Comparative anatomy of secretory structures of leaves in Hypericum L.

The structure, type, morphology and location of secretory structures in leaves of 43 species, 1 subspecies and 1 variety of 9 sections in Hypericum L. were comparatively studied using tissue clearing, paraffin sectioning and thin sectioning. The results have shown that the presence of secretory structures is a common feature of leaves in this genus. According to their anatomical characteristics, the secretory structures can be divided into nodules, secretory cavities (canals) and tiny secretory tubes. In their distribution in leaves the nodules fall into two types: the leaf edge type and the scattered type. According to the location of cavities in the cross sections of leaves, the cavities can be divided into 4 types: the median type which is situated between the palisade tissue and spongy tissue, the palisade tissue type, the spongy tissue type and the across-mesophyll type. Based on the location of cavities and nodules in leaves, the species in Hypericum can be divided into 3 groups: group Ⅰ , in which only cavities are present; group Ⅱ, in which only nodules are present; group Ⅲ, in which both cavities and nodules are present. The type, location, distribution density and morphology of secretory structures are of some taxonomic value at the level of species and of section in Hypericum L. From these observations, the evolutionary trends concerning the morphology and anatomy of secretory structures and the affinity among sections in the genus Hypericum ate dis-cussed.     

Morphology and development of secretory structures in Hypericum perforatum and H. richeri

Secretory organs are a specialised anatomical feature of plants, these tissues function in the production and/or storage of specific chemical substances, which often have pharmacological properties. The genus Hypericum is characterised by the presence of specialised secretory structures such as black nodules, translucent glands and secretory canals. The presence of these structures is not homogeneous for all the species or between the various floral and vegetative parts of the plants. In this study, we have compared the distributions, morphology and development of secretory structures in the leaves of H. perforatum and H. richeri. Whilst, black nodules occurred in both species, translucent glands followed different development stages depending on the leaf age.     

Characteristics of plant communities containing St. John’s wort (<Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.) in the Saratov Region

Analysis of 28 plant communities containing Hypericum perforatum L. in five different biotope types has been performed in 17 districts of the Saratov Region. A total of 325 species from 197 genera of 52 vascular plant species have been recorded, with plants characteristic of forest-edge, steppe, and anthropogenic biotopes prevailing in most communities. It has been shown that H. perforatum at the southeastern boundary of its range behaves as an explerent species. The main factor limiting its growth in biotopes of a certain type is the degree of disturbance in the structure of corresponding communities rather than by their taxonomic composition.     

Germination responses of three grassland species differ between native and invasive origins

The germination stage is critical in plant life-history and is also a key process during the expansion of species’ ranges into new environments. In this study we investigated the germination patterns of three plant species (Achillea millefolium, Hieracium pilosella and Hypericum perforatum) that are invasive to New Zealand (NZ) and native to Central Europe. We asked whether the species show differences in germination temperature requirements, germination speed and maximum germination rates, and thus, whether they display evidence of adaptation to different conditions in the invasive range. Seeds from three populations per species and region were subjected to three different temperature regimes to compare germination rates among origins and across temperature conditions. For Achillea millefolium and Hypericum perforatum, germination rates were significantly higher for invasive NZ provenances than for native German ones. Seeds from invasive populations of all three species displayed increased maximum germination at medium temperature conditions when compared to native populations, which indicates altered germination strategies in the invaded range. Changes in temporal development patterns were most conspicuous for invasive Hieracium pilosella and Hypericum perforatum populations. These findings imply that adaptation in germination patterns towards different climatic conditions in invasive populations has occurred. Our study emphasises the importance of the germination stage during plant invasion and its role in explaining range expansion of these species.     

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.     

杭白芷根中分泌道发生方式、分布及其挥发油积累过程研究

为探明杭白芷(Angelica dahurica var.formosana)根中分泌道发生方式、分布及其挥发油转运积累特征,该研究利用光镜及透射电子显微镜技术观察分泌道发生过程及挥发油转运特征,结合组织化学定位确定挥发油的主要积累部位。结果表明:杭白芷根中分泌道由中柱鞘细胞最先发生,次生结构中分泌道主要分布在韧皮部和皮层中;挥发油的合成不仅与分泌细胞中质体及细胞质有关,而且还与周围细胞关系密切;分泌细胞内高尔基体和内质网丰富,可能先通过形成小泡参与转运,再经由细胞壁向腔道内转移;相邻分泌细胞靠近角隅处的细胞壁分泌活动活跃,腔道内积累大量电子致密物质;成熟分泌道中分泌细胞及其腔道内积累大量油滴,因此挥发油主要积累场所为分泌细胞及其腔道。该研究明确了杭白芷根中分泌道的发生方式、分布及其挥发油积累部位,揭示了分泌道发育过程中挥发油的转运积累特征,为进一步阐明分泌组织生长发育与有效成分积累关系提供了理论依据。     

