maternally expressed gene1 Is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Abstract

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.

Figure 1.

Autoradiograph of an AMD Gel. Arrows highlight maternal allelic expression of meg1. F2 selfed (lane 1); Mo17 selfed (lane 2); F2 × Mo17 (lane 3); Mo17 × F2 (lane 4); A69Y selfed (lane 5); F2 selfed (lane 6); A69Y × F2 (lane 7); F2 × A69Y (lane 8).

Figure 2.

Expression Analysis of meg1 and Related Sequences. (A) RT-PCR of meg1 and five related cDNAs using RNA samples isolated from a range of tissues and amplified with gene-specific primers (see supplemental data online). Dark and light coloration indicate high and intermediate signal, respectively, and open boxes indicate no expression. (B) mRNA localization of meg1 in developing seeds. Left, 4 DAP; right, 12 DAP. Em, embryo; En, endosperm; PC, pedicel. Scale bars = 100 μm.

Figure 3.

Parent-of-Origin Expression and Methylation Analysis of meg1. (A) Allele-specific RT-PCR analysis of meg1 in the endosperm. Top left, meg1 sequence polymorphism detected in endosperms from selfed and reciprocally crossed F2 and A69Y (69) lines. Note that endosperms resulting from reciprocal crosses show expression of the maternal meg1 allele at 4 DAP. Top right, meg1 expression is biparental in 12 DAP endosperms resulting from F2 and A69Y reciprocal crosses. Bottom, meg1 sequence polymorphism in F2 diploid (2n) and W23 tetraploid (4n) endosperms and showing monoallelic maternal expression in 4 DAP endosperms resulting from reciprocal interploidy crosses. (B) Methylation analysis of meg1 in 6 DAP embryos and endosperms. Embryo samples (lanes 1 to 4): W22 selfed (lane 1); A69Y selfed (lane2); W22 × A69Y (lane 3); A69Y × W22 (lane 4). Endosperm samples (lanes 5 to 8): W22 selfed (lane 5); A69Y selfed (lane 6); A69Y × W22 (lane 7); W22 × A69Y (lane 8). Closed circles, W22-specific fragment. Open circles, A69Y-specific fragment.

Figure 4.

MEG1 Protein Analysis. (A) Amino acid conservation of predicted MEG polypeptides in maize (Zm), barley (Hv), and wheat (Ta). Arrowhead indicates putative cleavage site of transit peptides. Stars denote the putative glycosylation sequon. Arrows mark the positions of conserved Cys, whereas closed circles highlight the conserved Tyr residues. Note that MEG3 has two predicted polypeptides (MEG3a and MEG3b). (B) Homology-based model of MEG1 structure. Left, comparison of the predicted model of MEG1 and the crystal structure of SCR/SP11. Disulfide bonds are shown in yellow; single N-glycan is shown as a yellow ball-and-stick model. β-Strands are represented by broad arrows and the α helix by a helical ribbon. Right, spatial distribution of conserved Tyr residues on MEG1 predicted structure, shown as red balls and sticks (arrows).

Figure 5.

MEG1 Protein Localization. (A) Immunolocalization of MEG1 proteins. Left, 12 DAP basal endosperm; right, magnification of basal transfer cells showing cell wall localization of MEG1. En, endosperm; PC, pedicel. Scale bars = 100 μm. (B) Protein gel blot of proteins isolated from 10 DAP endosperms after subcellular fractionation in a 20% SDS-PAGE. Cytoplasmic fraction I (lane 1); cytoplasmic fraction II (lane 2); cell wall fraction I (lane 3); cell wall fraction II (lane 4). Arrows, proteins detected with anti-MEG1 antibody. Arrowhead denotes the presence of an ∼25-kD protein(s). (C) Protein gel blot of MEG1 proteins before and after treatment with exoglycosidases. Proteins were separated on a 12% SDS-PAGE. Arrowhead, putative glycosylated form of MEG1 detected in a partially purified cell wall fraction. Arrows, nonglycosylated MEG1 protein(s).

Figure 6.

Analysis of the meg1 Promoter. (A) Promoter sequence of meg1 showing four conserved regions also present in the promoters of BETL1, BETL4, and AE1. Putative TATA motifs are underlined. Transcription start site (TSS) is labeled with an arrow, and the first codon (ATG) is boxed. (B) Deletion analysis of the meg1 promoter. Left, schematic representations of each promoter-deletion construct used for cobombardment with pMON-MRP1. Right, results obtained from the cobombardment assay; plus sign indicates the presence of GUS staining (transactivation); minus sign denotes absence of GUS staining (lack of transactivation).

Figure 7.

Pattern of Maternal and Paternal pMEG1-GUS Expression during Endosperm Development. Saggital section of kernels resulting from reciprocal crosses between A188 and transgenic plants carrying a transgene with the promoter sequence of meg1 fused to uidA and analyzed by GUS staining. Arrowhead marks region of GUS staining. Em, embryo; En, endosperm; N, nucellus. (A), (C), (E), (G), and (I) Seeds from plants carrying the pMEG1-GUS transgene after crossing with A188 pollen. (B), (D), (F), (H), and (J) Seeds from A188 plants crossed with pollen from transgenic plants bearing the pMEG1-GUS transgene. (A) and (B) 4 DAP. (C) and (D) 6 DAP. (E) and (F) 10 DAP. (G) and (H) 15 DAP. (I) and (J) 20 DAP.