Role of glutathione and its precursors during ZYMV-infection

The agricultural use of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca GREB.) has a historical tradition in Styria (Austria) and plays an important regional role in agriculture since the European Union protected the Styrian oil pumpkin oil as a selected European specialty. Since 1996 the Zucchini yellow mosaic virus (ZYMV), causes severe losses of up to 60 % in Styrian pumpkin production every year. With the transmission electron microscope (TEM) it is possible to detect ZYMV-induced changes in plant cells even before symptoms are visible from the outside. Such investigations can be used to develop strategies and products to protect plants against pathogen attack. Increased concentrations of the antioxidant glutathione are known to protect plants against reactive oxygen species, which are formed in response to environmental stress situations such as pathogen attack. An accumulation of glutathione during various infections with fungal and bacterial pathogen has been reported for different plants. However, information about changes in glutathione contents during compatible and incompatible virus attack is rare and there is no information available about changes in the subcellular distribution of glutathione precursors during pathogen attack. Therefore, the aims of the present project proposal are to investigate changes in the subcellular distribution of glutathione and its precursors cysteine, glycine and glutatmate within roots, younger and older leaves of control, resistant (incompatible) and non resistant (compatible) ZYMV-infected Cucurbita plants. Differences in contents of glutathione and its precursors between resistant and non resistant plants will provide new insights into how glutathione synthesis and degradation is involved in the development of resistance. Immunogold labeling will be performed by using selective primary antibodies against glutathione, cysteine, glutamate and glycine and gold conjugated secondary antibodies which will be detected with the TEM. Together with the results we obtained in the previous project about changes in the subcellular distribution of glutathione during compatible ZYMV-infection, the results gained during these investigations will contribute towards a deeper understanding of the mechanisms behind the protective roles of glutathione during compatible and incompatible virus infection. This knowledge could help to protect Styrian pumpkin plants against severe epidemics of ZYMV- disease and, in turn, save the Styrian pumpkin industry from severe crop losses in the future. In collaborative studies we will apply the above described methods to transgenic and non-transgenic model plants (Arabidopsis thaliana and Nicotiana tabacum), which will enable us to compare the data from our studies with studies made on those model plants by other research teams. Cysteine-overexpressing tobacco plants from the lab of Prof. R. HELL, Germany will be used to study the impacts of cysteine-overexpression on the subcellular distribution of glutathione and will reveal into which cell compartments the excess of cysteine is translocated. -glutamylcysteine synthetase (-ECS)-overexpressing Arabidopsis thaliana plants, which show an increase in glutathione contents of about 1.5-fold based on the whole leaf, will be used from the lab of Prof. F. MAUCH, Switzerland. The obtained data will be compared with controls and Arabidopsis thaliana mutants that show a reduction in glutathione contents of about 75% based on the whole leaf. Differences in labeling density of glutathione and its precursors should reveal the impact of -ECS-overexpression on subcellular glutathione and its precursor levels. These collaborative studies will strongly advance our understanding of glutathione metabolism and redox regulation and will help to develop new scientific questions for further projects.

During the project novel methods were developed, that allow the visualization and quantification of glutathione and its precursors (cysteine, glutamate and glycine) in all cell compartments simultaneously in one experiment at a high level of resolution. Glutathione is an antioxidant and involved in the detoxification of reactive oxygen species, which are commonly formed during environmental stress situations, and lead to the destruction of biological membranes, proteins, RNA and DNA leading to mutation, cancer and eventually cell death. Changes in glutathione contents are therefore commonly used as stress markers during investigations in most fields of plant sciences. Nevertheless, as glutathione metabolism involves highly compartment specific pathways possible limitations in the ability of glutathione to protect the plant against stress situations can only be detected if glutathione contents are analyzed at the subcellular level. For this purpose an immunogold cytohistochemical approach was developed and adapted to different plant material in order to detect and quantify subcellular glutathione and its precursors with computer-supported transmission electron microscopy. These studies showed that the distribution of glutathione is similar in different plant species (Arabidopsis thaliana, Cucurbita pepo, Nicotiana tabacum, Beta vulgaris). Surprisingly, plastids, which have been described before as major site of glutathione accumulation, contained the lowest amounts of glutathione. Highest glutathione contents were always found in mitochondria, while glutathione-labeling in the cytosol, nuclei and peroxisomes was intermediate. No glutathione was detected in vacuoles and the apoplast. The accuracy of the glutathione-labeling method was supported by different observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles to background levels. Second, the overall glutathione-labeling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. These experiments also showed that mitochondria maintained high and stable glutathione levels during situations of glutathione deficiency which indicates that glutathione in mitochondria plays an important role in plants for cell survival. In further studies we were able to demonstrate that glutathione synthesis during pathogen attack is limited by the availability of glutathione precursors. Whereas a tolerant pumpkin species (which showed a stronger increase in glutathione contents than the susceptible one) contained increased levels of glutathione precursors during virus infection the susceptible one showed generally decreased levels. These results also indicated that cysteine might be the limiting factor for glutathione synthesis during compatible virus-infection. In further experiments we were able to demonstrate that the artificial elevation of cysteine led to a strong increase in glutathione contents and subsequently to a suppression of symptom development during compatible virus infection in Styrian oil pumpkin plants which was correlated with decreased amounts of viral particles within leaves and roots. Summing up, the development and application of these novel methods revealed that glutathione precursors (especially cysteine) limit the operation of glutathione metabolism during pathogen attack. The modification (increase) of cysteine contents in plants during pathogen attack resulted in a strong increase in glutathione contents and subsequently in a higher stress tolerance during virus infection in Styrian oil pumpkin plants. Thus these studies and methods can now be used for the development of new defense strategies for agricultural use in the future, and can protect farmers from possible crop losses induced by environmental stress situations in the future.