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

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.

Plant cells produce hydrogen peroxide (H2O2) in many sub-cellular locations where it initiates signal transduction in response to many environmental or developmental cues. Despite its major importance in plant biology, the accurate measurement of H2O2 in vivo both temporally and spatially at the sub-cellular level has not been possible. However recently, a synthetic protein nanosensor has been developed called HyPer, which is a circular permuted yellow fluorescent protein harbouring the H2O2 binding domain of the E.coli transcription factor, OxyR. HyPer fluorescence at 516nm can be measured ratiometrically from two excitation wavelengths of 488nm and 405nm. Upon H2O2 binding, the F488nm/F405nm HyPer fluorescence ratio rises in proportion to H2O2 concentration. HyPer has been shown to accurately report oscillations in H2O2 levels in human cell lines and zebra fish embryos. We have shown that HyPer works in root and leaf cells of Arabidopsis seedlings. The main project aim is to construct chimaeric genes coding for HyPer that can be targeted to 12 sub-cellular locations in plant cells. This will allow researchers for the first time to determine the rate and location of H2O2 accumulation and disappearance. The expression of HyPer will be under the control of an inducible promoter to avoid gene silencing, which we have observed when HyPer is constitutively expressed. By minimising expression time, using a codon optimised version, co-inducing the P19 suppressor of gene silencing and using mutants defective in transgene silencing, HyPer expression will be optimised. This strategy allows for a range of outcomes and will maximise the uptake of the technology. The project will refine protocols for using a range of imaging platforms to measure HyPer fluorescence over the life of the plant, in deep tissues and under a range of environmental conditions. An active programme of dissemination will ensure a rapid uptake of this technology.

While this project is targeted to a specific, largely academic global community of plant science researchers, there are potential beneficiaries or impacts that could occur outside this immediate sphere and these are listed below. First of these is the global agricultural-biotech industry and biotech supply sector. Researchers in industry are as aware and as interested in the development of specific tools to aid their research as the academic sector. Second, industry needs skilled personnel. A key element in the knowledge transfer process is the training of researchers at all levels. The proposed research will enable training of two postdoctoral researchers to acquire or enhance their skills in highly advanced recombinant DNA technology and cell biology, project management, writing, presentation and web-based communication. Finally, SMEs supply both the academic and commercial biotech sector with specialised reagents, kits and services. It is possible that the extended range of plasmids generated by this project could be held and distributed under a suitable commercial licence, freeing up the Essex and Exeter groups from having to distribute the materials beyond the life of the project. The second group of beneficiaries is the General Public and in particular students. The outputs of this project will include many highly informative images and videos, which would be particularly useful to our outreach work with local Colchester and Exeter A-level and AS-level students. Our programme of dissemination planned for this project to ensure awareness within the academic community will apply equally to researchers in the agri-biotech industry. Within the first 6 months of the project we shall construct a website which will act as a forum, source of data, images and protocols. The website will be the contact point to build up a network of researchers globally engaged in the development of nanosensors. No later than 6 months after the project has ended, we shall run an international workshop that will bring in participants and speakers primarily interested in plant-based small molecule nanosensors. We would specifically issue invites to all our industrial contacts (made through the network) and encourage them to participate, alongside academic colleagues. Finally, we shall run a practical summer school, which will provide training to a selected group of no more than 15 early career researchers. Again, this will be open to those from industry as well as academia. If there was a mix of applicants we would ensure some participation from industrial researchers. At Essex and Exeter 'Science in Society' schemes organise talks to schools, colleges and the public to highlight the range of research activities in the biological sciences. These schemes encourage participation and contribution of stakeholders' views on current and future research, and facilitates environmental and research driven activities for local, regional and national schools, organizations and other interested parties. The potential for commercial exploitation of research will be assessed by experts from both University's Research and Enterprise Offices (REOs). Upon the award of a grant, REO staff will meet regularly with the research team at each site to assess potential commercial impact, to identify relevant organisations, partners and research. The PIs, Co-Is and the PDRAs will undertake the impact activities with the support of specialist support staff from the Universities of Essex and Exeter. Communications outside the scientific community will be directed in a coordinated manner by both universities' Communications Offices.