Molecular characterization of the glauce mutant: a central cell-specific function is required for double fertilization in Arabidopsis

Abstract

Double fertilization of the egg cell and the central cell by two sperm cells, resulting in the formation of the embryo and the endosperm, respectively, is a defining characteristic of flowering plants. The Arabidopsis thaliana female gametophytic mutant glauce (glc) can exhibit embryo development without any endosperm. Here, we show that in glc mutant embryo sacs one sperm cell successfully fuses with the egg cell but the second sperm cell fails to fuse with the central cell, resulting in single fertilization. Complementation studies using genes from the glc deletion interval identified an unusual genomic locus having homology to BAHD (for BEAT, AHCT, HCBT, and DAT) acyl-transferases with dual transcription units and alternative splicing that could rescue the sterility defect of glc. Expression of these transcripts appears restricted to the central cell, and expression within the central cell is sufficient to restore fertility. We conclude that the central cell actively promotes its own fertilization by the sperm cell through a signaling mechanism involving products of At1g65450. Successful fertilization of the egg cell is not blocked in the glc mutant, suggesting that evolution of double fertilization in flowering plants involved acquisition of specific functions by the central cell to enable its role as a second female gamete.

Figure 1.

The Double Fertilization Process in glc Mutant Embryo Sacs. Representative fluorescence microscopy micrographs of glc embryo sacs expressing GFP markers in the SCs (DD31) ([A] and [B]), EC (DD45) ([C] to [E]), and CC (DD65) ([F] and [G]). In all micrographs except (C), the green and red fluorescent channels were merged and basal autofluorescence was captured to ensure visualization of the embryo sac. Image (F) is part of a single confocal section (captured by a MRC-1024 confocal microscope). The complete Z-stack images can be found in Supplemental Figure 3 online. Emasculated glc/GLC flowers were pollinated with pollen carrying the HTR10 sperm-specific RFP marker. Images were captured at least 24 HAP. White dashed lines indicate the late zygote stage in (D) and the central cell vacuole (CCV) in (F). White arrows throughout the figure point to sperm nuclei (SN; note that in [A], the white arrow points to four sperm nuclei that belong to two pollen tubes arriving at the funiculus); in (F), white arrows points to the probable location of the central cell nucleus (CCN) and to a central cell cytoplasmic protrusion at the micropylar end of the embryo sac called the synergid hook (SH). An embryo is shown in (F). n = 40 ovules for DD31 in (A) and (B) each, 70 for DD45 in (C), 30 for (D) and (E) each, and 40 for DD65. Occurrence percentages are presented in white, at the top right-hand side of the images. Bars = 100 μm in (C), 50 μm in (A) and (B), 25 μm in (D), (E), and (G), and 20 μm in (F).

Figure 2.

Fine Mapping of the glc Deletion Region. (A) The 215-kb deleted region on glc chromosome 1 (Ngo et al., 2007). (B) Exact position of the JAtY BACs used in this study and of the reported hot deletion mutant (Kaya et al., 2000). (C) Close-up of the right border of BAC JAtY 63O08 and gene model of At1g65450, adapted from The Arabidopsis Information Resource. Black arrows indicate localization of T-DNA in mutant lines. (D) Genomic DNA regions cloned in this study indicated by horizontal black lines. UTR, untranslated region. (E) cDNAs cloned in this study are indicated by orange bars. (F) Intergenic regions (putative promoters) cloned in the study are indicated by horizontal black lines. Bar at the base of the figure is in kilobases and shared by (C) to (F).

Figure 3.

Tissue-Specific Expression Profiles of At1g65450 and Other Arabidopsis BAHD Members. (A) Gene expression in mature nonfertilized flower. Cross-section cartoons are presented, obtained from the BAR microarray database. (B) Gene expression in the different embryo sac cell types of mature unfertilized ovules, extracted from the Arabidopsis female gametophyte transcriptome (Wuest et al., 2010). Shown are three replicates per cell type (CC, EC, and SC). (C) Gene expression of 54 Arabidopsis BAHD members in the different embryo sac cell types of mature unfertilized ovules using data extracted from Wuest et al. (2010). Shown are three replicates per cell type (CC, EC, and SC) derived from the Arabidopsis female gametophyte transcriptome, as in (B). Red boxes in (B) and (C) indicate 450.2 and 450.3.

Figure 4.

Rescue of the glc Semisterility Phenotype by Genomic DNA of At1g65450.2 (450.2) and At1g65450.3 (450.3). Opened siliques of wild-type (wt; Ler), glc, and glc plants transformed with genomic DNA of 450.2 and 450.3. White arrows indicate aborted ovules (more aborted ovules are present underneath the seeds). At least 10 siliques per genotype were used for the measurements presented in Table 3.

Figure 5.

Amino Acid Sequences of At1g65450 cDNAs. The amino acid sequences of the three At1g65450 cDNAs (Ler). At1g65450.1 product (50.5 kD) corresponds to the combined cDNA, while the products of At1g65450.2 (18.7 kD) and At1g65450.3 (32.3 kD), which are derived from the nonoverlapping 5′ and 3′ mature transcripts, are shown aligned. Highlighted in yellow are regions of similarity between the amino acid sequences of At1g65450.2 and At1g65450.3 (asterisk = identity, period = hydrophobicity or size similarity, and colon = both hydrophobicity and size similarity). The conserved BAHD motifs (bold blue text) are found only in At1g65450.1 and At1g65450.3 products. There are residues (bold red text) that are unique to At1g65450.1 and At1g65450.3. The extra amino acids in the At1g65450.3 product contain a putative canonical NLS sequence (underlined) that is absent from the other products due to alternative splicing. The extra residues in the At1g65450.1 product are a short stretch of amino acids linking the front and back ends of the combined product.

Figure 6.

Alignment of At1g65450.1 Amino Acid Sequence to Members of Clade I of the BAHD Transferase Family. Two subsections of an amino acid sequence alignment (ClustalW), containing the HXXXDG and DFGWG motifs, is shown (right panel) with At1g65450.1 (top) and other members of clade I of the BAHD transferase family (Yu et al., 2009). The sequences used were for the plants Selaginella moellendorffii (Sm), Physcomitrella patens (Pp), Populus trichocarpa (Pt), Zea mays (Zm), Oryza sativa (Os), and Arabidopsis (At), and only those that were complete. A rooted cladogram (left panel), with At5g23940 as the outgroup, consists of members that represent a subtree from the BAHD transferase family and was made using a t-coffee alignment of complete protein sequences (available as Supplemental Data Set 1 online). It shows three clades correlated with variation in both conserved motifs. The divergence of the DFGWG motif, which has occurred for At1g65450 and a related poplar sequence, have residue differences for positions 2 and 3 highlighted (green and purple).

Figure 7.

GFP Translational Fusions of the Different Gene Products from At1g65450 in Wild-Type Unfertilized Ovules. Provided are fluorescence micrographs (bottom panel) that were merged with their bright-field images (top panel). The construct used is given on top of each pair of vertical images. White arrows indicate position of the nucleus. Bar = 50 μm.