The control of specificity in guard cell ROS-based signalling

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Over the millennia plants have evolved mechanisms that allow then to adapt to changing environmental conditions. At the heart of these responses are systems that enable plants to detect changes in their environment and then to formulate the appropriate response to the changed conditions. At the level of the single cell changes are detected by receptors and then a complex intracellular machinery is responsible for the elicitation of the appropriate intracellular response. This process is known as stimulus-response coupling (or intracellular signalling). At the heart of this machinery are Reactive Oxygen Species (ROS). When a cell reacts to an external stimulus the concentration of the ROS inside the cell increases. This acts as an intermediate, or trigger, leading to the generation of the final response. The ubiquity of ROS as intermediaries involved in the responses to a plethora of different stimuli raises an important question and this is; how can the increase in ROS elicit specific responses? This nature of the problem can be readily understood by considering one example.Stomata are pores on the surfaces of leaves that open and close in response to changing environmental conditions. The stomatal pore is formed by two guard cells. When these shrink the pore closes whereas swelling results in opening. Stomata are important because they control carbon dioxide uptake and water loss. In guard cells stimuli that bring about swelling (opening) or closure (shrinking) both use intracellular signalling pathways that involve an increase in ROS. How does this work? Unravelling how response specificity is controlled in a single cell is one of the big and unresolved questions in plant cell signalling. Previously making measurements of ROS inside cells has been problematic, however in this application we are making use of step-change advances in technology developed in one of our labs to provide an unprecedented understanding of ROS dynamics in single cells. We believe that we have an international lead in this area, accordingly this is a very timely application, bringing together the new technology from Exeter and the biological system (stomata) in Bristol to find answers to a major unresolved question. Our hypothesis, backed by our preliminary data, is that different stimuli generate unique patterns of ROS inside cells. We call these ROS signatures. These are then decoded by the intracellular machinery inside the cell to produce the specific response. In this application, we will use our new technology to test this hypothesis. We will also find out the origin(s) of the ROS increase and whether these differ between stimuli and we will also investigate what parts of the stomatal opening and closure mechanism are controlled by increases in ROS. Finally we will investigate the interaction between another intracellular signal (calcium) to reveal the extent to which response specificity is controlled by the interaction of ROS and Ca signalling pathways.