A comparison of native and invasive populations of three clonal plant species in Germany and New Zealand

Aim Our aim was to test for changes in growth patterns of three clonally growing plant species (Achillea millefolium, Hieracium pilosella and Hypericum perforatum) between native and invaded regions. We addressed the hypotheses that with differing important life‐history traits, invasive populations perform better than native populations, and that this expected better performance is linked to weakened trade‐offs between individual growth and sexual and clonal reproduction. Location Germany and New Zealand. Methods We conducted field surveys for the three above‐mentioned species in both native German and invasive New Zealand populations, and collected data at both population and individual levels. Results At the population level, the proportion of flowering plants, population size and population density were all higher in invasive populations. Similarly, at the individual level, the number of stolons per plant, stolon–biomass ratio and population crowdedness (local plant density in a specified area around a target plant) were significantly higher in New Zealand. Plant height did not differ between countries, and plant biomass was lower in New Zealand than in Germany for Achillea millefolium and Hypericum perforatum. These two species showed significant trade‐offs between individual growth and sexual and clonal reproduction. Achillea millefolium exhibited a weakened trade‐off in its invaded range, where the same proportion of flowering plants was sustained at much higher levels of population crowdedness than in its native range. Main conclusions The apparent invasion success of the three study species is generally due to better overall performance in their respective invaded ranges. In respect of both Achillea millefolium and Hypericum perforatum, this is driven primarily by increased vegetative reproduction. In contrast, Hieracium pilosella seems to benefit more from increased sexual reproduction in its invaded range. Shifts in trade‐offs as a general trend seem to be of minor importance.     

元宝草的分泌结构与其次生产物的研究

利用整体透明,石蜡掉片和组织化学方法对元宝草不同器官中分泌结构的类型和分布进行研究。结果表明：其分泌结构根据形态结构特点可分为分泌细胞团,分泌囊及分泌道3类,并指出了3类分泌结构在各器官的分布位置和密度,组织化学试验表明,前者含金丝桃素等,后二者含挥发油,为此种药用植物的开发利用提供了依据。同时为该属植物的系统分类研究提供一些解剖学依据。     

Shoot anatomy and secretory structures in Hypericum species (Hypericaceae)

The anatomy and ultrastructure of internodes, leaves and petals were compared in Hypericum elegans, H. inodorum, H. olympicum, H. forrestii and two genotypes of H. perforatum. Internode anatomy was variable between species with respect to the structure of the cortical and pith parenchyma, including the presence of secretory reservoirs. Also, the secondary growth was more extensive in shrubs, i.e. H. inodorum and H. forrestii. In leaves, phloem secretory reservoirs were formed in all species, mesophyll secretory reservoirs were absent only in H. elegans and internal nodules were present only in H. elegans and H. perforatum. The petals differed between species in the mesophyll structure and the occurrence and location of secretory structures. The phloem secretory reservoirs lacked sheaths, whereas these were distinct in the mesophyll reservoirs. Other ultrastructural traits of the reservoirs were similar in all the species studied, with the exception of the leucoplast ultrastructure. In internal nodules, the inner cells vs. sheath cells differed in the number of vesicles and other membranous structures and plastid ultrastructure. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 70–86.     

Local performance of six clonal alien species differs between native and invasive regions in Germany and New Zealand

Exotic plant invasions are widely observed to have strong biogeographic patterns with invasive species occurring at higher abundances in their introduced range when compared with their native range. However, only few field studies have validated this assumption by comparing plant populations of multiple species in their native and introduced ranges and have evaluated to what extent changes in sexual and clonal reproduction potentially have contributed to the success of plant invasions. Here, we present the results of a comparative field study in both the native (Germany) and the introduced (New Zealand, NZ) ranges of six clonal plant species with different invasive status: Achillea millefolium L., Pilosella officinarum Vaill., Hypericum perforatum L., Prunella vulgaris L., Leucanthemum vulgare Lam. and Lotus pedunculatus Cav. We hypothesized that all six species show better performance in introduced NZ than in native German populations and tested if population structures investigated at different scales provide a useful tool to identify differences between native and introduced occurrences. In 10 populations per species and country we assessed plant density and flowering proportion at the population scale and around individual plants, thereby identifying the ‘crowdedness’ of the populations. Furthermore, we collected individual plants and determined the number of attached clonal organs and plant biomass. For all six species crowdedness in NZ populations was higher than in German populations. Additionally, overall population density of four species and the production of clonal organs (expressed as total number or per biomass ratio) of three species were higher in NZ than in Germany. When measured around individual plants, the flowering proportion was higher in native German populations of Pilosella officinarum, Hypericum perforatum and Leucanthemum vulgare. Although the study species differed in their invasive status, our findings show that for all six species performance was better in introduced than in native populations. Furthermore, this study emphasizes that multiple measures of plant performance, different spatial scales and differences among species should be taken into account when trying to identify biogeographic differences in the performance of weed species.     

