Classification of EA1-box proteins and new insights into their role during reproduction in grasses.

KEY MESSAGE: EA1-box protein classification. Success in reproduction and vegetative development in flowering plants strongly depends on precise cell-to-cell signaling events mediated by secreted peptides.A small peptide family named as EA1-like (EAL) has been first described 10 years ago and includes EA1 involved in pollen tubes attraction by the female gametophyte and EAL1-regulating germ cell identity in maize. EALs consist of an N-terminal endoplasmic reticulum-targeting motif, the highly conserved EA1-box and a short C-terminal alanine-rich domain. Whereas EAL peptides are exclusively found in the Gramineae, the EA1-box is widely distributed throughout the plant kingdom. Based on in silico analysis and subcellular localization studies, we report here a new classification of EA1-box proteins in flowering plants. They can be distinguished into three protein classes: the already defined EAL proteins, the EAG (EA1-box glycine-rich) proteins and the EAC (EA1-box containing)proteins. While fusion proteins of EAL and EAC classes locate to the secretory pathway, EAGs are cytoplasmic and locate also to the nucleus. Moreover, we further show that the third EAL protein of Zea mays, EAL2, appears to be also involved in processes related to late embryogenic development as its peptide level increases after formation of leaf primordia. Immunohistochemical studies indicate its presence in the scutellar parenchyma and around the vasculature, where it is secreted to the extracellular space. In conclusion, the members of the maize EAL family possess very diverse functions during reproduction and it will now be exciting to elucidate the functions of EAGs and EACs in plants.

Identifying plant cell-surface receptors: combining 'classical' techniques with novel methods.

Cell-cell communication during development and reproduction in plants depends largely on a few phytohormones and many diverse classes of polymorphic secreted peptides. The peptide ligands are bound at the cell surface of target cells by their membranous interaction partners representing, in most cases, either receptor-like kinases or ion channels. Although knowledge of both the extracellular ligand and its corresponding receptor(s) is necessary to describe the downstream signalling pathway(s), to date only a few ligand-receptor pairs have been identified. Several methods, such as affinity purification and yeast two-hybrid screens, have been used very successfully to elucidate interactions between soluble proteins, but most of these methods cannot be applied to membranous proteins. Experimental obstacles such as low concentration and poor solubility of membrane receptors, as well as instable transient interactions, often hamper the use of these 'classical' approaches. However, over the last few years, a lot of progress has been made to overcome these problems by combining classical techniques with new methodologies. In the present article, we review the most promising recent methods in identifying cell-surface receptor interactions, with an emphasis on success stories outside the field of plant research.

Overcoming hybridization barriers by the secretion of the maize pollen tube attractant ZmEA1 from Arabidopsis ovules.

A major goal of plant reproduction research is to understand and overcome hybridization barriers so that the gene pool of crop plants can be increased and improved upon. After successful pollen germination on a receptive stigma, the nonmotile sperm cells of flowering plants are transported via the pollen tube (PT) to the egg apparatus for the achievement of double fertilization. The PT path is controlled by various hybridization mechanisms probably involving a larger number of species-specific molecular interactions. The egg-apparatus-secreted polymorphic peptides ZmEA1 in maize and LURE1 and LURE2 in Torenia fournieri as well as TcCRP1 in T. concolor were shown to be required for micropylar PT guidance, the last step of the PT journey. We report here that ZmEA1 attracts maize PTs in vitro and arrests their growth at higher concentrations. Furthermore, it binds to the subapical region of maize PT tips in a species-preferential manner. To overcome hybridization barriers at the level of gametophytic PT guidance, we expressed ZmEA1 in Arabidopsis synergid cells. Secreted ZmEA1 enabled Arabidopsis ovules to guide maize PT in vitro in a species-preferential manner to the micropylar opening of the ovule. These results demonstrate that the egg-apparatus-controlled reproductive-isolation barrier of PT guidance can be overcome even between unrelated plant families.