Quantitative sensors for phytohormone signalling systems

Microbiosensors are very small probes used to measure the concentrations of important signals in living tissue and in real time. Their small size is advantageous because the data collected can be associated with more precise tissue placements and less trauma is induced during sensor placement. In order to be useful, biosensors need to be selective to the signal being measured and highly sensitive in order to record the low concentrations of these signals in living samples. Advances in electrochemistry technologies at Warwick have recently allowed the development of good microbiosensors for some neurotransmitters, signals important for brain function. Some plant hormones are part of the same family of chemical signals as the neurotransmitters. The cytokinins are phytohormones which, amongst other roles, help determine the development and size of the storage tissue in seeds. We have made a prototype microbiosensor for cytokinins using the same Warwick fabrication technologies. An enzyme that occurs naturally in plants known as cytokinin dehydrogenase (CKX) reacts with cytokinins to denature them. This enzyme has been purtified and encased in a permable glass layer on a coated microelectrode. When dipped into cytokinins the consequent electrical signal is dependent on both the presence of cytokinins and on their concentration. These prototype biosensors work well, but need improving. This project is about how we will test for improvements in their sensitivity. Our microbiosensors for cytokinin may be improved by using alternative forms of the enzyme with much higher activity, all available to us through our collaborators in Olomouc, Czech Republic. We will test different electrode coatings in order to improve connectivity and test methods to give nanostructure to the electrode to increase its surface area without increasing its size. Having optimised the cytokinin biosensor we will test it on plant samples. Initially we will dip it into sap droplets which form when the stem is cut off a root system. Known concentrations of cytokinin can be added to validate calibrations. A more exacting test will be to insert one of these microsensors into the storage tissue of a seed. A microincision into the seed coat will give access and we will evaluate the performance of both off-the-shelf ATP sensors in this new environment, and then the cytokinin sensors to record real-time changes in hormone signal strength with time and relate this to seed size. We know from other work that high seed cytokinins give bigger seeds, by understanding how and when the signal arrives we may be able to inform strategies for improving food security. Not all plant tissues are as soft and pulpy as seed storage tissue. We will evaluate how to make these biosensors more rugged so that they can be deployed directly into plants. We will test tungsten wires (stronger than platinum), microcatheters for delivering the sensor tip from a protected cover, and evaluate the prospects of making the sensors in glass micrcapillaries - which are known to be good for plant cell impalements, including single-cell measurements. Development of a microbiosensor for cytokinins will be a singularly impressive advance. Ruggedised biosensors based on the proven ATP framework will illustrate that this biosensor technology can be applied to address many other outstanding questions in plant biology. Our experiments will also illustrate how cytokinins drive seed filling and how we might use this information for food security. In short, the biosensors will provide the type of precision data needed to inform current and future studies on manipulating plant seed size and yield potential. Other sensors will follow.

The Dale lab at Warwick has developed unique biosensor technologies for measuring neuroactive purines in real time both in vitro and in vivo, in particular ATP and adenosine. The Napier lab has brought the prospect of using cytokinin dehydrogenase (CKX2) as a sensor for the purine-based cytokinins. Cytokinins are phytohormones crucial for cell division and, in the developing seed, the development of the endosperm. Endosperm is not only a rich storage tissue for the seed, but it is in constant communication with the embryo to coordinate development. Part of this communication is via cytokinins and the Guttierez-Marcos lab has shown that cytokinin concentrations help determine seed size and, hence, yield potential. This key role of cytokinins in seed development make it essential that we understand these signalling exchanges. Cytokinin biosensors will provide vital information for crop improvement programmes as nations address the challenges of future food security. Our pilot study has demonstrated the feasibility of a reagentless cytokinin biosensor based on CKX2. This project will improve the prototype. We will test alternative CKX isoforms offering over ten-fold higher specific activity, test alternative mediators and unglycosylated CKX to improve sensitivity. If necessary we will also test nanostructured electrodes. The biosensors will be tested in living plant tissues. Early tests will use root-pressure-driven sap drops. The main test will use the seed endosperm after a microincision through the seed coat, initially with the proven Dale ATP sensors (ATP is an extracellular signal in plants), then with the cytokinin sensors. Calibration and longevity will be assessed, and parallel samples validated by mass spectrometry. Methods for ruggedizing the biosensors for routine and direct deployment in plants will be tested, including tungsten wires, microcatheters and redesigning the sensors into glass microcapillaries.

Who will benefit from this research? The immediate impact will be realised by the academic communities of plant biology and biosensor electrochemists. The exploitation of reagentless biosensor will encourage and inspire electrochemists around the globe. The deployment of real-time, quantitative biosensor for cytokinins working in living tissues will fire the interest and imaginations of the developmental plant biology community. It will also capture the attention of those in systems biology who need more physiological data with which to train and test their models. The project is being undertaken by a lab that has previously launched a successful spin-out company from their technology developments, Sarissa Biomedical Ltd. Currently dealing with biosensors developed and used widely by the animal and biomedical research communities, Prof Dale is ideally placed to evaluate the potential of the new tools and resources being developed, the reagentless platform and phytohormone biosensors. It is likely to take more than a single year of exposure and exploitation to generate a new market within the plant community, but Sarissa (Dale and Tian) have recently (Sept 2010) held their first training workshop. Attended by users/potential users from across the world, this was a great success. They plan further events in future years. We will evaluate the potential for a plant sensor workshop once we have published the credentials of the new tools in refereed journals - therefore this will be past the lifetime of this short project. The PI is already in correspondence with other potential academic partners about the focus of future collaborative project proposals. As noted above, development of the tools alone will not be sufficient to convert the plant developmental biology community to biosensor measurements. We will need to demonstrate and validate the microbiosensors in a range of challenges. There is no shortage of interest. Dr Tian will benefit directly as his career portfolio of techniques and exposure to quite different biological problems (from neurobiology to plat developmental biology) are enlarged. How will they benefit from this research? There is one specific biological question we address in this project, that of CK concentration measurement in the endosperm during seed development. One Co-I (Guttierez-Marcos) has data suggesting that seed size is CK-dependent. Our experiments with the early sensors will help validate this, will help add the values of temporal resolution and CK quantitation in situ to other data. As this story is completed, this will attract public interest and will contribute to future strategies for food security. The PI will make sure that, when appropriate (incl after acceptance by a peer review process) this story will be presented to the widest audiences. This part of the work may have a considerable impact on public policy decisions and the development of strategic plans to exploit this knowledge. IP will be protected, as appropriate. By this route, there is a good chance that the data collected during this project, and by the tools we develop after this project, will enhance the quality of life and health of many people. It may take some further years to engineer or select food lines in elite crop varieties that will exploit the new knowledge, but once realised the benefits will accrue for many years to come.