The control of specificity in guard cell ROS-based signalling

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.

How is response specificity controlled in plant ROS-based signalling systems? We will use the guard cell as a model to investigate this question because stimuli leading to stomatal opening AND closure use increases ROS in their stimulus response coupling pathways. Previous work on ROS in plant cell signalling has been hampered by the lack of probes able to report ROS dynamics with high spatial and temporal accuracy. Through BBSRC-funded work we have developed recombinant ROS indicators that we will target to relevant cellular compartments and which will report ROS dynamics with unprecedented fidelity. We will use this breakthrough to test our new hypothesis: whether specificity in plant ROS-based signalling systems results from the generation of stimulus specific (in spatial and temporal terms) increases in ROS that we call ROS signatures. We will investigate, the origin(s) (location and mechanism) of the ROS signatures and find out whether they differ between different stimuli and investigate which sub-modules in the guard cell signalling network are controlled by the ROS system. We will also investigate whether there is cross-talk between the ROS and Ca2+ signalling systems.

We will answer the following questions:

1. Do light, ABA, ATP, increased CO2 and reduced relative humidity (RH) induce stimulus-specific guard cell ROS signatures?
2. What are the cellular origin(s) of the increases in ROS induced by light, ABA, ATP, increased CO2 and reduced RH?
3. What is the role of ROS removal mechanisms in generating response specificity?
4. Does the Ca2+ signature modify stimulus-induced increases in ROS?
5. Which guard cell signalling sub-modules are regulated by ROS?

We have an international lead in this area as we have developed new ROS imaging technology and proposed a new hypothesis. Accordingly we feel that our work is both timely and highly internationally competitive because it will answer an important unanswered question in plant biology.

Impact Summary

Who will benefit from this research?
Apart from the community of plant science researchers interested in fundamental questions concerning stimulus-response coupling in cells, there are two other communities who will benefit from the results of this work. Because stomatal research impacts on water use efficiency it is likely that crop breeders and industrialists will benefit from the work. Producing more water use efficient plants is an international target and insights from the work described here are likely to be of value because they will lead in the ability to impose fine -level control of stomatal aperture. Secondly, in this application we will be using new technology. This is likely to be of real interest to industry as it is highly applicable to use in high-throughput screens for, for example novel herbicides. The other group who should benefit from this work are climate change modellers. Stomatal dynamics are not well incorporated in any future climate models. This is important because of the clear involvement of stomata in the global carbon dioxide and water cycles.

How will they benefit?
Crop breeders and industrialists are likely to benefit because 1. we will generate new insights into the fine control of stomatal opening and closure 2. our characterization of how ROS signals control stomatal and in particular are involved in specific responses may result in the identification of new targets for agrochemical intervention / improvement through conventional breeding 3. our technological advance should allow the development of new high-throughput screens for the identification of valuable agrochemicals and 4. climate change modellers will benefit because our work will highlight the highly dynamic behaviour of stomatal aperture and how it responds to environment change; our results should provide the modellers with the justification and incentive for seeking more robust parameters to account for stomatal behaviour and inserting these into the underlying equations on which their models / simulations are based.