The maternally expressed polycomb group gene OsEMF2a is essential for endosperm cellularization and imprinting in rice

Abstract

Cellularization is a key event in endosperm development. Polycomb group (PcG) genes, such as Fertilization-Independent Seed 2 (FIS2), are vital for the syncytium-to-cellularization transition in Arabidopsis plants. In this study, we found that OsEMF2a, a rice homolog of the Arabidopsis PcG gene Embryonic Flower2 (EMF2), plays a role similar to that of FIS2 in regard to seed development, although there is limited sequence similarity between the genes. Delayed cellularization was observed in osemf2a, associated with an unusual activation of type I MADS-box genes. The cell cycle was persistently activated in osemf2a caryopses, which was likely caused by cytokinin overproduction. However, the overaccumulation of auxin was not found to be associated with the delayed cellularization. As OsEMF2a is a maternally expressed gene in the endosperm, a paternally inherited functional allele was unable to recover the maternal defects of OsEMF2a. Many imprinted rice genes were deregulated in the defective hybrid seeds of osemf2a (♀)/9311 (♂) (m9). The paternal expression bias of some paternally expressed genes was disrupted in m9 due to either the activation of maternal alleles or the repression of paternal alleles. These findings suggest that OsEMF2a-PRC2-mediated H3K27me3 is necessary for endosperm cellularization and genomic imprinting in rice.

Keywords: PRC2; cellularization; endosperm; genomic imprinting; osemf2a; rice.

Figure 1

Phenotype of the osemf2a mutants. (A) Generation of the osemf2a mutants in the Zhonghua11 (ZH11) background via a CRISPR/Cas9 approach. T1 and T2 indicate the targets used for gene mutation. Both mutant lines had a single-base insertion to cause premature stop codons. (B) Morphology of the wild-type (WT) (left), osemf2a-1 (middle), and osemf2a-2 (right) plants at the heading stage. (C) Morphology of the WT (left) and osemf2a-1 (right) flowers. The inset panels show the pollen activities of WT (left) and osemf2a-1 (right) after staining with I₂-IK solution. (D) Morphology of the seeds produced by the WT (upper) and osemf2a-1 (lower) at 10 days after fertilization (DAF). (E and F) Morphology of the WT (E) and osemf2a-1 caryopses (F) at 10 DAF. (G) Bare-handed sections that show the starchy endosperm of the WT (upper) and the jelly-like endosperm of OsEMF2a-1 (lower). (H) Morphology of the caryopsis produced by the WT (upper) and osemf2a-1 (lower) at the mature stage. (I) Setting rate of the WT and osemf2a-1. (J and K) H3K27me3 modifications in the chromatin of the endosperm (J) and leaf (K) of the WT (the first lane of each figure) and osemf2a-1 (the second lane of each figure). (L) Cumulative percentages of the non-fertilized ovules and enlarged, autonomous seeds in the WT, osemf2a-1, and osemf2a-2. (M) Morphology of autonomous seeds produced by osemf2a-1 at the mature stage. (N) Confocal laser scanning microscopy (CLSM) observation of an artificially fertilized osemf2a-1 caryopsis at 7 days after pollination. The red arrowhead and the yellow arrow show the embryo and endosperm, respectively. Scale bar, 100 μm. (O–R) CLSM observation of the autonomous endosperm of osemf2a-1(O and P) and osemf2a-2(Q and R) caryopses at 7 days after emasculation. Embryos were not observed in the autonomous seeds. (P) and (R) are magnified images of the boxed regions in (E) and (Q), respectively. Scale bars, 100 μm.

Figure 2

Endosperm development and embryogenesis of osemf2a. (A–H) Transverse sections of the syncytial or cellularized endosperm cells of the WT (A–D) and osemf2a-1(E–H) at 2 (A and E), 3 (B and F), 4 (C and G), and 5 DAF (D and H). Scale bars, 50 μm. (I and J) CLSM images of the cellularized endosperm cells of the WT at 3 DAF (I) and the syncytial endosperm cells of osemf2a-1 at 4 DAF (J). Scale bars, 50 μm. (K–S) CLSM images of the WT (K–N) and osemf2a-1(O–S) embryos at 2 (K and O), 3 (L and P), 4 (M and Q), and 10 DAF (N–S). The embryo development of osemf2a-1 ceased at the globular stage (O–R). Very few embryos of osemf2a-1 developed into a further stage (S) and lagged behind the WT at the same time after fertilization (N). Detached 10-DAF-old embryos of the WT were used for microscopy. Scale bars, 100 μm. The arrows indicate the embryos.

Figure 3

Storage compound accumulation defects in osemf2a. (A and B) Transverse sections of 7-DAF-old caryopses of the WT (A) and osemf2a-1(B). The sections were stained with toluidine blue. Pe, Al, and En indicate pericarp, aleurone, and cellularized endosperm cells, respectively. Scale bars, 50 μm. (C and D) I₂-IK staining of 7-DAF-old caryopses of the WT (C) and osemf2a-1(D). Pe, pericarp; En, cellularized endosperm cells. Scale bars, 50 μm. (E and F) Transmission electron microscopy analysis of 7-DAF-old endosperm cells of ZH11 (E) and osemf2a-1(F). The yellow arrows indicate starch granules in the endosperm cells. Scale bars, 10 μm. (G) Total starch content in the endosperm of the WT and osemf2a-1 at the mature stage. ∗∗p < 0.05, Student's t-test for statistical analysis. The error bars indicate standard deviations; three biological replicates were used for the analysis. (H–J) Sucrose (H), fructose (I), and glucose (J) contents of the WT and osemf2a-1 caryopses at 10 DAF. ∗∗p < 0.05, Student's t-test. The error bars indicate standard deviations; three biological replicates were used for the analysis. (K) Heatmap of the expression of key genes involved in starch biosynthesis. The expression level is indicated by the reads per kilobase per million mapped reads (RPKM) value. The color bar indicates the log₂(RPKM).

