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Pollen tube guidance precedes the double fertilization of flowering plants. Here, we report the identification of a small maize protein of 94 amino acids involved in short-range signaling required for pollen tube attraction by the female gametophyte. ZmEA1 is exclusively expressed in the egg apparatus, consisting of the egg cell and two synergids. Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. Transgenic down-regulation of the ZmEA1 gene led to ovule sterility caused by loss of close-range pollen tube guidance to the micropyle.
In contrast to most animal and many lower plant species, sperm cells of flowering plants are nonmotile and are transported from the stigma to the female gametophyte (embryo sac) via pollen tube growth to allow double fertilization (1). Genetic and physiological studies have shown the involvement of both female sporophytic and gametophytic tissues in pollen tube guidance of different plant species (2). Molecules involved in sporophytic guidance have been identified as γ-aminobutyric acid (GABA), arabinogalactans, and small secreted proteins (3-6), but little is known about the molecules produced by the female gametophyte required for pollen tube guidance. The synergids have been identified as the source of producing a short-range pollen tube attractant or attractants in Torenia fournieri, but the molecular nature of the attractant(s) is still unknown (7).
We report the identification of ZmEAl (Zea mays EGG APPARATUS!) from an unfertilized maize egg cell cDNA library (8) and its role for short-range pollen tube attraction by the female gametophyte. ZmEA1 is produced by the cells of the egg apparatus and represents a highly hydrophobic small protein of 94 amino acids with a predicted transmembrane domain (Fig. 1A). Tissue and single-cell reverse transcription polymerase chain reaction (RT-PCR) analyses showed that ZmEA1 is exclusively expressed in the maize egg apparatus (Fig. 1, B and C) before fertilization. Lower RNA levels were detected in zygotes after in vitro fertilization and at an even lower level in two-celled proembryos. Expression of ZmEA1 was no longer detectable at later embryo stages, which suggests a rapid down-regulation of the gene after fertilization. In situ hybridization of the maize ovary (Fig. 2B) and of female gametophyte isolated...