MATERNALLY EXPRESSED PAB C-TERMINAL, a novel imprinted gene in Arabidopsis, encodes the conserved C-terminal domain of polyadenylate binding proteins

Abstract

Parental imprinting is important for seed development, but few imprinted genes have been identified in plants. The four known imprinted genes in Arabidopsis thaliana encode transcriptional regulators. Here, we describe a novel imprinted gene, MATERNALLY EXPRESSED PAB C-TERMINAL (MPC), which encodes the C-terminal domain of poly(A) binding proteins (PABPs). PABPs play roles in mRNA stability and translation. MPC interacts with proteins that also interact with the C-terminal domain of typical PABPs, suggesting that MPC may regulate translation by modulating PABP activity. In the endosperm, MPC is expressed only from the maternal allele. Reduction of MPC expression affects seed development. In dna methyltransferase1 (met1) mutants, MPC is ectopically expressed, and the paternal allele is active in the endosperm. CGs in the 5' flanking region and gene body of MPC lose methylation in a met1 background. Both regions are required to confer imprinted reporter expression, suggesting that the gene body contains imprinting control region elements. In Arabidopsis, DEMETER (DME) activates expression of maternal alleles. MPC expression is reduced in flowers and seeds in a dme-4 mutant but only after fertilization in dme-1. We conclude that other factors along with DME promote MPC expression and that DME has indirect effects on imprinted gene expression in endosperm.

Figure 1.

Expression of the MPC Gene. (A) Allele-specific sequencing of MPC and control cDNAs from Col × Ler and Ler × Col siliques at 5 DAP. Only the maternal alleles of MPC and FWA are represented in the sequence for both crosses (polymorphic nucleotide in black box). The nonimprinted control gene, αVPE, shows a double peak at the polymorphic nucleotide. (B) RT-PCR and restriction analysis of cDNA from seeds dissected into embryo and endosperm+seed coat fractions at 7 DAP. MPC is expressed only from maternal alleles in endosperm+seed coat, but biallelic in embryo. The imprinted FWA gene is a positive control for the dissection, and αVPE is a nonimprinted control. (C) RT-PCR analysis of MPC cDNA from vegetative and reproductive organs. MPC is primarily expressed in flower buds, female reproductive organs of open flowers, and young siliques. GAPC, control; WP, whole plant; R, root 5 weeks old; RL, rosette leaf; CL, cauline leaf; S, stem; SL, seedling; BL, late floral buds; OF, open flowers; OF-P, open flowers without pistils; MS, mature stamens; S3, siliques at 3 DAP; S5, siliques 5 DAP; S7, siliques 7 DAP; SE3, seed 3 DAP; SW3, silique wall 3 DAP; SE5, seed 5 DAP; SW5, silique wall 5 DAP; Em7, embryo dissected from 7 DAP seed; En7, endosperm+seed coat dissected from 7 DAP seed.

Figure 2.

MPC Expression in the Seed Is Endosperm Specific, and a Translational but Not Transcriptional Reporter Is Imprinted. (A) to (C), (E), and (F) Nuclear-targeted translational MPC reporter containing the MPC promoter and gene body fused to GFP (proMPC-MPC-GFP). (A) The reporter is active in the central cell of an unfertilized ovule (arrow and inset). (B) Reporter expression increases after fertilization; this seed from a self-fertilized proMPC-MPC-GFP plant shows the four nuclei resulting from the first two endosperm divisions. The arrow points to one of the nuclei that lies in a different focal plane to the other three and is therefore fainter. (C) proMPC-MPC-GFP × proMPC-MPC-GFP. (D) Wild-type Col (Col × Col). (E) and (F) The translational reporter is imprinted. (E) proMPC-MPC-GFP (♀) × Col (♂). (F) Col (♀) × proMPC-MPC-GFP (♂). (G) and (H) Nuclear-targeted transcriptional reporter (proMPC-GFP). Confocal images with GFP in green and chlorophyll autofluorescence in red. Some images show green autofluorescence in the seed coat. The transcriptional reporter is expressed when contributed by either parent. All seeds in (C) to (H) are at 4 DAP. (G) proMPC-GFP (♀) × Col (♂). (H) Col (♀) × proMPC-GFP (♂). Bars = 2 μm in (A) and (B) and 100 μm in (C) to (H).

