MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis

Abstract

The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process, including guidance of pollen tube growth to the female gametophyte. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. MYB98 is a member of the R2R3-MYB gene family, the members of which likely encode transcription factors. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development.

Figure 1.

Depictions of the Female Gametophyte and Synergid Cells. (A) Depiction of the Arabidopsis female gametophyte. Modified from Drews et al. (1998). (B) Depiction of the synergid cells. This view is perpendicular to that in (A). The cytoplasm is gray, vacuoles are white, and nuclei are black. ac, three antipodal cells; cc, central cell; ch, chalazal region of the ovule; ec, egg cell; f, funiculus; fa, filiform apparatus; mp, micropyle; sc, synergid cell; sn, synergid cell nucleus; sv, synergid cell vacuole.

Figure 2.

RT-PCR Analysis of MYB98 Expression. (A) Expression of MYB98 in wild-type pistils (WP) and dif1-2/dif1-2 pistils (DP). Expression of the ACTIN2 gene was used as a control. (B) Real-time RT-PCR analysis of MYB98 expression in pistils (WP), roots (R), floral stems (S), young leaves at <5 mm in length (YL), older leaves at 10 to 20 mm in length (L), flowers at stages 1 to 11 (YF), and siliques at 1 to 2 d after pollination (SL). Each bar represents an average of four to five independent reactions. Error bars indicate sd. The number above each bar represents RNA level relative to pistil (WP). (C) Expression of MYB98 in wild-type pistils (WP), myb98-1/myb98-1 pistils (98-1), and myb98-2/myb98-2 pistils (98-2). Expression of the ACTIN2 gene was used as a control.

Figure 3.

Expression of the Pro_MYB98:GFP and Pro_MYB98:GUS Genes. (A) and (B) Expression of Pro_MYB98:GFP (A) and Pro_MYB98:GUS (B) in female gametophytes at the terminal developmental stage (stage FG7). Expression is detected only in the synergid cells. The ovule in (A) is oriented like that in Figure 1A, and the ovule in (B) is oriented like that in Figure 1B. The ovule in (B) was stained in 1 mM 5-bromo-4-chloro-3-indolyl-β-glucuronic acid (X-Gluc) for 30 min. (C) Expression of Pro_MYB98:GFP in the synergid cells in a female gametophyte at late stage FG5. (D) Expression of Pro_MYB98:GUS in a fertilized embryo sac at 12 h after pollination. Expression is detected only in the persistent synergid cell. This ovule was stained in 1 mM X-Gluc for 4 h. (E) Expression of Pro_MYB98:GUS in a fertilized embryo sac at 12 h after pollination. Expression is detected in the persistent synergid cell and endosperm (arrowheads). This ovule was stained in 5 mM X-Gluc for 4 h. (F) Expression of Pro_MYB98:GFP in a fertilized embryo sac at 24 h after pollination. Expression is detected in the persistent synergid cell and endosperm (arrowheads). (G) Expression of Pro_MYB98:GUS in the trichomes of young (<5 mm) leaves. This leaf was stained in 1 mM X-Gluc for 16 h. (H) Opened siliques from wild-type (left) and myb98-1/+ (right) plants at 10 d after pollination. Siliques from mutant plants contain desiccated ovules (arrows). psc, persistent synergid cell; sc, synergid cell.

Figure 4.

Structure of the MYB98 Protein. (A) MYB98 gene structure. Black boxes represent coding sequence, and arrowheads represent the T-DNA insertion sites in myb98-1 (40 bp downstream of the ATG) and myb98-2 (195 bp upstream of the ATG). nt, nucleotide. (B) MYB98 protein structure. MYB98 contains a predicted nuclear localization signal (NLS) between amino acids (aa) 196 and 212 (gray box). The R2 and R3 repeats are represented by black boxes. (C) Comparison of the R2 and R3 Myb repeats between Arabidopsis MYB98 and Mus musculus c-Myb. Conserved amino acids are shaded in gray. The asterisks denote the conserved Trp residues. The arrow designates the conserved Ala residue in the second helix of R2 that is often a Pro residue in plant R2R3 proteins (Jia et al., 2004). The arrowheads denote the seven amino acids in c-Myb that contact the DNA bases.

Figure 5.

Microscopic Analysis of myb98 Female Gametophytes. (A) and (B) CLSM images of female gametophytes at the terminal developmental stage (stage FG7) in the wild type (A) and myb98-1 (B). (C) and (D) Light micrographs of synergid cells from thick plastic sections of wild-type (C) and myb98-1 (D) ovules. (E) to (L) Transmission electron microscopy images of wild-type ([E], [G], and [I]), myb98-1 ([F], [H], and [J]), and myb98-2 ([K] and [L]) synergid cells. The asterisks in (D), (F), and (H) denote the abnormal micropylar structure in myb98-1 synergid cells. Within the filiform apparatus of the wild type ([G] and [I]) are electron-translucent (light gray patches) and electron-dense (medium gray patches marked with arrowheads) regions. The dark gray regions are pockets of cytoplasm. cw, cell wall between the synergid cells; ed, electron-dense region; en, egg cell nucleus; et, electron-translucent region; ev, egg cell vacuole; fa, filiform apparatus; sc, synergid cell; sn, synergid cell nucleus; sv, synergid cell vacuole. Bars = 10 μm in (A) and (B), 5 μm in (C) to (F), 3 μm in (G), (H), and (K), and 1 μm in (I), (J), and (L).

Figure 6.

Pollen Tube Guidance Is Affected in myb98 Female Gametophytes. (A) Wild-type pollen tube on a wild-type ovule at 12 h after pollination. (B) Wild-type pollen tube on a myb98-1/myb98-1 ovule at 12 h after pollination. (C) Wild-type pollen tubes on a myb98-1/myb98-1 ovule at 24 h after pollination. Four pollen tubes (pt1 to pt4) are present on the ovule. (D) Wild-type pollen tubes on a myb98-1/myb98-1 ovule at 24 h after pollination. Four pollen tubes (pt1 to pt4) are present on the ovule. One of the pollen tubes (pt1) is in the micropyle. All images are composites composed of CLSM micrographs of the pollen tubes merged with bright-field images of ovules. All images are projections of multiple 1-μm sections. f, funiculus; mp, micropyle; pt, pollen tube; t, tip of pollen tube. Bars = 25 μm.