Evolution of gametophytic communication systems by analyzing RALF and ROS signalling

The evolution of reproductive organs and gametes of land plants was accompanied by numerous changes and adaptations of gametic/gametophytic communication systems. While gametes of opposite mating types of single celled algae like Chlamydomonas communicate in a watery environment, bryophytes and ferns generate mobile sperm cells, which are attracted by egg cells in a moist environment. Adaptations to dry environments in gymnosperms and angiosperms were accompanied by loss of sperm cell mobility and the invention of pollen grains (male gametophyte), that protect and carry sperm cells by wind or animals over long distances to female flower organs. Moreover, pollen grains form a tube that growths deeply into the maternal flower organs to release its sperm cell cargo for fertilization inside the embryo sac (female gametophyte). The complexity of gametic cross-talk in single celled algae towards gametophytic cross-talk in angiosperms has enormously increased during land plant evolution. By using the angiosperm model plants Arabidopsis and maize, a lot of progress has been made in the past 15 years to elucidate molecules and mechanisms involved in gametophyte communication. Especially secreted polymorphic cysteine-rich peptides (CRPs) appear to play a central role during pollen tube germination, growth, guidance and reception. Among these, pollen tube-specific RALF (Rapid ALkalinization Factor) peptides were recently shown to regulate pollen tube integrity and sperm release in Arabidopsis. While single celled algae do not contain RALF genes, an ancient RALF clade consisting of a few genes exists in mosses, and the number of RALF genes was strongly amplified during evolution to 37 genes, for example, in Arabidopsis. Until now, to our knowledge, only RALF genes related to the ancient clade have been functionally characterized and activate ROS production in Arabidopsis. The aim of this project therefore is to contribute to the understanding of the evolution of gametophytic cross-talk by studying two gene families and their interaction(s). (i) We will study the evolution, diversification and specification of members of the RALF gene family including functional studies of ancient RALFs in a moss and a novel clade in maize lacking the typical RALF peptide signature. Studies will also include complementation analyses of Arabidopsis and maize pollen mutants using RALFs from different land plant. Interaction studies will be performed with known receptors. (ii) We will further investigate and compare the conservation of RALF-triggered ROS production in mosses and angiosperms in collaboration, and (iii) study major ROS-producing RBOH genes in papilla hair cell of maize after pollen adhesion.