VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract

Abstract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.

Figure 1.

Phenotype of the vgd1 Mutant Compared with the Wild Type. (A) A vgd1 mutant plant with reduced fertility indicated by the smaller siliques with a few seeds. (B) A vgd1 silique showing that the seeds were produced only in the upper part of the silique. (C) A wild-type plant with full fertility indicated by the siliques with a full seed set. (D) A wild-type silique showing the full seed set. (E) A vgd1 flower with normal floral organs and pollen grains. (F) A wild-type flower with normal floral organs and pollen grains. sd, seeds. Bars = 1 cm in (A) and (C) and 1 mm in (B) and (D) to (F).

Figure 2.

Electronic Microscopic Observation of vgd1 Pollen Tubes Compared with the Wild-Type Pollen Tubes. (A) Scanning electron microscopy of vgd1 pollen tubes on wild-type stigma. (B) Scanning electron microscopy of wild-type pollen tubes on wild-type stigma. The arrow indicates the invasion of a pollen tube into a stigmatic cell. (C) TEM of a papillary cell transversal section, showing a vgd1 pollen tube inside the cell wall of the papillary cell. (D) TEM of a papillary cell transversal section, showing a wild-type pollen tube inside the cell wall of the papillary cell. (E) TEM of a style transversal section, showing a vgd1 pollen tube inside the transmitting tract. (F) TEM of a style transversal section, showing a wild-type pollen tube inside the transmitting tract. cw, cell wall; pc, papillary cell; pg, pollen grain; pt, pollen tube; stg, stigmatic cell; tt, transmitting tract. Bars = 10 μm in (A) and (B), 2 μm in (C) and (D), and 1 μm in (E) and (F).

Figure 3.

Growth Patterns of the vgd1 Pollen Tubes in Female Floral Tissues Compared with Those of Wild-Type Pollen Tubes. (A) Growth pattern of the wild-type pollen tubes in the wild-type female organs 4 hap, showing that the pollen tubes had penetrated through the style tissue and reached the upper end of transmitting tract. (B) Growth pattern of the vgd1 pollen tubes in the wild-type female organs 4 hap, showing that the pollen tubes had not been able to penetrate through the style tissue. (C) Growth pattern of the wild-type pollen tubes in the wild-type female organs 12 hap, showing that the pollen tubes almost had reached the base of the transmitting tract. (D) Growth pattern of the vgd1 pollen tubes in the wild-type female organs 24 hap, showing that the pollen tubes had reached only approximately one-fifth of the transmitting tract length. (E) Growth pattern of the vgd1 pollen tubes in the wild-type female organs 48 hap, showing that the pollen tubes had reached approximately half of the transmitting tract length. st, style; tt, transmitting tract. The arrows indicate the pollen tubes. Bars = 200 μm.

Figure 4.

Morphology and in Vitro Germination of vgd1 Pollen Compared with Wild-Type Pollen. (A) Scanning electron microscopy of wild-type pollen grains. (B) Scanning electron microscopy of vgd1 pollen grains. (C) to (E) Growing vgd1 and wild-type pollen tubes. (C) The unusual shape of the vgd1 pollen tube. (D) A wild-type pollen tube. (E) A burst of a vgd1 pollen tube. pt, pollen tube; pg, pollen grain. Bars = 10 μm in (A) and (B) and 20 μm in (C) to (E).

Figure 5.

The Genomic Organizations of VGD1 and the Structure of VGD1 Protein. (A) Genomic organization of the VGD1 gene. The numbers over the vertical lines indicate the positions of the start and end nucleotides of the fragment in the BAC clone. The boxes indicate the positions of exons, and the intervening lines indicate the positions of introns. The numbers in the boxes indicate the sizes of the fragments. The arrow points out the Ds insertion site. (B) The structure of VGD1, showing the positions of the secretive signal peptide (S), PMEI, and PME domains. The arrow indicates the Ds insertion site. (C) Genomic organization of the VGD1 locus and the Ds insertion. The numbers under the vertical lines indicate the positions of start nucleotides of the fragment in BAC clone F14M4. The numbers in the boxes indicate the sizes of the transcribed regions.

Figure 6.

The Alignment of VGD1, At2g47030, and At3g62170 Proteins. The black boxes indicate the identical amino acids. The arrowhead points out the Ds insertion site in VGD1.

Figure 7.

Promoter Activities of VGD1, At2g47030, and At3g62170 Monitored by GUS Activity and Subcellular Localization of VGD1-GFP. (A) Expression of VGD1, At2g47030, and At3g62170 in the wild-type and vgd1 flowers revealed by RNA gel blot hybridization, showing that VGD1 was expressed only in wild-type floral tissue (3), whereas At2g47030 and At3g62170 were expressed in both vgd1 and wild-type floral tissues (1 and 2), respectively. Thirty micrograms of total RNA was loaded in each lane. The bottom panels are the loading controls showing the 18S RNA. WT, wild-type flower total RNAs; vgd1, vgd1 flower total RNA; WM, RNA molecular weight markers; 1, hybridized with At2g47030-specific probe; 2, hybridized with At3g62170-specific probe; 3, hybridized with VGD1 (At2g47040)-specific probe. (B) The P_VGD1-GUS-T_NOS transgenic pollen grains, showing the GUS activity in the pollen grains. (C) The P_At2g47030-GUS-T_NOS transgenic pollen grains, showing the GUS stain in pollen grains. (D) The P_At3g62170-GUS-T_NOS transgenic pollen grains, showing the GUS stain in pollen grains. (E) The P_VGD1-GUS-T_NOS transgenic pollen tubes, showing the GUS stain in the transgenic pollen tubes germinating in the wild-type stigmatic tissues. (F) The P_At2g47030-GUS-T_NOS transgenic pollen tubes, showing the GUS stain in the transgenic pollen tubes germinating in the wild-type stigmatic tissues. (G) The P_At3g62170-GUS-T_NOS transgenic pollen tubes, showing the GUS stain in the transgenic pollen tubes germinating in the wild-type stigmatic tissues. (H) A transgenic pollen tube (arrow) showing that the VGD1-GFP fusion protein is distributed throughout the whole pollen tube. The picture was merged from a GFP signal and an image using transmitted light. pg, pollen grain. (I) A section of a transgenic pollen tube showing the localization of VGD1-GFP fusion protein in the wall of the pollen tube after plasmolysis. The picture was scanned for GFP signal without light transmission. cw, cell wall; cp, cell plasma. The arrow indicates the pollen tube wall. Bars = 100 μm in (B) to (G) and 25 μm in (H) and (I).