Mitochondrial GFA2 is required for synergid cell death in Arabidopsis

Abstract

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

Figure 1.

Developmental Stages Affected in the FG Mutants. Megagametogenesis has been described previously and divided into seven stages (Christensen et al., 1997). The haploid megaspore defines stage FG1. The megaspore undergoes three rounds of mitosis without cytokinesis, giving rise to an eight-nucleate syncytium (early-stage FG5). Immediately after the third mitosis, cell walls form (late-stage FG5). The central cell inherits two nuclei, called the polar nuclei, which fuse to form the homodiploid secondary nucleus (stage FG6). Finally, the antipodal cells degenerate, producing a mature FG (stage FG7). Early during the fertilization process, one of the synergid cells is induced to undergo cell death (data not shown). Category designations show the developmental stages affected in the FG mutants. In this figure, cytoplasm is shown in light gray, vacuoles are shown in black, nuclei are shown in dark gray, and nucleoli are represented as the white areas within the nuclei. ac, antipodal cell; cc, central cell; ec, egg cell; emb, embryo; end, endosperm; pn, polar nucleus; sc, synergid cell; sdc, seed coat; sn, secondary nucleus.

Figure 2.

Phenotypic Analysis of the FG Mutants. (A) and (B) Wild-type FG at the terminal developmental stage (stage FG7). The egg is pear shaped, has a nucleus at its chalazal end, and has a single large vacuole occupying the remainder of the cell. Both synergid cells are present, and the secondary nucleus is undivided. (C) Wild-type (ecotype Ws) seed at 7 HAP containing a single-celled zygote, an undivided endosperm nucleus (primary endosperm nucleus), one degenerated synergid cell, and one persistent synergid cell (data not shown). (D) and (E) Wild-type (ecotype Ws) seed at 15 HAP. The zygote is relatively round, has several small vacuoles, and has a centrally located nucleus. One of the synergids is degenerate, and the primary endosperm nucleus has divided. (F) Wild-type (ecotype Ws) seed at 48 HAP containing a four-celled proembryo (two cells are not projected in this image) and multiple peripheral endosperm nuclei. (G) fem16 FG that has collapsed. (H) fem12 FG arrested at the one-nucleate stage (stage FG1). (I) fem12 FG arrested at the four-nucleate stage. (J) fem12 FG containing three micropylar nuclei and one chalazal nucleus. (K) fem13 FG containing two nuclei in the position of the egg nucleus. (L) fem5 FG containing two large nuclei. (M) fem5 FG containing four micropylar nuclei and two chalazal nuclei. (N) fem7 FG with reduced cytoplasm associated with the nuclei and fuzzy nuclear margins. The second synergid was present but is not projected in this image. (O) fem8 FG containing a large number of small vacuoles in the central cell cytoplasm and a disordered egg apparatus. (P) gfa2 FG with unfused polar nuclei. (Q) and (R) gfa2 FG at 24 HAP. Both synergids are intact, the egg cell appears to be unfertilized (it is pear shaped, has a nucleus at its chalazal end, and has a single large vacuole), and the polar nuclei appear to be unfertilized (they are unfused and remain near the chalazal end of the egg cell). gfa2 embryo sacs at 7 to 15 HAP have similar morphology. (S) and (T) ctr1-2 seed at 48 HAP showing early signs of embryogenesis. This seed has three endosperm nuclei, a single-celled zygote, a degenerated synergid, and a persistent synergid. (A), (C), (D), (F) to (Q), and (S) are CLSM images. (B), (E), (R), and (T) are depictions. All CLSM images are projections of several optical sections taken at the terminal developmental stage or at the HAP indicated. In the CLSM images, cytoplasm is shown in gray, vacuoles are shown in black, and nucleoli are shown in white. In the depictions, cytoplasm is shown in light gray, vacuoles are shown in black, nuclei are shown in dark gray, and nucleoli are shown in white. In all images, the micropylar pole is at the bottom and the chalazal pole is at the top. cn, chalazal nuclei; dsc, degenerate synergid cell; ec, egg cell; ecn, egg cell nucleus; en, endosperm nucleus; fg, female gametophyte; mn, micropylar nuclei; n, nucleus; pe, proembryo; pen, primary endosperm nucleus; pn, polar nucleus; sc, synergid cell; scn, synergid cell nucleus; sn, secondary nucleus; su, suspensor; WT, wild type; z, zygote; zn, zygote nucleus.

Figure 3.

GFA2 Gene Sequence. Exons are indicated with uppercase letters. A 33-bp deletion associated with the site of the T-DNA insert is indicated by strikethrough. The position of the T-DNA left border is indicated above the strikethrough. Plant DNA on the other side of the T-DNA insert is adjacent to the internal T-DNA sequence. The predicted GFA2 protein contains two in-frame ATG codons (marked in boldface and underlined) in its N terminus. The first Met codon is expected to initiate translation because it satisfies the consensus sequence criteria for a translation initiation codon (Kozak, 1991). Some of the exon-intron boundaries derived from the cDNA sequence differ from the annotation of the At5 g48030 locus shown in the MIPS Arabidopsis thaliana database (MATDB; http://mips.gsf.de/proj/thal/db/index.html). Miscalled exons and splice sites relative to the annotation for At5 g48030 are underlined. The underlined regions are indicated as exons or introns according the structure of the GFA2 cDNA.

Figure 4.

Alignment of the Predicted Protein Sequences for Arabidopsis GFA2, Yeast Mdj1p, and E. coli DnaJ. The J domain (residues 90 to 156) and the zinc-finger domain (residues 238 to 294) are double underlined. The Gly/Phe-rich domain (residues 157 to 237) lies between these two domains. Sequence similarity is indicated by shading. Relative to DnaJ, the predicted GFA2 sequence contains an 89-residue extension at the N terminus that has several characteristics of a mitochondrial targeting sequence: an overrepresentation of Arg, Ala, and Ser residues (25 of 89 residues); a paucity of Asp and Glu residues (3 of 89 residues); and an ability to form an amphiphilic α-helix (residues 7 to 16 may form an amphiphilic α-helix region) (Gavel et al., 1988; Roise, 1997).

Figure 5.

Reverse Transcriptase –PCR Analysis of GFA2 Expression.

Figure 6.

GFA2::GUS Expression in the Wild Type, GFA2-GFP Protein Localization in the Wild Type, and FIE::FIE-sGFP Expression in the gfa2 Mutant. (A) and (B) Histochemical assay for the expression of the GFA2::GUS transgene in the whole plant (A) and the flower (B). Longer histochemical staining showed expression in the petals. (C) Trichome from a transgenic plant expressing 35S::GFP. (D) Trichome from a transgenic plant expressing 35S::GFA2-GFP. (E) to (G) Mitotracker-stained root hair from a transgenic plant expressing 35S::GFA2-GFP. (E) Mitotracker fluorescence. (F) GFP fluorescence. (G) Merger of red and green channel fluorescence. (H) and (I) Expression of the FIE::FIE-GFP construct in the wild type (H) and the gfa2 mutant (I). pn, polar nucleus; sn, secondary nucleus.

Figure 7.

GFA2 Complementation of the Yeast mdj1 Mutant Growth Defect. Cultures of the four possible combinations of the MDJ1 or mdj1::HIS3 allele and the expression vectors pRS416-MET25-GFA2 or pRS416-MET25 were diluted serially (1:10) and grown at 25, 30, and 37°C on dextrose.