The role of glutathione during virus infections

The agricultural use of the Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca GREB.) has a historical tradition in Styria (Austria) and plans an important regional role in agriculture since the European Union protected the Styrian pumpkin oil as a selected European speciality. Since 1996 the zucchini yellow mosaic virus (ZYMV), causes severe losses of up to 60 % in Styrian pumpkin production every year. The ZYMV, which was first described in 1981, belongs to the genus of potyvirus and is one of the most widespread and destructive viral agents throughout the world and causes severe economic loss to cucurbit crops every year. Viral infections are changing the ultrastructure of cells before visible symtoms occur an the plant. With the transmission electron microscope it is possible to detect such early cellular changes which help us to understand the development of virus induced visible damages. With this background it is possible to develop new plant protectants and new strategies to fight viral diseases. Till this day there is no information available an how the ZYMV changes the ultrastructure of the pumpkin cells and in which parts of the plants and cells the strongest ZYMV induced alterations occur. Certain antioxidants like vitamin A, vitamin E or the reduced forte of glutathione (GSH) protect the plants during viral infections. GSH for example has been found responsible for the removal of toxic metabolites and for the protection of intact cells from oxidative damage after a pathogen attack in plants. lt has also been shown that GSH is able to inhibit the replication of human viruses which makes GSH important as a medical product and as an additive for pharmaceuticals. However the mechanisms behind the protective roles of GSH during a viral infection in humans, animals and plants are not yet clear. Therefore the aim of this project is to localize GSH within the pumkin cells and to investigate changes in GSH concentrations by using different microscopical techniques (fght-, fluorescence- and eleetron microscopy). This should help us to understand where elevated GSH concentrations occur during viral infections and how GSH fights the virus induced oxidative stress. Additionally the research should clear whether artificially elevated GSH concentrations suppress the ZYMV- disease in Styrian pumpkin plants as it has been shown in other plant species. The results obtained from the present research will provide new and relevant information an cellular changes and an the protective role of GSH in ZYMV infected plants, which could help to protect the Styrian pumpkin industry from severe crop losses in the Future.

The present study was aimed to investigate the effects of Zucchini Yellow Mosaic Virus (ZYMV) infection on the ultrastructure of single cells and on glutathione contents in Styrian oil pumpkin plants. Studies with the transmission electron microscope (TEM) revealed that ZYMV-infection led to severe ultrastructural changes in single leaf cells (cylindrical inclusions, proliferated endoplasmatic reticulum) and to a strong decrease in the amounts of chloroplasts. As chloroplasts are the main centers of photosynthesis, these changes can lead to the development of symptoms such as yellowing, stunting etc. Even though virus particles were found in roots, cells of this organ did not show ultrastructural alterations. Biochemical and cytohistochemical studies were used to investigate possible ZYMV-induced changes in glutathione contents in leaves and roots to find possible protective roles of glutathione in Styrian oil pumpkin plants during ZYMV-infection. With the TEM it was possible for the first time to study the distribution of glutathione within single cells. Single intact cells of ZYMV-infected leaves showed strongly increased glutathione-contents even though levels of glutathione were decreased in the whole organ. In older leaves, which developed strong symptoms of ZYMV-disease glutathione contents were strongly decreased in comparison to younger ones. Roots from ZYMV-infected plants also showed strongly decreased glutathione contents when compared to uninfected plants. Therefore the hypothesis was tested whether the artificial elevation of glutathione can lead to an increased protection of Styrian oil pumpkin plants against ZYMV-disease. The artificial elevation of glutathione leads to a massive reduction and a total absence of symptoms in ZYMV- infected Styrian oil pumpkin plants. Three weeks after ZYMV-infection 60% of plants with artificial elevated glutathione contents showed no symptoms and 40% of those plants showed mild symptom of ZYMV-disease. All plants that did not show artificial elevated glutathione contents developed strong and medium symptoms of ZYMV-diseases. Six weeks after ZYMV-infection all of these plants developed strong symptoms. At this time 20% of plants with artificial elevated glutathione contents showed no symptoms and only 30% showed mild symptoms. The present study revealed that elevated glutathione contents protect plants against virus attack and lead to the absence and suppression of symptom development. The results of the present investigations can now be used to develop new resistant strategies against virus infections. The present studies lead to numerous publications and international presentations and the "Erzherzog-Johann-Forschungspreises 2005" was awarded to Bernd Zechmann by the Styrian government.