The influence of some elicitors on growth and morphogenesis of Hypericum perforatum L. callus cultures

We studied the effects of elicitors, such as mannan, gβ-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.     

The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.

Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.     

Morphogenetic and diurnal variation of hypericin in some Hypericum species from Turkey during the course of ontogenesis

The genus Hypericum has received considerable interest from scientists, as it is a source of a variety of biologically active compounds including the hypericins. The present study was conducted to determine ontogenetic, morphogenetic and diurnal variation of the total hypericins content in some species of Hypericum growing in Turkey namely, Hypericum aviculariifolium subsp. depilatum var. depilatum (endemic), Hypericum perforatum and Hypericum pruinatum. The Hypericum plants were harvested from wild populations at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages four times a day. Plants were dissected into stem, leaf and reproductive tissues, which were dried separately, and subsequently assayed for total hypericin content. The density of dark glands on leaves at full flowering plants was determined for each species. Floral parts had the highest hypericin content in all species tested. But diurnal fluctuation in the hypericin content of whole plant during the course of ontogenesis varied among the species. It reached the highest level at floral budding and tended to increase at night in H. aviculariifolium subsp. depilatum var. depilatum and H. pruinatum, whereas in H. perforatum hypericin content was the highest at full flowering and no diurnal fluctuation was observed. In general, hypericin content of leaves and whole plant was higher in H. aviculariifolium subsp. depilatum var. depilatum whose leaves had more numerous dark glands than those of the two other species.     

Oleoresin glands in copaíba (<Emphasis Type="Italic">Copaifera trapezifolia</Emphasis> Hayne: Leguminosae), a Brazilian rainforest tree

Although studies have addressed the chemical analysis and the biological activity of oleoresin in species of Copaifera, the cellular mechanisms of oleoresin production, storage, and release have rarely been investigated. This study detailed the distribution, ontogeny, and ultrastructure of secretory cavities and canals distributed in leaf and stem, respectively, of Copaifera trapezifolia, a Brazilian species included in a plant group of great economic interest. Axillary vegetative buds, leaflets, and portions of stem in primary and secondary growth were collected and processed in order to study the anatomy, histolocalization of substances, and ultrastructure. Secretory cavities are observed in the foliar blade and secretory canals in the petiolule and stem. They are made up of a uniseriate epithelium delimiting an isodiametric or elongated lumen. Biseriate epithelium is rarely observed and is a novelty for Leguminosae. Cavities and canals originate from ground meristem cells and the lumen is formed by schizogenesis. The content of the cavities and canals of both stem and leaf is oily and resinous, which suggests that the oleoresin could be extracted from the leaf instead of the stem. Phenolic compounds are also detected in the epithelial cell cytoplasm. Cavities and canals in the beginning of developmental stages have polarized epithelial cells. The cytoplasm is rich in smooth and rough endoplasmic reticula connected to vesicles or plastids. Smooth and rough endoplasmic reticulum and plastids were found to be predominant in the epithelial cells of the secretory cavities and canals of C. trapezifolia. Such features testify the quantities of oleoresin found in the lumen and phenolic compounds in the epithelial cell cytoplasm of these glands. Other studies employing techniques such as correlative light electron microscopy could show the vesicle traffic and the compartmentalization of the produced substances in such glands.     

Optimization of Protein Extraction from <Emphasis Type="Italic">Hypericum perforatum</Emphasis> Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.     

