ROS Bursts in plant growth and fertility: evolution and diversification of the NADPH oxidase family

Land plant evolution has shaped fundamental changes in the alternation of generations and the fertilization mode. Whereas bryophytes possess a dominant haploid generation and fertilization occurs between flagellated spermatozoids and egg cells in archegonia on gametophytes, gametophytes in angiosperms are reduced to a low number of cells. Here, fertilization has become independent of water, but requires the interaction of tip-growing pollen tubes with the embryo sac enclosed in the ovule. The local generation of reactive oxygen species (ROS) is involved in cell wall remodelling and signalling processes on both the male and female side of reproduction. In angiosperms, Respiratory Burst Oxidase Homologs (RBOHs) have been identified as the source of specific extracellular ROS generation. The first plant RBOHs appeared before the transition to land in green algae and the protein family has continuously expanded during land plant evolution, suggesting functional diversification. The precise role of RBOH isoforms and local generation of extracellular ROS for plant growth and reproduction remains unknown in an evolutionary perspective. This project will investigate (1) the ancestral biological function of RBOH plant homologs and (2) the sub- and neo-functionalisation of RBOH within model species and between evolutionary distant plant model species. We will generate null mutants for the two RBOH isoforms of the liverwort Marchantia polymorpha and the four RBOH homologs of the moss Physcomitrium patens via reverse genetics and quantify the resulting phenotypes regarding plant growth and fertility. In order to specify and quantify ROS-dependent fertility defects, we will use and further develop intracellular and extracellular dynamic sensing of local ROS generation via genetically encoded redox biosensors. In addition, we will investigate expression domains and subcellular localisation of the four P. patens isoforms via reporter constructs. Complementation studies with algal and bryophyte RBOH homologs in A. thaliana rboh mutants with reduced tip growth and fertility will allow us to conclude on functional diversification across large evolutionary distances. Thus, this project will specify the role and functional evolution of RBOHs in local ROS generation in the apoplast during sexual reproduction in different land plant lineages. This will allow a better understanding of the complex redox processes occurring during angiosperm fertilization, as well as reveal the evolutionary roots of RBOH function in plants.