ABNORMAL POLLEN TUBE GUIDANCE1, an Endoplasmic Reticulum-Localized Mannosyltransferase Homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in Yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in Human, Is Required for Arabidopsis Pollen Tube Micropylar Guidance and Embryo Development

Abstract

The perception and response of pollen tubes to the female guidance signals are crucial for directional pollen tube growth inside female tissues, which leads to successful reproduction. In pursuing the mechanisms underlying this biological process, we identified the Arabidopsis (Arabidopsis thaliana) abnormal pollen tube guidance1 (aptg1) mutant, whose pollen tubes showed compromised micropylar guidance. In addition to its male defect, the aptg1 mutant showed embryo lethality. APTG1 encodes a putative mannosyltransferase homolog to human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast (Saccharomyces cerevisiae) GLYCOSYLPHOSPHATIDYLINOSITOL10 (GPI10), both of which are involved in the biosynthesis of GPI anchors. We found that APTG1 was expressed in most plant tissues, including mature pollen, pollen tubes, mature embryo sacs, and developing embryos. By fluorescence colabeling, we showed that APTG1 was localized in the endoplasmic reticulum, where GPI anchors are synthesized. Disruption of APTG1 affected the localization of COBRA-LIKE10, a GPI-anchored protein important for pollen tube growth and guidance. The results shown here demonstrate that APTG1 is involved in both vegetative and reproductive development in Arabidopsis, likely through processing and proper targeting of GPI-anchored proteins.

Figure 1.

Pollen viability, germination, and tube growth of the heterozygous aptg1/+ mutant and wild-type pollen. A, Schematic representation of APTG1 genomic organization and the T-DNA position in SALK_080854. Exons of APTG1 are represented by black boxes and introns by lines. The location of the T-DNA insertion of SALK_080854 is indicated with the triangle. Primer LB1.3 is for the left border of the T-DNA, and RP1 and LP1 are gene-specific primers at upstream and downstream locations, respectively. B to E, Mature pollen grains of the wild type (B and D) and aptg1/+ (C and E) with Alexander (B and C) and DAPI (D and E) staining. F and G, Mature pollen grains of the wild type (F) and aptg1/+ (G) observed by SEM. H and I, Wild-type (H) and aptg1/+ (I) pollen germination in vitro. J and K, Pollen germination ratio and pollen tube length in vitro for 4 h, showing no significant differences between the wild type and the aptg1/+ mutant (Student’s t test, P > 0.05). Bars = 20 µm.

Figure 2.

Guidance of the heterozygous aptg1/+ and wild-type pollen tubes analyzed with Aniline Blue staining. Emasculated wild-type pistils were pollinated with limited amounts of wild-type pollen (A) or aptg1/+ pollen (B–E) and observed 24 h after pollination. A, Wild-type normal pollen tube guidance. B, aptg1/+ pollen tube winding among ovules without entering a micropyle. C, aptg1/+ pollen tube twisting around the micropyle but without entering it. D, aptg1/+ pollen tube twisting around the micropyle but eventually entering it. E, Two aptg1/+ pollen tubes grow toward the micropyle. One pollen tube (pt1) enters the micropyle, but the other (pt2) fails to reach the micropyle. F, Percentage of normal and abnormal guidance of pollen tubes in the wild type and the aptg1/+ mutant. The pollen tubes showing abnormal guidance in aptg1/+ include two types: growth on the surfaces of the ovules but missing the micropyle (nontargeting) and twisted growth around the micropyles before finally entering them (targeting). The numbers at the ends of the black bars indicate the percentages of different types of pollen tubes. Statistical analysis of pollen tube guidance revealed that there were no differences between normal and abnormal guidance in the aptg1/+ mutant (Student’s t test, P > 0.05). Data were collected from three independent experiments. Arrowheads indicate the pollen tubes, and arrows indicate the growing apices of pollen tubes. Bars = 100 µm.

Figure 3.

Early embryo development in the aptg1/+ pistil. A to F, Embryo development 3 d after limited pollination using aptg1/+ pollen and emasculated aptg1/+ pistils. A, Globular embryo showing normal development. B to F, Putative homozygous mutant embryos showing abnormal development at different stages, including retarded development at the two-cell stage (B), amorphous zygotic tissue (C), retarded apical cell division (arrow; D), abnormal suspensor cell (arrow) at the globular stage (E), and a degenerating globular embryo (F). G to K, Embryo development 4 d after limited pollination using aptg1/+ pollen and emasculated aptg1/+ pistils. G, Heart-shaped embryo showing normal development. H to K, Putative homozygous mutant embryos showing abnormal development at different stages, including a retarded and degenerating two-cell embryo (arrow; H), abnormal transverse division (arrows) of the apical cell (I), abnormal oblique division (arrows) of the apical cell (J), and a degenerating globular embryo (arrow; K). Pseudocolor was used to depict the young embryos in A to E and G. Bars = 20 µm.

