Genetic interaction of an origin recognition complex subunit and the Polycomb group gene MEDEA during seed development

Abstract

The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.

Figure 1.

The orc2 Mutant. (A) Developing seeds of orc2/+ plants showing the typical seed abortion phenotype (white arrowheads) and the less common translucent phenotype (yellow arrowhead). (B) Predicted gene structure of AtORC2 and flanking genes. The insertion site of the Ds transposable element in the orc2 mutant allele is indicated. The genomic fragment used to complement the orc2 mutant is shown by a black bar.

Figure 2.

Microscopic Analysis of the orc2 Zygotic Lethal Phenotype. (A) to (D) Thin-sectioned seeds of orc2/+ plants stained with toluidine blue. Wild-type ([A] and [C]) and mutant ([B] and [D]) seeds from the same siliques. In (B), the arrow points to an enlarged endosperm nucleus. In (D), the arrows show aberrant cell divisions, and the inset shows an enlarged endosperm nucleus from the same seed. (E) to (H) Wild-type (E) and mutant ([F] to [H]) seeds that were cleared, whole mounted, and viewed with Nomarski optics. (F) Typical orc2 phenotype in a seed from the same silique as (E). Arrows point to enlarged endosperm nuclei. (G) Normal embryo but abnormal endosperm (arrow). (H) Rare almond-shaped embryo. Arrows in (A), (C), and (E) point to normal endosperm nuclei. Bar = 10 μM.

Figure 3.

Analysis of AtORC2 Expression. (A) Assay for AtORC2 and actin11 transcripts by RT-PCR: lane 1, DNA size markers; lane 2, large PCR product from plasmid p1300-ORC2 that contains a genomic fragment spanning the complete AtORC2 gene; lane 3, control (no CDNA); lanes 4 to 6, PCR products from cDNA of seedlings (lane 4), mature leaves (lane 5), and inflorescences (lane 6). (B) to (E) In situ hybridization experiments. CC, central cell; E, endosperm; EC, egg cell; S, synergid. (B) Female gametophyte, antisense probe. (C) Female gametophyte, sense probe. (D) Preglobular embryo, antisense probe. (E) Preglobular embryo, sense probe. (F) and (G) GUS activity assays on tissue from orc2/+ plants. (F) GUS activity in the trichome. (G) GUS activity in pollen tubes from an orc2/+ plant on a wild-type stigma.

Figure 4.

Phenotypes of Developing Seeds in orc2/+; mea/+ Plants. Cleared, whole-mounted seeds from siliques at the same developmental stage. (A) The wild type. (B) mea^M phenotype with large heart-stage embryo and aggregated endosperm. (C) Typical orc2/orc2 phenotype. (D) mea^M-like phenotype but with no large embryo; arrow points to small orc2/orc2-like embryo. (E) orc2/orc2-like phenotype but bigger seed and more cells in embryo than ever seen in orc2/+ plants. Bar = 10 μM.