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First published online 1 November 2006
doi: 10.1242/dev.02683

Development 133, 4761-4769 (2006)
Published by The Company of Biologists 2006
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Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

Kiera von Besser1,2, Aubrey C. Frank3, Mark A. Johnson3,* and Daphne Preuss1

1 Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Il 60637, USA.
2 Medical Scientist Training Program, The University of Chicago, Il 60637, USA.
3 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.


Figure 1
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  		Fig. 1. hap2 disrupts pollen tube guidance. (A) In vitro grown hap2/HAP2 pollen tubes stained to reveal GUS expression in hap2 pollen tubes. (B) Quantitative analysis of ovule targeting. ms1 pistils were hand-pollinated with hap2/HAP2 or control pollen heterozygous for LAT52:GUS. The number of ovules receiving GUS activity from the pollen tube was counted for each and is plotted as a percentage (±s.d.) of the total number of ovules. (C-F) ms1 pistils stained for GUS activity 14 hours after pollination. (C) An ovule that has received a GUS+ control pollen tube. (D) An ovule that has received a GUS+ hap2 pollen tube. GUS is released into synergids from tubes that successfully enter the micropyle and burst (arrowheads in C and D). (E,F) hap2 pollen tube tips that fail to enter the micropyle (arrows). m, micropyle; f, funiculus. Scale bars: 40 µm in A; 20 µm in C-F.

 

Figure 2
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  		Fig. 2. hap2 blocks egg fertilization and central cell fertilization. Siliques from self-fertilized plants heterozygous for a control T-DNA insertion (A) or hap2/HAP2, which contain aborted ovules (B, arrowheads). (C) An unfertilized ovule showing nuclei of FG cells. (D-I) Ovules following targeting by HAP2 (D,E,I) or hap2 (F,G,H) pollen tubes at either 48 (D-G) or 24 hours (H,I) after pollination. Early embryo and endosperm development are apparent in ovules targeted by HAP2 (D,E,I), but not hap2 (F,G,H) pollen tubes. Ovules stained for GUS activity (D,F) show that they have been targeted by either HAP2 (D, no GUS activity) or hap2 (F, GUS activity present in synergid) pollen tubes and that hap2 pollen tubes have burst in the targeted ovule. ccn, central cell nucleus; ecn, egg cell nucleus; scn, synergid cell nucleus; emb, embryo; arrowheads, endosperm nuclei. Arrows (C,F,G,H) denote an unfertilized central cell. Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. hap2 pollen contains a normal male germ unit. (A-D) Pollen tetrads imaged with bright field (A,C) or epifluorescence (B,D) following DAPI staining. The morphology of pollen grains, vegetative nuclei (arrows) and sperm nuclei (arrowheads) were indistinguishable in tetrads from wild-type (A,B) and hap2/HAP2 plants (C,D). Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. HAP2 and its predicted protein structure. (A) HAP2 has 17 exons (black rectangles, untranslated regions are gray). hap2-1, hap2-2 and gcs1 (Mori et al., 2006Go) are T-DNA insertions in exons 15, 9 and 16, respectively. Black lines under the gene schematic indicate regions used for molecular complementation (HAP2tr), analysis of expression pattern (HAP2promoter:YFP) and subcellular localization (HAP2protein:YFP). (B) HAP2 encodes a 705 amino acid protein with a predicted 19 amino acid N-terminal signal sequence (S), a 23 amino acid transmembrane domain (T) and a C-terminus containing a 96 amino acid histidine-rich domain (HIS). (C) An alignment of the HAP2 HIS-rich region from Arabidopsis (A. thaliana), poplar (P. trichocarpa), lily (L. longiflorum) (Mori et al., 2006Go) and rice (O. sativa). The alignment includes the region of the HAP2 C-terminal to the transmembrane domain. HIS residues within the HIS-rich region are shaded gray. Conserved residues are indicated with a star. Conservative amino acid substitutions are indicated with a dot. (D) Pairwise analysis of amino acid identity between Arabidopsis HAP2 and HAP2 sequences from other organisms; only HAP2 sequences from angiosperms have a C-terminal HIS-rich region.

 

Figure 5
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  		Fig. 5. HAP2 expression is sperm specific. (A) RT-PCR of wild-type tissues, using primers spanning HAP2 exon 2 (25-40 cycles). (B) Northern blot of wild-type RNA probed with full-length HAP2 cDNA; ethidium bromide stained gel before blotting shows relative RNA amounts in each lane. The source of each RNA sample is indicated between A and B. (C) A pollen tetrad from a plant heterozygous for the HAP2promoter:YFP fusion; DAPI fluorescence (left), YFP fluorescence (center), merged image (right). (D) DAPI (left) and YFP (right) fluorescence in pollen tetrads from qrt and homozygous HAP2promoter:YFP transgenic plants. Uninucleate microspores (UNM, left panels), bicellular pollen (BCP, center panels) and mature pollen grains (right panels) were analyzed. Autofluorescence from the pollen surface is observed in wild-type and transgenic pollen; signal from YFP is only observed in the sperm of mature pollen grains from transgenic plants. Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. HAP2 protein is sperm-localized during pollen development and tube growth. (A) A pollen tetrad and (B) sperm nuclei from a plant heterozygous for the HAP2protein:YFP fusion; DAPI fluorescence (left), YFP fluorescence (center), merged image (right). (C) DAPI (left) and YFP (right) fluorescence in pollen tetrads from homozygous HAP2protein:YFP transgenic plants at three stages of pollen development; see Fig. 5D for comparison with qrt. (D) DAPI (center) and YFP (right) fluorescence in growing pollen tubes from qrt, HAP2protein:YFP and LAT52:GFP transgenic plants. DIC images of growing pollen tubes (left). Signal from YFP is observed only in the sperm (HAP2protein:YFP) or throughout the entire pollen tube cytoplasm (LAT52:GFP). Scale bars: 20 µm A and C; 1 µm in B; 5 µm in C.

 

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