Figure 4.

APTG1 encoding a mannosyltransferase. A, Predicted transmembrane domains of APTG1. aa, Amino acids. B, Phylogenetic analysis of APTG1 (NP_568305.1) with its homologs in A. lyrata (XP_002873694.1), Brachypodium distachyon (XP_003571962.1), Dictyostelium purpureum (XP_003285528.1), Drosophila sechellia (XP_002035230.1), G. max (XP_003542300.1), human (AAH17711.1; PIG-B), Macaca mulatta (XP_002804830.1), M. truncatula (XP_003610240.1), Oryza sativa (NP_001043404.1, Os01g0580100), Physcomitrella patens (XP_001758335.1), Rattus norvegicus (EDL84114.1), R. communis (XP_002513232.1), yeast (DAA07968.1; GPI10), V. vinifera (XP_002271446.1), Xenopus laevis (NP_001089512.1), and Zea mays (ACL54567.1). C, Alignments of APTG1 and its homologs in A. lyrata (XP_002873694.1), G. max (XP_003542300.1), Z. mays (ACL54567.1), human (AAH17711.1; PIG-B), and yeast (DAA07968.1; GPI10) showing their conserved motif, HKEXRF (underlined). D, Full-length protein sequence alignment between APTG1, PIG-B in human (AAH17711.1), and GPI10 in yeast (DAA07968.1). Identical amino acids are labeled in black. E, APTG1 complements the thermosensitivity of the yeast mutant gpi10. The yeast YGL142c thermosensitivity strain (gpi10 mutant) and the wild-type strain BY4741 could grow at 25°C. But the yeast YGL142c thermosensitivity strain could not grow at 37°C, although the BY4741 strain could. Compared with transformation with the empty vector pYES2, the yeast YGL142c TS strain transformed with pYES2 harboring APTG1 complementary DNA could grow at 37°C.

Figure 5.

Expression pattern of APTG1. A, qRT-PCR analysis of APTG1 expression in seedlings, leaves, roots, stems, flowers, siliques, and mature pollen grains. B to F, J, and K, GUS staining analysis in transgenic plants of Pro_APTG1::GUS showing the expression of APTG1 in the seedling (B), leaf (C), root tip (D), inflorescence (E), pistil (F), immature pollen (J), and mature pollen (K). The inset in K is a magnified mature pollen grain. G, H, I, L, and M, GFP fluorescence analysis in transgenic plants of Pro_APTG1::APTG1-GFP showing the expression of APTG1 in the embryo sac (G), torpedo embryo (I), and pollen tube (L). H and M are bright-field images of G and L, respectively. Bars = 10 µm (K inset), 50 µm (D, G, H, J–L, and M), 100 µm (F and I), 1 mm (B and E), and 5 mm (C).

Figure 6.

Subcellular localization of APTG1. A, Colocalization analysis of APTG1 and ER in the Arabidopsis meristem zone of root expressing 35S::APTG1-GFP and the ER marker ER-rb. A1, A2, and A3 show APTG1-GFP, ER-rb, and merged images, respectively. B, Colocalization analysis of APTG1-GFP and ER in Arabidopsis pollen tubes expressing Pro_APTG1::APTG1-GFP stained with ER-Tracker Red. B1, B2, B3, and B4 show APTG1-GFP, ER-Tracker Red, merged, and bright-field images, respectively. B5 shows the Pearson correlation coefficient (R) analysis. Bars = 10 µm.

Figure 7.

Localization of citrine-COBL10 in pollen tubes of the aptg1/+ mutant. A, Citrine-COBL10 showing normal PM localization (arrow) in the tip of the aptg1/+ pollen tube expressing Pro_COBL10:SP-citrine-COBL10. B, Citrine-COBL10 showing abnormal PM localization (arrow) in the tip of the aptg1/+ pollen tube expressing Pro_COBL10:SP-citrine-COBL10. C, Percentage of pollen tubes showing normal and abnormal PM localization of citrine-COBL10 (n = 453) in the wild type and the aptg1/+ mutant. D, Relative fluorescence intensity through the red lines in the tips of pollen tubes showing normal (A) and abnormal (B) PM localization expressing citrine-COBL 10 in aptg1/+. Bars = 10 µm.