FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex 2 Plays a Dual Role in Regulating Type I MADS-Box Genes in Early Endosperm Development

Abstract

Early endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modifications. In Arabidopsis (Arabidopsis thaliana), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-specific expression analysis, we also identified a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes.

Figure 1.

Stages of unfertilized seeds and early fertilized seeds encompassing the three phases of endosperm development used in the RT-qPCR assays. Cleared ovules or seeds at 3 DAE (A and H), 0 DAP (B and I), 1 DAP (C and J), 2 DAP (D and K), 3 DAP (E and L), 4 DAP (F and M), and 5 DAP (G and N) from Col-0 (A–G) and mea-3;swn-3 mutant (H–N) plants are shown. The embryo is false colored in yellow. The embryo sac or the part of the embryo sac excluding the embryo is false colored in blue. Bars = 50 µm.

Figure 2.

mRNA profiles of 53 MADS-box genes during early seed development in Col-0 and swn-3/−;mea-3/+ double-mutant plants. A, Centered average C_T values obtained from RT-qPCR analysis of mRNA levels in 3-DAE carpels (−3), 0-DAP carpels (0), and 1- to 5-DAP siliques (1–5). Values were subjected to hierarchical cluster analysis and are presented as a heat map to identify the three expression clusters C1, C2.1, and C2.2 based on the relative mRNA levels in both genetic backgrounds. Red denotes relatively high and blue denotes relatively low mRNA levels. B, Heat map depicting the differences in mRNA levels (ΔΔC_T) between Col-0 and swn-3/−;mea-3/+ mutant backgrounds. Genes are arranged in the same order as in A. Yellow denotes up-regulation in the mutant, and blue denotes down-regulation in the mutant. ΔΔC_T values below the statistical threshold are in black (|ΔΔC_T| > 2; P < 0.05, Student’s t test). C, Graphic representation of average mRNA profiles of C1, C2.1, and C2.2 genes.

Figure 3.

Expression of C2 type I MADS-box gene reporter lines during endosperm development. Promoter-GFP activities were analyzed at 11 stages (separated by dashed lines) during endosperm development, including the coenocytic phase (endosperm stages I–VIII) and cellularized phases (endosperm stage IX, early torpedo stage [et], and early bent-cotyledon stage [ebc]). Five distinct expression patterns are represented by AGL46 (A), AGL48 (B), AGL96 (C), PHE2 (D), and AGL91 (E). All seeds are oriented with the micropylar end toward the bottom left. Arrows indicate the position of the chalazal domain at endosperm stage VIII. Bars = 100 µm.

Figure 4.

Differential imprinted expression patterns of C2 type I MADS-box genes. The allele-specific expression of 14 C2 genes and control genes was analyzed in reciprocal crosses between Col-0 and Cvi-0 or Ler by single-base extension with fluorescently labeled ddNTPs (SBE) based on SNPs. Orange denotes maternal allele expression frequency, and blue denotes paternal allele expression frequency. In cross descriptions, the maternal parent is denoted first. FIS2 represents a MEG, and AGL62 represents a biallelically expressed gene. Three independent biological replicates were used. The threshold for determining MEGs and PEGs was set at and (where m denotes maternal transcripts and p denotes paternal transcripts measured by SBE) in both reciprocal crosses, as described previously (Hsieh et al., 2011). P values calculated using Student’s t test are provided in Supplemental Table S5.

Figure 5.

Allele-specific expression of C2 type I MADS-box genes in the endosperm. GFP or YFP signals were analyzed at 2 and 5 DAP. The AGL reporter transgenes, including the pAGL90:GFP promoter fusion (A), the PHE1:YFP full-length gene fusion (B), the pAGL48:GFP fusion (C), and the pAGL91:GFP fusion (D), were inherited either from the maternal (m) or paternal (p) parent, while the mutant allele (mea-3 and fis2-8) was always inherited from the maternal parent. Bars = 100 µm.