Final Report Summary - ARSEEDAS (Alternative splicing of NADPH-Oxidases as a mechanism for seed dormancy regulation) This interdisciplinary project brought together a top-class, young researcher from Germany, Dr. Kerstin Mueller, with two world-leading experts on complementary aspects of seed biology: Prof. Michael Holdsworth at Nottingham University, UK, and Prof. Allison Kermode at Simon Fraser University, Canada, to work on the molecular mechanisms of seed dormancy breakage and the transition to germination. This is a very active field for current research and this project has made significant contributions to scientific understanding of these vital processes.Seeds of many species, including economically important crops and trees, can enter a state of dormancy in which they fail to germinate, even under ideal conditions. Dormancy can be broken by species-specific methods such as after-ripening (air-dry storage at room temperature), or moist chilling. When dormancy is broken, seeds have the potential to germinate if they encounter favourable conditions. If they do not, dormancy is reinstated, and can then in turn be broken again. This is known as dormancy cycling. The mechanisms underlying this economically and environmentally important process were not well understood. This project investigated (1) the role of reactive oxygen species (ROS) in dormancy regulation and its interplay with plant hormone signalling, (2) the regulation of enzymes that produce ROS, in particular NADPH Oxidases (rbohs), on the epigenetic as well as the post-transcriptional level, and their effect on the transition from dormancy to germination, and (3) the role of ROS in the protein stability of key plant hormone signalling components. Three species were selected for this work: Arabidopsis thaliana, a model species with a sequenced genome and a large collection of resources available to the scientific community, Lepidium sativum (garden cress), a model system used specifically for the study of seed dormancy and seedling growth, and the conifer Callitropsis nootkatensis (yellow cypress, formerly called yellow-cedar), an economically important species whose very deep dormancy is pivotally affected by climate change, and on which Prof. Kermode's lab has worked in the past. (1) Our investigation of the effect of alterations in the expression of ROS producing enzymes and the resulting decrease in ROS production in garden cress revealed a novel connection between ROS and the plant hormone auxin and demonstrated its role in seedling root growth: Roots of garden cress seedlings which had been genetically modified to produce less ROS showed strong developmental phenotypes that we correlated with a reduced expression of auxin marker genes. This is supported by other current discoveries that point to a role of ROS in auxin signalling, and our work is at the forefront of publications providing experimental evidence for this phenomenon. In addition, our investigation of the water uptake and distribution in transgenic seeds of our model species led to a very fruitful collaboration with the company Bruker, a European manufacturer of high end analytical machines. We created a new protocol which allows magnetic resonance imaging (MRI) of these processes not only in cress seeds, but also in the much smaller seeds of Arabidopsis. Scientists had previously been unable to use MRI technology in the study of seeds this small, so this breakthrough constitutes a significant technical advance in extending the use of 1H-MRI to major model species..(2) Our research elucidated some of the different levels of regulation of Rbohs and seed dormancy genes. Our previous work had revealed that alternative splicing of rbohB in Arabidopsis contributed to seed dormancy release and this discovery prompted this part of our investigation. Extensive experiments on the post-transcriptional regulation of Rbohs in Arabidopsis and garden cress indicate that this alternative splicing seems to be a phenomenon unique to Arabidopsis. We concluded that post-transcriptional modifications of other members of the Rboh family in Arabidopsis, and in other species, do not play a major role in the regulation of dormancy and germination. Our creation and optimisation of a novel method for chromatin immunoprecipitation in seed material enabled us to be the first group to successfully investigate the epigenetic regulation of key dormancy regulators and ROS-producing enzymes during the transition from dormancy to germination. We found that modifications which histones undergo in response to environmental conditions contribute strongly to the regulation of the transcription of plant hormone related genes, and we hypothesise that they can serve as a memory mechanism for the environmental conditions to which a seed has been exposed. This process seems to be evolutionarily conserved between species as distant as yellow cypress and Arabidopsis, which indicates its key relevance in the plant life cycle. Further experiments showed that specific histone modifications are removed and added as the seed cycles in and out of dormancy. These results allowed us to narrow down the candidate mechanisms for the epigenetic memory of environmental conditions in seeds. The most commonly studied system for studying epigenetic memory in plants is currently the flowering process. This system has the disadvantage of being a one-way process. The seed dormancy cycling experimental system which we developed will be a very useful addition to this field of research , since it has the great advantage of being a cyclical process that can be controlled in laboratory conditions. (3) Based on recently published findings and the extensive experience of the Holdsworth group, we hypothesised that the stability of specific groups of proteins, among them many hormone signalling components relevant to the transition of seeds from dormancy to germination, might be altered by ROS produced by rbohs. These proteins are potential substrates of the N-end rule pathway, in which proteins are recognised, ubiquitinated and degraded based on their N-terminal amino acid sequence. Specifically, in proteins starting on methionine-cysteine (MC) the cysteine can be oxidised, which leads to the recognition of the protein by the N-end rule pathway and its degradation. We found that exposure of MC proteins in Arabidopsis seedlings to ROS (superoxide and hydrogen peroxide) alone is not sufficient to lead to protein degradation, but that the reactive nitrogen species NO is needed in addition to ROS. This adds a level of control and fine tuning to the reaction of the plants to their environment through the modification of the stability of key proteins.With the successful conclusion of this Marie Curie Outgoing Fellowship in 2014, we now have a comprehensive overview of the role of ROS and the regulation of ROS producing enzymes during the transition from seed dormancy to germination and early seedling growth. We have made three major technical advances that will benefit the plant science community: (1) the development and publication of our seed chromatin immune precipitation protocol, (2) the development of a protocol on small seed water uptake imaging, which will certainly be of interest to both industry and academic researchers, and (3) the establishment of seed dormancy cycling as a model system for epigenetic research. We have also contributed to the growing body of publicly available knowledge about seed dormancy and germination, which will be the basis of future developments in agriculture, forestry and plant conservation efforts.