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, 18 (8), 1862-72

AGL80 Is Required for Central Cell and Endosperm Development in Arabidopsis

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AGL80 Is Required for Central Cell and Endosperm Development in Arabidopsis

Michael F Portereiko et al. Plant Cell.

Abstract

During plant reproduction, the central cell of the female gametophyte becomes fertilized to produce the endosperm, a storage tissue that nourishes the developing embryo within the seed. The molecular mechanisms controlling the specification and differentiation of the central cell are poorly understood. We identified a female gametophyte mutant in Arabidopsis thaliana, fem111, that is affected in central cell development. In fem111 female gametophytes, the central cell's nucleolus and vacuole fail to mature properly. In addition, endosperm development is not initiated after fertilization of fem111 female gametophytes. fem111 contains a T-DNA insertion in AGAMOUS-LIKE80 (AGL80). FEM111/AGL80 is a member of the MADS box family of genes that likely encode transcription factors. An AGL80-green fluorescent protein fusion protein is localized to the nucleus. Within the ovule and seed, FEM111/AGL80 is expressed exclusively in the central cell and uncellularized endosperm. FEM111/AGL80 expression is also detected in roots, leaves, floral stems, anthers, and young flowers by real-time RT-PCR. FEM111/AGL80 is required for the expression of two central cell-expressed genes, DEMETER and DD46, but not for a third central cell-expressed gene, FERTILIZATION-INDEPENDENT SEED2. Together, these data suggest that FEM111/AGL80 functions as a transcription factor within the central cell gene regulatory network and controls the expression of downstream genes required for central cell development and function.