Antioxidant enzymes as biochemical defenses against phototoxin-induced oxidative stress in three species of herbivorous lepidoptera

Many secondary plant compounds are capable of photoactivation resulting in the production of toxic species of oxygen. One mechanism of defense for insects feeding on phototoxic plants may be the presence of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR). The activities of these enzymes were examined in larvae of three lepidoptera: Ostrinia nubilalis, Manduca sexta, and Anaitis plagiata. Highest levels of antioxidant enzyme activity were found in A. plagiata, a specialist feeder on Hypericum perforatum, which contains high levels of the phototoxin hypericin. Larvae of A. plagiata fed leaf discs treated with hypericin exhibited a short-term, concentration-dependent decline in enzyme activity. Longer term studies with A. palgiata fed either the photoxic H. perforatum, or the closely related but non-phototoxic H. calycinum, resulted in increased CAT and GR activity in larvae fed the phototoxic plant whereas SOD activity was not significantly different. These results suggest that CAT and GR may be inducible defenses against phototoxins.     

Positive correlations between hypericin and putative precursors detected in the quantitative secondary metabolite spectrum of Hypericum

A spectrum of eight pharmacologically important secondary compounds, all putatively belonging to the polyketide pathway (hypericin, pseudohypericin, emodin, hyperforin, hyperoside, rutin, quercetin, and quercitrin) were analyzed in several hypericin-producing species of Hypericum by LC–MS/MS. Different organs such as leaves, stems and roots of wild-grown plants of Hypericum hirsutum L., Hypericum maculatum Crantz s. l., Hypericum montanum L., Hypericum tetrapterum Fr. collected in Slovakia and of Hypericum perforatum L. collected in India were examined individually. Highest contents of hypericin, pseudohypericin, and emodin were found in H. montanum, suggesting that there are alternative species to H. perforatum with high pharmaceutical value. Amounts of hyperforin and quercetin were highest in H. perforatum, whereas highest contents of hyperoside and quercitrin were found in H. maculatum. A significant positive correlation between hypericin and pseudohypericin as well as between hypericin and emodin was observed by Kruskal’s multidimensional scaling (MDS), indicating a parallel enhancement of emodin as a common precursor in the biosynthetic pathways of hypericin and pseudohypericin. Furthermore, MDS combined with principal component analysis (PCA) revealed strong correlations in the occurrence of pseudohypericin and emodin, pseudohypericin and quercitrin, hypericin and quercitrin, emodin and quercitrin, hyperoside and quercitrin, rutin and quercetin, and, hyperforin and quercetin. On the other hand, rutin showed a negative correlation with emodin as well as with quercitrin. Furthermore, hierarchical agglomerative cluster analysis (HACA) clustered hypericin and pseudohypericin, grouping emodin at equal distance from both. Considerable infraspecific variability in secondary compound spectrum and load of different populations of H. maculatum from Slovakia underscores the need for detailed studies of genotypic variation and environmental factors in relation to polyketide biosynthesis and accumulation.     

Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow

Background and Aims Hypericum perforatum

(St. John''s wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixis. Here, hypotheses on the evolutionary origin of St. John''s wort are tested and contrasted with the subsequent history of interspecific gene flow.

Methods

Extensive field collections were analysed for quantitative morphological variation, ploidy, chromosome numbers and genetic diversity using nuclear (amplified fragment length polymorphism) and plastid (trnL-trnF) markers. The mode of reproduction was analysed by FCSS (flow cytometric seed screen).

Key Results

It is demonstrated that H. perforatum is not of hybrid origin, and for the first time wild diploid populations are documented. Pseudogamous facultative apomictic reproduction is prevalent in the polyploids, whereas diploids are predominantly sexual, a phenomenon which also characterizes its sister species H. maculatum. Both molecular markers characterize identical major gene pools, distinguishing H. perforatum from H. maculatum and two genetic groups in H. perforatum. All three gene pools are in close geographical contact. Extensive gene flow and hybridization throughout Europe within and between gene pools and species is exemplified by the molecular data and confirmed by morphometric analyses.

Conclusions Hypericum perforatum

is of a single evolutionary origin and later split into two major gene pools. Subsequently, independent and recurrent polyploidization occurred in all lineages and was accompanied by substantial gene flow within and between H. perforatum and H. maculatum. These processes are highly influenced by the reproductive system in both species, with a switch to predominantly apomictic reproduction in polyploids, irrespective of their origin.     

Karyotype Analysis of Hypericum Perforatum L.

A karyotype study was made on Hypericum perforatum using plants differentiated in vitro with different ploidy level. The chromosomes of this species are small, morphologically similar, median and submedian. In the basic chromosome set the most distinguishable is chromosome number 1 which was subjected to detail analysis. It was found that there are two types of this chromosome which contribute differentially in diploid, triploid and tetraploid plants.