ABSTRACT
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.
Subject(s)
Antioxidants/metabolism , Cold Temperature , Hypericum/physiology , Oxidative Stress , 3,3'-Diaminobenzidine/metabolism , Catalase/genetics , Catalase/metabolism , Chloroplasts/ultrastructure , Cryopreservation , Fluoresceins/metabolism , Gene Expression Regulation, Plant , Genotype , Hydrogen Peroxide/metabolism , Hypericum/enzymology , Hypericum/genetics , Hypericum/ultrastructure , Isoenzymes/genetics , Isoenzymes/metabolism , Oxidative Stress/genetics , Plant Cells/ultrastructure , Plant Shoots/enzymology , Plant Shoots/genetics , Reactive Oxygen Species/metabolism , Staining and Labeling , Stress, Physiological/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Time FactorsABSTRACT
Hypericum perforatum L. in vitro cultured shoot tips were characterised at the physiological, biochemical and molecular levels following recovery from cryogenic treatment using the plant vitrification solutions PVS2 and PVS3. This comparative study revealed an increase in recovery and regrowth of explants cryoprotected with PVS3. Among the physiological markers only lipid peroxidation in the regenerants treated with PVS2 significantly increased indicating membrane damage. Genotype-specific interactions were found in most characteristics studied, with some variation detected within control and cryopreserved samples. Analyses of metabolite biosynthesis and genetic stability showed no significant differences in hypericin content, RAPD and minisatellite amplification profiles between PVS2- and PVS3-treated explants. This study demonstrates and discusses the criteria selective for PVS3 to improve the cryopreservation of H. perforatum L.
