Cellular distribution of ascorbate, glutathione, salicyliate

During growth and development plants are constantly exposed to different environmental conditions. If these conditions have a negative impact on plant metabolism they can be defined as abiotic (e.g. excess light, exposure to heavy metals, drought, salt stress) or biotic (e.g pathogen attack) stress. Plants have developed various mechanisms to adapt to such stress conditions, which are essential for plant survival. One of the most important protection systems against environmental stress situations is the ascorbate-glutathione cycle that allows plants to detoxify reactive oxygen (ROS) and reactive nitrogen species (RNS), which are commonly formed during environmental stress situations, and could otherwise damage cellular components. Changes in the antioxidative system are therefore commonly used as stress marker in plant science. The most important antioxidants are ascorbate and glutathione. They play important protective roles by detoxifying ROS and RNS, which would otherwise lead to the destruction of biological membranes, proteins, RNA and DNA leading to mutation, cancer and eventually cell death. Glutathione is also involved in the detoxification of various herbicides, xenobiotics and heavy metals which are bound by glutathione, transported and sequestered in(to) vacuoles. Another important substance for plant defense is salicylic acid. It is well known as signaling molecule in establishing and signaling defense reaction against pathogens. The ability of salicylic acid to signal stress responses is also affected by ROS and therefore the homeostasis of ascorbate, glutathione and salicylic acid in different organs, tissues and cell compartments is extremely important for plant defense. Within plants ascorbate, glutathione and salicylic acid synthesis and degradation underlies highly compartment specific pathways. Nevertheless, changes of these components during abiotic and biotic stress conditions have mostly been measured in whole organs, rather than in single cells and organelles, and therefore its compartment specific importance during abiotic stress situations is largely unexplored. Therefore the aim of the present study is to use cytohistochemical and biochemical investigations (transmission electron microscopy, high performance liquid chromatography) to study changes in single cells and organelles and on the whole tissue level during selected abiotic and biotic stress situations (excess light, exposure to heavy metal, drought, salt stress, Pseudomonas infection). Additionally, the defense marker gene PR-1, phytochelatins and the subcellular accumulation of ROS will be measured during these stress conditions. In order to test the hypothesis whether different levels of ascorbate, glutathione and salicylic acid in different cell compartments play an essential role in plant defence during the above mentioned stress situations the data will be compared between different stress sensitive Arabidopsis mutants and the wildtype. Additionally, the hypothesis will be tested that high levels of glutathione in certain cell compartments (e.g. in plastids during high light conditions, drought and salt stress; in the cytosol during heavy metal exposure) are essential in the protection of plants against abiotic stress situations. The data that will be obtained from the described investigations should give a deeper insight into the importance of ascorbate, glutathione and salicylic acid in certain cell compartments for cell survival strategies during the proposed abiotic and biotic stress situations and could help to develop new defense strategies for agricultural use in the future.

The negative impact of climate change on plant yield and food production will be the biggest problem that society will have to solve in the next decades. Antioxidants such as ascobate and glutathione play important role in the protection of plants against environmental stress as they are involved in the detoxification of reactive oxygen species, redox signaling, the modulation of defense gene expression and important for the regulation of enzymatic activities. The aim of this project was to analyze the subcellular importance of ascorbate and glutathione during different abiotic stress conditions (high light, drought, high salinity and exposure to heavy metal) in order to characterize subcellular adaptation strategies of plants to environmental stress. The results of this project revealed that both ascorbate and glutathione play important roles in the detoxification of reactive oxygen species produced in chloroplasts and peroxisomes during high light, drought and salt stress. It revealed for the first time that hydrogen peroxide leaks into and accumulates in vacuoles and that ascorbate is the major antioxidant that detoxifies hydrogen peroxide in vacuoles under these stress conditions. Higher sensitivity of plants to heavy metals (e.g. cadmium) could be correlated to low subcellular glutathione levels rather than low ascorbate contents confirming the importance of glutathione in the detoxification of heavy metals. Peroxisomes could be identified as the hotspot of glutathione accumulation and the collapse of the antioxidative system in peroxisomes could be correlated with the progress of diseases development during infection of Arabidopsis with bacteria (Pseudomonas syringae). The data obtained from this project helped to characterize the compartment specific importance of ascorbate and glutathione for cell survival strategies during high light stress, drought, high salinity and exposure to heavy metals and can now be used to develop new defense strategies for agricultural use in the future.