The Egg Apparatus-Specific Gene ZmEA1 Encodes a Signalling Protein Required for Micropylar Pollen Tube Guidance in Maize (Zea mays L.)

The development and function of the female gametophyte (embryo sac) as well as double fertilisation are fundamental issues in plant reproductive biology. Although extensively studied at the cytological level, little is known about the molecular and genetic processes controlling embryo sac development and function. For example, the molecules secreted by the female gametophyte to attract the male gametophyte are unknown. The aim of this work was to identify genes highly and specifically expressed in the female gametophyte and to perform functional analysis with a gene encoding a possible secreted protein as a ligand for cell-cell communication processes. From 988 maize egg cells ESTs generated during this thesis, it was selected the novel and second highly expressed female gametophyte-specific gene ZmEA1 (Zea mays Egg Apparatus1). RT-PCR analyses with different maize tissues showed that ZmEA1 is exclusively expressed in the egg apparatus, consisting of the egg cell and two synergids. ZmEA1 does not contain introns and encodes a small protein of 94 amino acids with a predicted transmembrane domain. The ZmEA1 promoter (ZmEA1p) was isolated as 1570 base pairs of genomic sequence 5' of the AUG initiation codon. The specificity and functionality of the ZmEA1p was analysed in transgenic maize and rice plants containing the GUS coding sequence under the control of the ZmEA1p. Using histochemical GUS-assays, GUS activity was exclusively detected in the egg apparatus of unpollinated mature ovules in three independent functional ZmEA1p::GUS transgenic maize lines. GUS transcripts were also detected by RT-PCR in pistils of three different transgenic rice lines demonstrating that the ZmEA1 promoter is also functional in rice. A ZmEA1:GFP C-terminal fusion protein regulated by the ZmEA1p was secreted from the egg apparatus to the micropylar region of the nucellus of four independent transgenic lines in a manner dependent on floral developmental stage. GFP signals were faint in unfertilised young ovules, shortly after silk emergence and increased in unfertilised mature ovules (silk elongation ->10 cm) generating a gradient from the filiform apparatus (egg apparatus cell walls) towards the surface of the micropyle. Confocal laser scanning microscopy (CLSM) observations confirmed a presence of a cell wall localised ZmEA1:GFP fusion protein within the nucellus cells of the micropyle. 24 h after in vitro pollination (around 18 h after fertilisation), signals of the fusion protein were no longer detectable in the samples analysed. RNA interference (RNAi) and antisense (AS) approaches have been applied to study the function of ZmEA1 in maize. Most transgenic lines with complete transgene integrations showed a significant reduction of seed set upon selfing (0-75%), but full seed set (100%) after crossing pollen to wild type plants indicating that the female gametophyte but not the male gametophyte is affected. Four RNAi lines with incomplete transgene integrations displayed a seed set comparable to the wild type. In vitro pollination studies showed that the fertilisation efficiency of RNAi plants was significantly reduced (40-55%) compared to wild type plants (82%), although ovary, ovule and female gametophyte development appeared not to be affected. Detailed microscopic analyses showed that pollen tubes were visible at the surface of the inner integument of all transgenic ovules analysed, but the majority of them did not enter the micropyle and in most cases grew at random directions at the surface of the inner integument. These types of loss of pollen tube guidance were never observed in wild type ovules. Based on observed phenotypes, it can be concluded that reduction of ZmEA1 activity in the egg apparatus affects micropylar, but not long range pollen tube guidance in about half of the ovules. In summary, ZmEA1 represents the first gene discovered to be specifically expressed in the egg apparatus of a plant species and encodes for a short-range signalling protein required for micropylar pollen tube guidance in maize. Thus, ZmEA1 is also the first female gametophyte pollen tube attractant discovered so far.