Figure 4

Activation of cell cycles caused by overproduction of cytokinin in the caryopses of osemf2a. (A) Heatmap of the expression of cyclin genes in the caryopses of WT and osemf2a-1 at 10 DAF. The expression of three biological replicates of WT and the mutant are presented. The color bar indicates log₂(RPKM). (B) Expression of the cytokinin biosynthesis genes OsLOGL1 and OsIPT2, and the signaling genes OsRR5 and OsRR13. (C–F) Contents of trans-zeatin riboside (tZR) (C), zeatin (D), isopentenyladenine (iP) (E), and isopentenyladenine riboside (iPR) (F) in the WT and osemf2a-1 caryopses at 5 and 10 DAF. Three biological replicates were used for the analysis. Error bars indicate standard deviations. ∗∗p < 0.05, Student's t-test.

Figure 5

Overactivation of type I OsMADSs in osemf2a. (A) Heatmap of the expression of type I MADS-box genes in 10-DAF-old caryopses of the WT and osemf2a-1. The color bar indicates the RPKM values of the genes in three biological replicates. (B–F) Expression dynamics of OsMADS77(B), OsMADS79(C), OsMADS82(D), OsMADS87(E), and OsMADS89(F) in WT and osemf2a-1 caryopses of different ages. (G) Neighbor-joining tree of the type I MADS transcription factors of rice. PHE1, PHE2, and AGL62 in Arabidopsis are highlighted in red. (H and I) ChIP–PCR assay showed that OsMADS77(H) and OsMADS79(I) were highly enriched for H3K27me3 in the endosperm of WT compared with that in the endosperm of osemf2a-1 at 10 DAF. Six investigated regions (P1–P6) are indicated in the schematic figures for each gene. Black boxes and gray bars indicate the coding sequences and the promoters of the genes, respectively. Error bars indicate standard deviations (n = 3). The ChIP–PCR assay was performed twice with consistent results; one of the experiments is presented.

Figure 6

OsEMF2a Is a maternally expressed gene in rice. (A) Morphology of the seeds produced by the reciprocal crosses of ZH11 and osemf2a-1. Left: ZH11 (♀)/osemf2a-1 (♂) (Zm); right, osemf2a-1 (♀)/ZH11 (♂) (mZ). (B) Percentage of well-filled, partially filled, and empty seeds produced by ZH11 (♀)/ZH11 (♂) (ZZ), osemf2a-1 (♀)/osemf2a-1 (♂) (mm), Zm, and mZ. No empty seeds were observed in ZZ and Zm, and no well-filled seeds were found in mm. (C) Caryopsis morphology of the ZH11 (♀)/9311 (♂) (Z9), 9311 (♀)/ZH11 (♂) (9Z), osemf2a-1 (♀)/9311 (♂) (m9), and 9311 (♀)/osemf2a-1 (♂) (9m). From top to bottom: Z9, 9Z, m9, and 9m. (D) Verification of the parent-of-origin-dependent expression of OsEMF2a in the reciprocal crosses of ZH11 and 9311, and of osemf2a-1 and 9311. The SNP used to distinguish the parental origin of the transcripts is indicated by the red arrow.

Figure 7

Disrupted imprinting in osemf2a-1. (A and B) The proportions of the maternal transcripts in the total transcripts of each imprinted gene in the ZH11-9311 reciprocal crosses (Z9–9Z) (A) and their corresponding proportions in the osemf2a-9311 reciprocal crosses (m9–9m) (B). The black and red dots indicate the paternally expressed genes (PEGs) and maternally expressed genes (MEGs), respectively, identified from the Z9–9Z. The genes indicated in the figures were selected for validation of the disrupted imprinting in the hybrid endosperm of m9. (C and D) The imprinted genes identified from Z9–9Z showed a similar expression level in the hybrid endosperm of 9Z and 9m (C), but exhibited expression disorders in m9 when compared with Z9 (D). (E) Heatmap of the expression of the disrupted imprinted genes in the hybrid endosperm of Z9, 9Z, m9, and 9m, respectively. The expression level is indicated by RPKM values revealed by RNA-seq. The color bar indicates log₂(RPKM). (F–H) Parental expression of the upregulated disrupted PEGs (dPEGs) (F), downregulated dPEGs (G), and disrupted MEGs (dMEGs) (H) in m9 and Z9. Expression of the parental transcripts is indicated by the number of informative reads showing SNPs between the parents. Expression of the parental transcripts is indicated by the number of informative reads showing SNPs (SNP-counts) between the parents. If there were multiple SNPs within the transcripts, we used the means for analysis. M, maternal transcripts; P, paternal transcripts.