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, 20 (3), 635-47

The AGL62 MADS Domain Protein Regulates Cellularization During Endosperm Development in Arabidopsis

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The AGL62 MADS Domain Protein Regulates Cellularization During Endosperm Development in Arabidopsis

Il-Ho Kang et al. Plant Cell.

Abstract

Endosperm, a storage tissue in the angiosperm seed, provides nutrients to the embryo during seed development and/or to the developing seedling during germination. A major event in endosperm development is the transition between the syncytial phase, during which the endosperm nuclei undergo many rounds of mitosis without cytokinesis, and the cellularized phase, during which cell walls form around the endosperm nuclei. The molecular processes controlling this phase transition are not understood. In agl62 seeds, the endosperm cellularizes prematurely, indicating that AGL62 is required for suppression of cellularization during the syncytial phase. AGL62 encodes a Type I MADS domain protein that likely functions as a transcription factor. During seed development, AGL62 is expressed exclusively in the endosperm. During wild-type endosperm development, AGL62 expression is strong during the syncytial phase and then declines abruptly just before cellularization. By contrast, in mutant seeds containing defects in some FERTILIZATION-INDEPENDENT SEED (FIS) class Polycomb group genes, the endosperm fails to cellularize and AGL62 expression fails to decline. Together, these data suggest that AGL62 suppresses cellularization during the syncytial phase of endosperm development and that endosperm cellularization is triggered via direct or indirect AGL62 inactivation by the FIS polycomb complex.

Figures

Figure 1.
Figure 1.
Structures of the AGL62 Gene and AGL62 Protein. (A) AGL62 gene structure. Black boxes represent coding sequence, white boxes represent the 5′ (35 nucleotides) and 3′ (196 nucleotides) untranslated regions, and the horizontal line represents intron sequence. The insertion sites of the T-DNAs in the agl62-1, agl62-2, and agl62-3 mutants are marked by triangles. The T-DNA in agl62-1 is inserted into the intron, 474 nucleotides downstream of the start codon, and is associated with a 75-bp deletion in the intron. The T-DNA in agl62-2 is inserted in the second exon, 620 nucleotides downstream of the start codon, and is associated with a 9-bp deletion in the predicted second exon. The T-DNA in agl62-3 is inserted in the second exon, 1009 nucleotides downstream of the start codon, and is associated with a 12-bp deletion in the predicted second exon. (B) AGL62 protein structure. AGL62 contains a MADS domain (hatched box; amino acids 6 to 66).
Figure 2.
Figure 2.
Analysis of AGL62-GFP Expression and of agl62 Endosperm Development in GFP-Marked Endosperm. (A) to (C) and (G) to (I) Fluorescence images. (D) to (F) Fluorescence bright-field overlay images. (A) and (D) Expression of AGL62-GFP in a mature female gametophyte (stage FG7). The GFP signal is associated with the antipodal cells. (B), (C), (E), and (F) Expression of AGL62-GFP in seeds at stage II (two endosperm nuclei) ([B] and [E]) and stage VII (∼50 endosperm nuclei) ([C] and [F]) of endosperm development. Endosperm stages are described by Boisnard-Lorig et al. (2001). Expression is detected only in the endosperm nuclei. (G) and (H) Fluorescence images of wild-type (G) and agl62-1 (H) seeds at 36 h after pollination. Fluorescence is due to endosperm expression of ProDD19:GFP. In (H), cellularization in the micropylar chamber is obscured by the embryo present in this region. (I) Expression of AGL62-GFP in a fis2-8 seed at 6 d after pollination. In (A) to (H), seeds are oriented with the micropylar pole to the left and the chalazal pole to the right. ac, antipodal cells. Bars = 20 μm.
Figure 3.
Figure 3.
Real-Time RT-PCR Analysis of AGL62 Expression. Real-time RT-PCR was performed with cDNAs at 1 to 3 d after pollination from siliques (Si), leaves (L), roots (R), floral stems (St), anthers (A), and flower clusters (FC). Each bar represents an average of three independent reactions, including both biological and technical replicates. In all cases, AGL62 transcript levels were normalized to ACTIN2 levels. Error bars indicate sd.
Figure 4.
Figure 4.
Microscopy Analysis of Wild-Type and agl62-1 Seeds. All panels are CLSM images of unstained tissue. The confocal microscope detects autofluorescence. In these images, cytoplasm is gray, vacuoles are black, and nucleoli are white. Arrowheads point to syncytial nuclei. In all panels, the seeds are oriented with the micropylar pole to the left and the chalazal pole to the right. cze, chalazal endosperm; em, embryo. Bars = 20 μm. (A) Wild-type seed at 24 h after pollination. At this time point, the endosperm is uncellularized and contains four to eight nuclei, and the embryo is at the zygote stage. (B) agl62-1 seed at 24 h after pollination. At this time point, the endosperm is uncellularized and typically contains four to eight nuclei, and the embryo is at the zygote stage. (C) Wild-type seed at 36 h after pollination. At this time point, the endosperm is uncellularized and typically contains 16 to 30 nuclei, and the embryo typically is at the two-celled proembryo stage. (D) agl62-1 seed at 36 h after pollination. At this time point, the endosperm is cellularized and typically contains 14 nuclei, and the embryo is at the one- or two-celled proembryo stage. (E) Wild-type seed at 48 h after pollination. At this time point, the endosperm is uncellularized and contains >50 nuclei, and the embryo typically is at the quadrant stage. (F) agl62-1 seed at 48 h after pollination. At this time point, the endosperm is cellularized and typically contains 14 nuclei, and the seed is partially collapsed. (G) Wild-type embryo at 48 h after pollination. The embryo is at the quadrant stage. (H) agl62-1 embryo at 48 h after pollination. The embryo is at the two-celled proembryo stage.
Figure 5.
Figure 5.
Number of Endosperm Nuclei in Wild-Type and agl62-1 Seeds. Each point represents the average of nine seeds. Wild-type seeds at 48 h after pollination were not scored but typically contain >60 endosperm nuclei. Error bars indicate sd.
Figure 6.
Figure 6.
Expression of AGL62-GFP Expression in fis Seeds. Percentage of seeds expressing AGL62-GFP in siliques resulting from crosses of homozygous AGL62-GFP males with the wild type (white bars), fie-1/FIE (black bars), fis2-8/FIS2 (light-gray bars), and mea-3/MEA (dark-gray bars) females. Error bars indicate sd.

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