Annexin1-mediated calcium signalling controls plant responses to ozone

Levels of ozone as a ground-level pollutant threaten crop and pasture productivity. European crop losses are worth several billion Euros annually. The frequency of ozone "episodes" (when local conditions promote significant ozone elevation during the growing season) is set to increase. Ozone reduces shoot (and root) growth, causes leaf loss and affects grain development. Ozone can also exacerbate the effects of drought on plants. With drought affecting large areas of the UK, how plants defend against ozone must be understood to combat this problem.

Plants respond to ozone by generating an internal signal that causes changes in the genes that are expressed. It can also lead to cell death. An increase in calcium is an important component of this signal. We have discovered a mutant plant that cannot generate this calcium signal. By comparing the mutant with its non-mutated wild type, we can use the mutant as a tool with which to reveal the changes that occur in response to ozone from the genetic to whole plant level. We know the protein that is missing in the mutant and will test for its function in critical types of cell. We will also assess its importance to a plant's ability to regulate its water and gas relations and grow under ozone stress alone or combined with drought. These comparative studies will also identify genes likely to be of use in breeding programmes for ozone-resistant crops.

Ozone is a significant phytotoxic pollutant that is predicted to inflict increasing economic damage to agriculture in a changing climate. A transient increase in cytosolic free calcium that acts as a second messenger is one of the earliest plant response to ozone. We have identified an Arabidopsis calcium channel mutant in which the calcium signal is abolished in response to 300 ppb ozone. The calcium signal in the wild type is known to control production of reactive oxygen species and links to cell death. Our recent work has shown that the calcium signal also directs the transcriptional response to ozone in Arabidopsis.

In this project we will first establish which cells are responsible for generating the calcium signal in wild type Arabidopsis by using lines expressing aequorin or yellow cameleon 3.6 as cytosolic free calcium indicators. We shall then compare to the equivalent mutant lines in a dose-response study to determine the operational range of the channel in the ozone response. Electrophysiological studies will be undertaken for tractable cell types (supporting the calcium signal in wild type) to confirm that the channel is ozone-responsive. The effect of the mutation on the stomatal response to ozone will be determined under normal and droughted conditions. The consequences for water-use efficiency, gas exchange and productivity will also be determined. Leaf cell death assays will be undertaken to test whether the loss of calcium signal in the mutant protects against cell death. Finally, comparative transcriptomic analysis of wild type against mutant will delineate the extent to which the channel and calcium signal command the genetic response to ozone.

Ozone is an increasing threat to crop, pasture and forest productivity. This project addresses critical and fundamental events in the plant response to ozone pollution which might be exploited commercially for the development of crops with improved ozone tolerance. We will identify the mechanism by which an ozone-induced calcium signal is generated in plants and its role in the transcriptional response to ozone and the initiation of cell death. We shall also link calcium signalling to water-use efficiency and productivity. The outputs of this research will have an immediate impact on researchers and stakeholders engaged in plant cell biology, calcium signalling, stress responses, immunity and the effects of ozone on plant productivity. In the longer term, the overall outputs will have an impact for plant breeders, those in the agriculture, horticulture and forestry sectors and also policy makers concerned with the control of ozone pollution. The work is relevant to UK and European food security and highly relevant to the sustainability of crop production in Asia where farming practices put harvests at risk of ozone stress. We shall utilise our University Research Services plus our existing links to stakeholder networks, industry and policy makers to disseminate our findings, thus ensuring best possible impact in a timely manner whilst also protecting IP as appropriate.

How will academic and commercial stakeholders benefit?

We will identify which cells of the plant are important in sensing ozone and signalling its presence via calcium, allowing future researchers to use these as targets for manipulating the ozone tolerance of crops. These studies will benefit from the availability of a mutant which does not exhibit an ozone- calcium signal and which we can use to delineate the calcium-dependent transcriptomic response to ozone (without recourse to a pharmacological dissection). This will inform how the remodelling of the transcriptome by ozone might be exploited to improve crop ozone tolerance. The increased understanding of the routes by which ozone induces cell death resulting from our research will provide data that could be utilised in future to mitigate the deleterious effects of ozone. This also has relevance for the use of ozone fumigation in post-harvest protection of foodstuffs. The studies of stomatal responses to ozone under drought stress that we propose will address a new area of ozone research that is highly relevant for the development of predictive models for crop productivity under climate change and for the development of robust crops. In addition, the similarity between the responses triggered by ozone and plant immune responses highlight the potential for cross-over of the results of our research with research into plant defence and its applications. That our work is on Arabidopsis is an advantage in that we will be generating data from a tractable plant that provides a basis for crop improvement (Nature Biotechnology 30,360).

Wider impact

We shall engage a range of strategies to ensure that our research has an impact on the school community and general public, detailed in the "Pathways to Impact". In addition to standard outreach practices for local schools, we shall work with the Science and Plants for Schools project in order to contribute to national teaching resources. We shall disseminate our findings during National Science Week and also seek to engage the public through Royal Society outreach events.