves of Hypericum plants

DataSheet_1_Does phenotyping of Hypericum secondary metabolism reveal a tolerance to biotic/abiotic stressors?.docx

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.</p

Comparative transcriptome reconstruction of four Hypericum species focused on hypericin biosynthesis

Next generation sequencing technology (NGS) rapidly developed research applications in thefield of plant functional genomics. Several Hypericum spp. with an aim to generate andenhance gene annotations especially for genes coding the enzymes supposedly included inbiosynthesis of valuable bioactive compounds were analyzed. The first de novo transcriptomeprofiling of H. annulatum Moris, H. tomentosum L., H. kalmianum L. and H. androsaemumL. leaves cultivated in vitro was accomplished. All four species with only limited genomicinformation were selected on the basis of differences in ability to synthesize hypericins andpresence of dark nodules accumulating these metabolites with purpose to enrich genomicbackground of Hypericum spp. H. annulatum was chosen because of high number of the darknodules and high content of hypericin. H. tomentosum leaves are typical for the presence ofonly 1-2 dark nodules localized in the apical part. Both H. kalmianum and H. androsaemumlack hypericin and have no dark nodules. Four separated datasets of the pair-end reads weregathered and used for de novo assembly by Trinity program. Assembled transcriptomes wereannotated to the public databases Swiss-Prot and non-redundant protein database (NCBI-nr).Gene ontology analysis was performed. Differences of expression levels in the marginaltissues with dark nodules and inner part of leaves lacking these nodules indicate a potentialgenetic background for hypericin formation as the presumed site of hypericin biosynthesis isin the cells adjacent to these structures. Altogether 165 contigs in H. annulatum and 100contigs in H. tomentosum were detected as significantly differentially expressed (P<0.05) andupregulated in the leaf rim tissues containing the dark nodules. The new sequenceshomologous to octaketide synthase and enzymes catalyzing phenolic oxidative couplingreactions indispensable for hypericin biosynthesis were discovered. The presentedtranscriptomic sequence data will improve current knowledge about the selected Hypericumspp. with proposed relation to hypericin biosynthesis and will provide a useful resource ofgenomic information for consequential studies in the field of functional genomics, proteomicsand metabolomics

Bioactive xanthones from roots, hairy roots and cell suspension cultures of some Hypericum species and antifungal activity against Candida albicans

Members of the genus Hypericum L. are distributed worldwide but predominantly in the temperate zones of the Earth. Up to the present, almost 500 species of the genus were documented (Nürk et al., 2013, Mol Phylogenet Evol 66:1-16) but at least 40% of them have not been phytochemically characterized yet. Species of the genus Hypericum, a natural sources of bioactive compounds display several roles in multiple bioassays (reviewed by Wolfle et al. 2014, Planta Med 80:109-120). Among them, xanthones are attaining interest because of their powerful antioxidant, antibacterial, antitumor and antifungal activities (reviewed by Fotie & Bohle 2006, Anti-Infect Agents Med Chem 5: 15-31; El-Seedi et al., 2010, Curr Med Chem 17: 854–901). Xanthones are mostly accumulated in the roots. In vitro grown root cultures of Hypericum perforatum represent a suitable biotechnological tool for production of standardized extracts with high content of xanthones (Tocci et al., 2011, Appl Microbiol Biotechnol 91: 977-987; Tocci et al. 2012, Plant Physiol Biochem 57: 54-58). In addition to standard root cultures, Tusevski et al. (2013, Cent Eur J Biol 8: 1010-1022) determined significant quantities of xanthones in hairy roots of H. perforatum obtained by Agrobacterium rhizogenes transformation. The aim of this study was (i) to determine xanthone profile in root culture extracts of several Hypericum species not studied from this view point so far; (ii) to evaluate the capacity for xanthone production by H. tetrapterum Fries. and H. tomentosum L. hairy roots; (iii) to ascertain the effect of elicitation with chitosan and salicylic acid on xanthone content in cell suspension culture of H. perforatum L. and finally, (iv) to assess antifungal activity of these extracts against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin and mangiferin, was present in methanolic extracts of the root cultures. The highest total xanthone content with five identified xanthones was determined in untransformed H. pulchrum and H. annulatum root cultures. The composition of xanthones in hairy roots was species-specific. While the hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, hairy root cultures and controls of H. tomentosum contained toxyloxanthone B; 1,3,6,7 and 1,3,5,6 tetrahydroxyxanthone. Two xantnones, cadensin G and paxanthone, were found in cell suspension cultures of H. perforatum. Their content increased about twofold after elicitation with salicylic acid. Among untrasformed roots, inhibition of C. albicans ATCC 10231 growth was observed by methanolic extracts of H. annulatum roots cultured in the dark with MIC50 of 128 μg ml-1. Regarding hairy roots extracts, H. tomentosum (clone A4 and clone 7 ATCC 15834) and H. tetrapterum (clone 2 ATCC15834 and clone 2 A4), showed anti- Candida activity with the MIC50 range from μg.ml-1 and from 64 to 128 μg.ml-1, respectively. Extracts obtained by chitosan-elicitated cells did not show antifungal activity against C. albicans

Xanthones from roots, hairy roots and cell suspension cultures of selected Hypericum species and their antifungal activity against Candida albicans

Abstract Key message Highest xanthone contents were found in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Abstract Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin or mangiferin was identified in methanolic extracts of the untransformed root cultures. The highest total xanthone content, with five xanthones, was found in untransformed H. pulchrum and H. annulatum root cultures. Hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, while hairy root cultures and the corresponding controls of H. tomentosum contained toxyloxanthone B, 1,3,6,7- and 1,3,5,6-tetrahydroxyxanthone. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. Their content increased about two-fold following elicitation with salicylic acid. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml(-1). Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90 % of Candida growth with 256 μg ml(-1). Extracts from chitosan-elicitated cells did not show antifungal activity