In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Comparative analysis of geotypic variations in the proteome of Nostoc commune.

Cyanobacterium Nostoc commune is a filamentous terrestrial prokaryotic organism widely distributed, which suggest its high adaptive potential to environmental or abiotic stress. Physiological parameters and proteomic analysis were performed in two accession of N. commune with the aim to elucidate the differences of physiological trails between distant geotypes, namely Antarctic (AN) and central European (CE). The result obtained clearly showed that the AN geotype demonstrates elevated levels of total phenols, flavonoids, carotenoids, and phycobiliproteins, indicative of its adaptation to environmental stress as referred by comparison to CE sample. Additionally, we employed LC-MS analysis to investigate the proteomes of N. commune from AN and CE geotypes. In total, 1147 proteins were identified, among which 646 proteins expressed significant (up-regulation) changes in both accessions. In the AN geotype, 83 exclusive proteins were identified compared to 25 in the CE geotype. Functional classification of the significant proteins showed a large fraction involved in photosynthesis, amino acid metabolism, carbohydrate metabolism and protein biosynthesis. Further analysis revealed some defense-related proteins such as, superoxide dismutase (SOD) and glutathione reductase, which are rather explicitly expressed in the AN N. commune. The last two proteins suggest a more stressful condition in AN N. commune. In summary, our findings highlight biochemical processes that safeguard the AN geotype of N. commune from extreme environmental challenges, not recorded in CE accession, probably due to less stressful environment in Europe. This study brings the first ever proteomic analysis of N. commune, emphasizing the need for additional investigations into the climate adaptation of this species with rather plastic genome.

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.

Whole-genome sequence data of Hypericum perforatum and functional characterization of melatonin biosynthesis by N-acetylserotonin O-methyltransferase.

Hypericum perforatum is among the most commonly used herbal remedies and supplements. The aerial plant parts are often used to treat depression. Due to the lack of genomic information of H. perforatum, the gene networks regulating secondary metabolite synthesis remain unclear. Here, we present a high-quality genome for H. perforatum with a 2.3-Mb scaffold N50. The draft assembly covers 91.9% of the predicted genome and represents the fourth sequenced genus in the order Malpighiales. Comparing this sequence with model or related species revealed that Populus trichocarpa and Hevea brasiliensis could be grouped into one branch, while H. perforatum and Linum usitatissimum are grouped in another branch. Combined with transcriptome data, 40 key genes related to melatonin, hyperforin, and hypericin synthesis were screened and analyzed. Five N-acetylserotonin O-methyltransferases (HpASMT1-HpASMT5) were cloned and functionally characterized. Purified HpASMT3 protein converted N-acetylserotonin into melatonin with a Vmax of about 1.35 pkat/mg protein. HpASMT1 and HpASMT3 overexpression in Arabidopsis mutants caused 1.5-2-fold higher melatonin content than in mutant and wild-type plants. The endogenous reactive oxygen species (ROS) in transgenic plants was significantly lower than ROS in mutant and wild-type plants, suggesting higher drought tolerance. The obtained genomic data offer new resources for further study on the evolution of Hypericaceae family, but also provide a basis for further study of melatonin biosynthetic pathways in other plants.

Computational screening of miRNAs and their targets in leaves of Hypericum spp. by transcriptome-mining: a pilot study.

MAIN CONCLUSION: Our work provides a survey of mature miRNAs, their target genes and primary precursors identified by in-silico approach in leaf transcriptomes of five selected Hypericum species. MiRNAs are small non-coding RNA molecules found in animals, terrestrial plants, several algae and molds. As their role lies in the post-transcriptional gene silencing, these tiny molecules regulate many biological processes. Phyto-miRNAs are considered the important regulators of secondary metabolism in medicinal plants. The genus Hypericum comprises many producers of bioactive compounds, mainly unique naphtodianthrones with a great therapeutic potential. The main goal of our work was to identify genetically conserved miRNAs, characterize their primary precursors and target sequences in the leaf transcriptomes of five Hypericum species using in-silico approach. We found 20 sequences of potential Hypericum pri-miRNAs, and predicted and computationally validated their secondary structures. The mature miRNAs were identified by target genes screening analysis. Whereas predicted miRNA profiles differed in less genetically conserved families, the highly conserved miRNAs were found in almost all studied species. Moreover, we detected several novel highly likely miRNA-mRNA interactions, such as mir1171 with predicted regulatory role in the biosynthesis of melatonin in plants. Our work contributes to the knowledge of Hypericum miRNAome and miRNA-mRNA interactions.

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.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.

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.

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.

Oxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature treatment.

Extreme low temperatures cause plants multiple stresses, among which oxidative stress is presumed to be the major component affecting the resultant recovery rate. Plants of Hypericum perforatum L., which are known especially for the photodynamic activities of hypericins capable of producing reactive oxygen species under exposure to visible light, were observed to display a substantial increase and persistence in active oxygen production at least two months after recovery from cryogenic treatment. In an effort to uncover the causative mechanism, the individual contributions of wounding during explant isolation, dehydration and cold were examined by means of antioxidant profiling. The investigation revealed activation of genes coding for enzymatic antioxidant catalase and superoxide dismutase at both the transcript and protein levels. Interestingly, plants responded more to wounding than to either low-temperature associated stressor, presumably due to tissue damage. Furthermore, superoxide dismutase zymograms showed the Cu/Zn isoforms as the most responsive, directing the ROS production particularly to chloroplasts. Transmission electron microscopy revealed chloroplasts as damaged structures with substantial thylakoid ruptures.