Mannosyltransferase ABNORMAL POLLEN TUBE GUIDANCE1 Is Essential For Arabidopsis Pollen Tube Micropylar Guidance And Early Embryo Development

Pollination is an important process for plant sexual reproduction and is also the premise for producing seeds. In the process of plant pollination, following pollen grains landing on the stigma, the germinated pollen tubes grow through the stigma and style, and reach the ovules where they release sperms into the embryo sac for double fertilization. The pollination processes include a number of important scientific problems, but a little is known about them, such as how the pollen tubes perceive and response to the female attraction signals, then grow to the ovules.To understand molecular mechanism of pollen tube guiding growth in pistil, we isolated an Arabidopsis transfer DNA(T-DNA) insertion mutant showing abnormal pollen tube guidance which was named abnormal pollen tube guidance1(aptg1). The T-DNA was inserted in the fifth intron of the genomic sequence of AT5G14850 in this mutant. The progeny genotypes of the heterozygous mutant plants were identified, but we did not find homozygous plants in these self-progenies, and the genotype ratio between the wild type and heterozygous plants was nearly 1:1. We made reciprocal crosses between the wild-type plant and the heterozygous mutant and examined the genotypes of the progeny. We found that 96.9% of the mutant allele was transmitted via the female gametophytes, while only 8.3% of transmission occurred via the male gametophytes. These results suggested that the male gametophytes in this mutant line were defective. No difference of mature pollen grains in viability or morphology was detected between the aptg1/+ mutant and the wild-type plants. And the aptg1 mutation does not result in abnormal pollen germination or pollen tube growth in vitro. When the emasculated wild-type pistils were pollinated with limited amounts pollen of aptg1/+, about half of pollen tubes showed abnormal guidance near the micropyle. These pollen tubes showing abnormal guidance can be arbitrarily divided into two categories: growth on the surfaces of the ovules or the funiculus but missing the micropyle, and twisted growth around the micropyles before finally entering them. These results suggest that mutant pollen tubes were compromised in micropylar guidance. In the aptg1/+silques 3 and 4 d after limited self-pollination, the most of the early embryos reached the globular or heart-shaped stages, but the development of about 11.2% of embryos was retarded at the globular embryo or earlier stages, indicating that aptg1 mutant resulted in abnormal embryo development at early stages.qRT-PCR and GUS staining analysis showed that APTG1 was expressed in most of tissues, such as mature pollen grians, pollen tubes, mature embryo sacs, and developing embryos. The expression of APTG1 in mature pollen grains was much higher than that in young ones. Fluorescence co-labeling analysis revealed that APTG1 protein was localized in the endoplasmic reticulums.Phylogenetic analysis and protein sequence alignments indicated that APTG1 is a homolog of human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast GLYCOSYLPHOSPHATIDYLINOSITOL10(GPI10), both of which are mannosyltransferases involving in the biosynthesis of glycosylphosphatidylinositol(GPI) anchor. The CDS of APTG1 could complement the thermosensitive phenotype of the gpi10 mutant, indicating that APTG1 is a functional homolog of yeast GPI10, and may be involved in the biosynthesis of GPI anchors.Arabidopsis COBRA-LIKE10(COBL10) is a GPI-anchored protein localized in apical plasma membrane and cytoplasm in growing pollen tubes, and it is important for pollen tube growth and guidance in pistil tissue. We found that disruption of APTG1 led to abnormal localization of COBL10 in the apical plasma membrane of pollen tube, implying that the APTG1 involved-biosynthesis of GPI anchor is important for the COBL10 localization in apical plasma membrane of growing pollen tubes.The Arabidopsis seedling growths were inhibited remarkably when the expression of APTG1 suppressed by using artificial microRNA technology. This result implies that APTG1 is important for the vegetative development of Arabidopsis.In conclusion, our results suggested that APTG1 function in pollen tube guidance, embryo development and seedling growth in Arabidopsis, likely through the process of GPI-anchored proteins.