Development of plant-based hydrogen peroxide YFP nanosensors targeted to multiple sub-cellular locations

点击翻译按钮获取中文摘要

The oxygen (O2) we breathe is produced by plants when they photosynthesise. However, for cells that produce and/or consume O2 (by respiration) as a key part of their metabolism, there is an inherent danger and that is the production of reactive oxygen species (ROS). ROS arise as an inevitable consequence of O2 chemistry and if they accumulate, they cause oxidative damage to cell components (in particular chloroplasts and mitochondria) and can trigger the death of the cell. This is why plants make antioxidants, to limit the accumulation of ROS. One ROS, hydrogen peroxide (H2O2), is relatively stable while still being a powerful oxidant. H2O2 is used as a bleaching agent because of its powerful oxidising activity. H2O2 is made in plants as a bi-product of photosynthesis, respiration and many other chemical reactions that plant cells carry out. If it accumulates then, as with other ROS, it will cause oxidative damage. Evolution, though, has a habit of turning the potentially damaging into something useful. This is the case for H2O2. The accumulation of H2O2 in different parts of cells, before it attains damaging levels, acts to alter the expression of hundreds of genes by stimulating cellular signalling systems. H2O2 is an important cellular signalling molecule in bacteria, animal cells and especially plant cells. H2O2 stimulates cell signalling both internally and from cell-to-cell in response to many changes in the plant's environment, such as changes in light levels, wounding by herbivores and attack by pathogens. H2O2 is also used to regulate growth and development in plants, such as the development of secondary roots, the growth of pollen tubes and the hardening of cell walls. The intimate involvement of H2O2 in many aspects of plants' lives means it is imperative that we are able to locate and determine the changes in the level of H2O2 in different parts of the plant from the tissue down to the sub-cellular level. Until very recently this has not been possible. Knowing where, when and how much H2O2 accumulates is important in understanding if a plant is suffering oxidative damage or is actively signalling. The lack of technology for measuring H2O2 in real time, non-invasively and accurately means there are serious gaps in our understanding of how plants grow, reproduce and interact with their environment. Our aim is to provide the plant science community with means to locate and measure H2O2 at different sub-cellular locations in plant cells in real time. We can do this because a novel technology has been developed in which H2O2 can be specifically detected in cells using a genetically encoded protein sensor called HyPer. HyPer is a novel artificial protein which consists of a part (called a domain) of a bacterial protein called OxyR which changes shape when it specifically binds H2O2 .This OxyR domain is linked to a greatly modified fluorescent protein from a jellyfish, which changes its fluorescence characteristics in response to the change in shape of the OxyR domain. This fluorescence change, in response to H2O2, can be visualised by one of several types of microscope which allows the researcher to locate and measure changes in H2O2 concentration over time. HyPer has been shown to work in animal cells, bacteria and fish embryos. We have shown that HyPer works in exactly the same way in cells of roots and leaves of young seedlings. We aim to construct HyPer variants that will go to different locations in the cell so that researchers can build up a comprehensive picture of H2O2 accumulation in different tissues and conditions. However, HyPer expression is silent in older plants, which is common with other types of fluorescent sensors in plants. We have provided a number of solutions to this problem which will be deployed in this project to allow maximum and rapid uptake of this technology by the global plant science community, advancing knowledge of plant functions on a wide front.