Analysis of cell-cell communication during reproductive processes by EA1-like peptides in maize

Signaling processes mediated by secreted peptides are of eminently importance in cell-cell-communication of plants. This work focuses on extracellular signaling peptides containing the EA1-box and their involvement in control of reproductive processes in maize (Zea mays L.). In maize, the EA1-box protein ZmEA1 was already demonstrated to act as a secreted signaling peptide in micropylar pollen tube guidance, whereas the closely related peptide ZmEAL1 is necessary for cell fate determination of the female gametophyte. To extent our knowledge about EA1-box proteins, several plant genomes of monocotyledonous and dicotyledonous plants were searched for this motif and EA1-box proteins could be identified in all of the analyzed plant species. Based on similarities of structural features and subcellular localization, a new classification and phylogenetic analysis is presented, dividing these proteins into three classes, the EAL, EAG and EAC proteins. The EA1-like (EAL) proteins, including ZmEA1 and ZmEAL1, consist of less than 200 amino acids and are predicted to be secreted using either an N-terminal or internal signal sequence. Besides the EA1-box, they contain the so-called P-box and a C-terminal alanine-rich region as conserved motifs. All analyzed EAL proteins were demonstrated to enter the secretory pathway in transiently transformed plant suspension cells. In contrast, the EA1-box containing glycine-rich (EAG) proteins show cyto-plasmic localization and are expected to act as intracellular components. The third class, the EA1-box containing (EAC) proteins, includes EA1-box proteins that could not be classified as EALs or EAGs and contains proteins with up to several transmembrane domains. The expression pattern of rice EALs was analyzed and comparison with maize EALs indicated that these proteins might act as orthologs. Furthermore, the final member of the small maize EAL family, ZmEAL2, was analyzed in detail and was demonstrated to exhibit a broader expression pattern compared to ZmEA1 and ZmEAL1, and showed a remarkable strong expression pattern in the embryo during later stages of development. Using immunohistochemical analysis, ZmEAL2 was localized in the scutellar parenchym and surrounding the vascular system of the embryo. Taken together, these results suggest a role of ZmEAL2 during embryogenesis. To gain deeper insights into the role of ZmEA1 during micropylar pollen tube attraction, it was expressed as GFP-fusion protein in the synergids of Arabidopsis thaliana. Interestingly, dissected transgenic Arabidopsis ovules placed on solid media were demonstrated to attract in vitro germinated maize pollen tubes, whereas no impact on tube growth direction could be detected for wild-type ovules. As this indicates the direct binding of secreted ZmEA1 protein to the pollen tube, germinated maize pollen was further incubated with predicted mature ZmEA1 (sEA1), labeled with a fluorophore to visualize the interaction. The peptide bound to the surface of the pollen tube apex and was internalized quickly, probably for degradation. To identify the interaction partner(s) located on the pollen tube surface binding to sEA1, a large number of pull-down experiments were performed. Immunoblot analysis of the isolated frac-tions indicated binding to one or more interaction partners, which should be identified by Orbitrap mass spectrometry. Three different protein sequences were identified as candidates for interaction with sEA1 and named as WHY SO LATE (WSL) proteins. Contrary to the expected membranous proteins, WSL proteins represent putative secreted cysteine-rich peptides. They were shown to enter the secretory pathway in transiently transformed tobacco leaves. WSL1 is encoded by two genes which were named as WSL1a and WSL1b. WSL1a/b and WSL3 protein sequences are highly similar, therefore they were classified to form a small protein family sharing structural similarities to defensin-like proteins (DEFLs). All WSL peptides are strongly and specifically expressed in pollen and are exclusively found in maize and the closely related grass Sorghum bicolor. First attempts were performed to confirm the interaction of WSL peptides with sEA1. Taken together, potential factors acting in the pathway of ZmEA1-signaling could be identified and were made available for further research. In case, the direct binding between ZmEA1 and WSL peptides as well as the involvement of this complex in micropylar pollen tube guidance can be demonstrated in future, this would be the first report of a heterodimeric signaling ligand generated both by male and female gametophytes, which would provide a highly specific interaction representing a prerequisite for a molecular mechanism contributing to reproductive isolation barriers in plants