Figures

Figure 1.
Figure 1.
Microscopic Analysis of Wild-Type and fem111 Female Gametophytes. (A) and (D) Depictions of the Arabidopsis female gametophyte at the terminal developmental stage (stage FG7) in the wild type (A) and fem111 (D). Modified from Drews et al. (1998). In these panels, cytoplasm is light gray, vacuoles are black, nuclei are dark gray, and nucleoli are white. Only one of the two synergid cells is depicted in this orientation. (B), (C), and (E) to (I) CLSM images. In these images, cytoplasm is gray, vacuoles are black, and nucleoli are white. (B), (E), and (G) Wild type (B) and fem111 ([E] and [G]) female gametophytes at the terminal developmental stage (stage FG7). The wild-type female gametophyte in (B) and the fem111 female gametophyte in (E) are from ovules at 24 h after emasculation of a stage 12c flower. The fem111 female gametophyte in (G) is from an ovule at 42 h after emasculation of a stage 12c flower. The vacuole and nucleolus are smaller in fem111 ([E] and [G]) compared with the wild type (B). In the wild type (B), the egg cell is elongated along the micropylar/chalazal axis and has a single vacuole at the micropylar end and its nucleus at the chalazal end. (C) and (F) Wild-type (C) and fem111 (F) seeds at 18 h after pollination. In (C), only two of the four endosperm nuclei can be seen. The zygote, in contrast with the egg cell, is rounded, has the nucleus in the center of the cell, and contains many smaller vacuoles that surround the nucleus on all sides. In (F), the zygote, degenerating synergid cell, and degenerating central cell are in focus and endosperm nuclei are not observed. (H) and (I) Wild-type (H) and fem111 (I) seeds at 40 h after pollination. Wild-type seeds (H) contain embryos at the globular stage. fem111 seeds (I) contain zygote-like structures. cc, central cell; ch, chalazal pole; cn, central cell nucleolus; cv, central cell vacuole; dcc, degenerating central cell; dsc, degenerating synergid cell; e, embryo; ec, egg cell; ev, egg cell vacuole; mp, micropylar pole; sc, synergid cell; sn, secondary nucleus; sv, synergid cell vacuole; z, zygote; zls, zygote-like structure. Bars = 10 μm.
Figure 2.
Figure 2.
Size of the Central Cell Nucleolus in Wild-Type and fem111 Female Gametophytes. Black bars, nucleolus size in wild-type plants; gray bars, nucleolus size in fem111/FEM111 plants. The two sets of bars for fem111/FEM111 plants represent two distinct classes of female gametophytes within the pistil. For the wild type at stages FG6 and FG7, n = 100 for each stage. For fem111/FEM111 at stage FG6, n = 235; for fem111/FEM111 at stage FG7, n = 250. Error bars indicate sd.
Figure 3.
Figure 3.
Structures of the FEM111/AGL80 Gene and FEM111/AGL80 Protein. (A) FEM111/AGL80 gene structure. The black box represents coding sequence, and the white boxes represent the 5′ (51 nucleotides [nt]) and 3′ (109 nucleotides) untranslated regions. The insertion site of the T-DNA in the fem111 mutant is marked by an arrowhead. (B) FEM111/AGL80 protein structure. FEM111/AGL80 contains a MADS domain (amino acids 9 to 60; black box), a predicted nuclear localization signal (NLS) (amino acids 23 to 26; light gray box), and a His-rich domain (amino acids 275 to 291; dark gray box).
Figure 4.
Figure 4.
AGL80-GFP Expression. (A) Expression of AGL80-GFP in female gametophytes before fusion of the polar nuclei (late stage FG5). Expression is detected only in the two polar nuclei. (B) Expression of AGL80-GFP in female gametophytes at the terminal developmental stage (stage FG7). Expression is detected only in the secondary nucleus. (C) Expression of AGL80-GFP in a fertilized female gametophyte at 18 h after pollination. Expression is detected only in the endosperm nuclei. Only three of the four endosperm nuclei can be seen in this focal plane. All images are composites of CLSM micrographs of AGL80-GFP expression merged with bright-field images of ovules. en, endosperm nuclei; pn1 and pn2, the two polar nuclei before fusion; sn, secondary nucleus of the central cell.
Figure 5.
Figure 5.
Real-Time RT-PCR Analysis of FEM111/AGL80 Expression. AGL80 transcript levels were normalized to ACTIN2 levels using the formula ΔCT = CT(AGL80) – CT(ACTIN2). Real-time RT-PCR was performed with cDNAs from anthers harvested from flowers at stages 10 to 13 (A; ΔCT = 7.3 ± 1.5), flowers at stages 1 to 10 (FC; ΔCT = 8.4 ± 0.3), leaves (L; ΔCT = 11.1 ± 0.6), ovaries from unpollinated flowers (O; ΔCT = 9.8 ± 0.4), roots (R; ΔCT = 9.4 ± 0.3), siliques at 1 to 3 d after pollination (Si; ΔCT = 11.4 ± 0.8), and stems (St; ΔCT = 11.0 ± 0.8). Each bar represents an average of four independent reactions.
Figure 6.
Figure 6.
Expression of DME, FIS2, and DD46 in Wild-Type and fem111 Female Gametophytes. (A) Expression of DME-GUS in a wild-type female gametophyte. Expression is detected in the central cell nucleus. The weak signal in the integuments is an artifact of the staining protocol and is not associated with DME-GUS expression. (B) Expression of FIS2-GFP in a wild-type female gametophyte. Expression is detected only in the central cell (cc). (C) and (D) Expression of ProDD46:GFP in wild-type (C) and fem111 (D) female gametophytes. In the wild type (C), expression is detected in the central cell (cc) and synergid cells (sc). In fem111 (D), expression is detected only in the synergid cells. (E) Bar graph of the percentage of ovules expressing DME-GUS, FIS2-GFP, and ProDD46:GFP in wild-type (black bars) and fem111/FEM111 (gray bars) plants. Percentage expression is based on an average of the number of ovules expressing the reporter construct per plant analyzed. For each reporter construct, a minimum of three plants and two siliques per plant were analyzed. Error bars indicate sd. The reporter construct was homozygous for all plants scored. FGs, female gametophytes.

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