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.

The role of secreting structures position on the leaf volatile organic compounds of Hypericum androsaemum.

Hypericum androsaemum L. presents typical translucent, essential oil producing glands, which are distributed on the leaf along both margins (margin glands) and on the lamina (lamina glands). The gland secretion was studied by histochemical and chemical analysis; the gland content was sampled directly from the secretory glands, and the volatile organic compounds (VOC) of the margin and lamina glands were separately analyzed. The lipophilic fraction of the lamina glands had as main components: (E)-2-hexenal (15.5%), hexadecanoic acid (14.7%), beta-caryophyllene (11.2%), germacrene B (11.0%) and gamma-himachalene (9.8%). The lipophilic fraction of the margin glands had as its main components: beta-pinene (22.0%), limonene (17.6%), (E)-beta-ocimene (6.1%), methyl linoleate (5.7%), terpinolene (5.4 %), (E)-2-hexenal (4.9%) and alpha-pinene (4.1%).

[The development of secondary cells in the callus of Hypericum perforatum L. and hypericins accumulation].

Hypericum perforatum L. is a kind of traditional herbal medicine that has been used as an anti-depression medicine in Europe for centuries. One of its biological active compound, hypericins, is stored in the special secondary structure,black nodules,which located in the stems, leaves and flowers. Most researches focus on the development of the black nodules in vivo and how to culture the plant to produce more hypericins. We studied the process of de-differentiation from explants to callus and the pathway of hypericins biosynthesis in callus and cells of H. perforatum L. which reflected the relationship between the cell development and secondary metabolites accumulation. The morphogenesis of cells development and hypericins accumulation were studied by electron microscopy and histological technologies. Hypericins began to accumulate in a bunch of secondary cells located on the surface of the callus in late development period. Hypericins initially produced in the cytoplasm and were transported into the vacuole and then accumulated. E.R. took apart in the process of hypericins production. Theses results supplied the gap of hypericins production and accumulation in vitro and gave some useful information regarding mass-production hypericins by tissue and cell culture technology.

The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure.

In vitro shoot cultures of Hypericum perforatum derived from wild populations grown in Armenia have a wide variation of hypericin and pseudohypericin metabolite content. We found that a germ line denoted as HP3 produces six times more hypericin and fourteen times more pseudohypericin than a second line labeled HP1. We undertook a structural comparison of the two lines (HP1 and HP3) in order to see if there are any anatomical or morphological differences that could explain the differences in production of these economically important metabolites. Analysis by LM (light microscopy), SEM (scanning electron microscopy), and TEM (transmission electron microscopy) reveals that the hypericin/pseudohypericin-containing black glands located along the margins of the leaves consist of a peripheral sheath of flattened cells surrounding a core of interior cells that are typically dead at maturity. The peripheral cells of the HP3 glands appear less flattened than those of the HP1 glands. This may indicate that the peripheral cells are involved in hypericin/pseudohypericin production. Furthermore, we find that these peripheral cells undergo a developmental transition into the gland's interior cells. The fact that the size of the peripheral cells may correlate with metabolite production adds a new hypothesis for the actual site of hypericin synthesis.