Figure 3.

The Predicted MPC Protein Has Homology to the C-Terminal Domain of Poly(A) Binding Proteins and Can Bind CID Proteins That Also Interact with a Full-Length Arabidopsis PABP. (A) Alignment of the complete predicted MPC (At3g19350) protein with the C-terminal domains of the major human PABP and three Arabidopsis PABPs. Conserved amino acid residues are shaded. (B) The structure of MPC compared with a typical PABP. (C) A yeast two-hybrid assay indicates that PAM2-containing fragments of CID1, CID7, CID8, or CID12 interact with MPC in a similar manner as with PAB2. The plates show streaks of two representative yeast colonies for each one of the two-hybrid interactions tested. In the left half of the plate are the interaction with PAB2 and in the right half with MPC (At3g19350). Colonies were streaked on synthetic complete (SC) medium to select for both two-hybrid clones and on SC supplemented with 5 mM 3-amino-1,2,4-triazole (SC-His + 3AT) to assess the interaction. The position of the interactions in the plate is indicated on the slices of the diagram to the right of the figure; vector represents the empty cloning vector.

Figure 4.

Reducing MPC Expression Affects Seed and Trichome Development. (A) Trichomes on rosette leaves of wild-type Col have three branches. (B) Trichomes on rosette leaves of MPC RNAi plants have one to two branches. (C) A proMPC-MPC:GUS translational reporter transformed into wild-type Col is expressed in trichomes. (D) and (E) Mature seeds from wild-type Col (left) and MPC RNAi (right) plant. (F) to (J) Differential contrast images of developing seeds with embryos pseudocolored green and chalazal endosperm pseudocolored red. (F) and (G) Wild-type Col at 5 DAP (F) and 7 DAP (G). (H) to (J) MPC RNAi seeds at 7 DAP showing delayed embryogenesis, abnormal embryo morphology, and enlarged chalazal endosperm ([H] and [J]). Bars = 1 mm in (A) to (C), 250 μm in (D) and (E), and 100 μm in (F) to (J).

Figure 5.

Loss of MET1 Function Causes Ectopic MPC Expression. (A) RT-PCR of MPC RNA from mature stamen and rosette leaf of met1 mutant and wild-type Col plants. Ectopic expression of FWA is a control for hypomethylation in the met1-9 background. GAPC, control. (B) Allele-specific sequencing of MPC RNA extracted from siliques at 5 DAP. When the pollen parent is wild-type, only the maternal MPC allele is represented in the sequence (left), but when the pollen parent is homozygous for met1-9, both maternal and paternal alleles are detected (right). (C) RT-PCR and restriction analysis of MPC RNA from embryo and endosperm+seed coat fractions of seeds dissected at 7 DAP shows the paternal MPC allele is ectopically expressed in endosperm when contributed by a met1-9 pollen parent (cf. Figure 1B, which shows the paternal band is absent in endosperm+seed coat when the pollen parent is wild-type). The imprinted FWA gene is a control for the dissection, and αVPE is a nonimprinted control.

Figure 6.

MPC Is Methylated in the 5′ Flanking Region and in the Gene Body. (A) RT-PCR of MPC and surrounding regions treated with the methylation-sensitive restriction endonuclease McrBC (+) or mock treated (–). McrBC digests methylated DNA; the presence of a PCR product indicates a lack of methylation in that genomic fragment. Genomic DNA was extracted from rosette leaf of met1-6 or wild-type Col plants. The start codon is at position +1, and the positions of other gene features are indicated in the schematic below. (B) CG methylation in multiple clones of the upstream region and gene body of MPC determined by bisulfite sequencing of rosette leaf DNA in met1-9 and wild-type Col backgrounds. A black circle indicates a methylated cytosine in CG context, an open circle is an unmethylated cytosine, and a gray circle is a methylated cytosine in a CN context where N is an unknown base. There are no CG sites between +25 and +45.

Figure 7.

MPC Expression in dme Mutant Backgrounds. qRT-PCR analysis of MPC and FWA in buds, flowers, and siliques from dme-1 and dme-4. Each bar represents the log2 ratio of expression in the dme mutant to its wild-type background (Ler for dme-1 and C24 for dme-4). Error bars = se.