Yeonhee Choi

DEMETER, a DNA Glycosylase Domain Protein, Is Required for Endosperm Gene Imprinting and Seed Viability in Arabidopsis

We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm development. For the DEMETER (DME) gene, seed viability depends only on the maternal allele. DME encodes a large protein with DNA glycosylase and nuclear localization domains. DME is expressed primarily in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEDEA (MEA) Polycomb gene in the central cell and endosperm. Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. Thus, a DNA glycosylase activates maternal expression of an imprinted gene in the central cell.

DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

Mutations in the FIE and MEA Genes That Encode Interacting Polycomb Proteins Cause Parent-of-Origin Effects on Seed Development by Distinct Mechanisms

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus, which replicates to generate the endosperm, a tissue that supports embryo development. The FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) genes encode WD and SET domain polycomb proteins, respectively. In the absence of fertilization, a female gametophyte with a loss-of-function fie or mea allele initiates endosperm development without fertilization. fie and mea mutations also cause parent-of-origin effects, in which the wild-type maternal allele is essential and the paternal allele is dispensable for seed viability. Here, we show that FIE and MEA polycomb proteins interact physically, suggesting that the molecular partnership of WD and SET domain polycomb proteins has been conserved during the evolution of flowering plants. The overlapping expression patterns of FIE and MEA are consistent with their suppression of gene transcription and endosperm development in the central cell as well as their control of seed development after fertilization. Although FIE and MEA interact, differences in maternal versus paternal patterns of expression, as well as the effect of a recessive mutation in the DECREASE IN DNA METHYLATION1 (DDM1) gene on mutant allele transmission, indicate that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms.

Imprinting of the MEA Polycomb Gene Is Controlled by Antagonism between MET1 Methyltransferase and DME Glycosylase

The MEA Polycomb gene is imprinted in the Arabidopsis endosperm. DME DNA glycosylase activates maternal MEA allele expression in the central cell of the female gametophyte, the progenitor of the endosperm. Maternal mutant dme or mea alleles result in seed abortion. We identified mutations that suppress dme seed abortion and found that they reside in the MET1 methyltransferase gene, which maintains cytosine methylation. Seeds with maternal dme and met1 alleles survive, indicating that suppression occurs in the female gametophyte. Suppression requires a maternal wild-type MEA allele, suggesting that MET1 functions upstream of, or at, MEA. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell. MET1 is required for DNA methylation of three regions in the MEA promoter in seeds. Our data suggest that imprinting is controlled in the female gametophyte by antagonism between the two DNA-modifying enzymes, MET1 methyltransferase and DME DNA glycosylase.

Imprinting and seed development.

Imprinted genes are expressed predominantly from one allele in a parent-of-origin–specific manner. The endosperm, a seed tissue that mediates the transfer of nutrients from the maternal parent to the embryo, is an important site of imprinting in flowering plants. Imprinted genes have been

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.

Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development

The epigenetic regulation of the floral repressor Flowering Locus C (FLC) is one of the critical factors that determine flowering time in Arabidopsis thaliana. Although many FLC regulators, and their effects on FLC chromatin, have been extensively studied, the epigenetic resetting of FLC has not yet been thoroughly characterized. Here, we investigate the FLC expression during gametogenesis and embryogenesis using FLC::GUS transgenic plants and RNA analysis. Regardless of the epigenetic state in adult plants, FLC expression disappeared in gametophytes. Subsequently, FLC expression was reactivated after fertilization in embryos, but not in the endosperm. Both parental alleles contributed equally to the expression of FLC in embryos. Surprisingly, the reactivation of FLC in early embryos was independent of FRIGIDA (FRI) and SUPPRESSOR OF FRIGIDA 4 (SUF4) activities. Instead, FRI, SUF4 and autonomous-pathway genes determined the level of FLC expression only in late embryogenesis. Many FLC regulators exhibited expression patterns similar to that of FLC, indicating potential roles in FLC reprogramming. An FVE mutation caused ectopic expression of FLC in the endosperm. A mutation in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 caused defects in FLC reactivation in early embryogenesis, and maintenance of full FLC expression in late embryogenesis. We also show that the polycomb group complex components, Fertilization-Independent endosperm and MEDEA, which mediate epigenetic regulation in seeds, are not relevant for FLC reprogramming. Based on our results, we propose that FLC reprogramming is composed of three phases: (i) repression in gametogenesis, (ii) reactivation in early embryogenesis and (iii) maintenance in late embryogenesis.

Growth habit determination by the balance of histone methylation activities in Arabidopsis.

In Arabidopsis, the rapid‐flowering summer‐annual versus the vernalization‐requiring winter‐annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter‐annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)‐specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36‐ and H3K4‐specific histone demethylase (HDM) activities of autonomous‐pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter‐annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36‐specific HMT and HDM activities.

A novel extensin gene encoding a hydroxyproline-rich glycoprotein requires sucrose for its wound-inducible expression in transgenic plants.

A novel hydroxyproline-rich glycoprotein (SbHRGP3) that consists of two different domains is encoded by an extensin gene from soybean. The first domain (domain 1) located at the N terminus is composed of 11 repeats of Ser-Pro4-Lys-His-Ser-Pro4-Tyr3-His, whereas the second domain (domain 2) at the C terminus contains five repeats of Ser-Pro4-Val-Tyr-Lys-Tyr-Lys-Ser-Pro4-Tyr-Lys-Tyr-Pro-Ser-Pro5-Tyr-Lys-T yr- Pro-Ser-Pro4-Val-Tyr-Lys-Tyr-Lys. These two repeat motifs are organized in an extremely well-ordered pattern in each domain, which suggests that SbHRGP3 belongs to a new group of proteins having the repeat motifs of two distinct groups of dicot extensins. The expression of the SbHRGP3 gene increased with seedling maturation, and its expression was relatively high in the mature regions of the hypocotyl and in the root of soybean seedlings. An SbHRGP3-beta-glucuronidase (SbHRGP3-GUS) chimeric gene was constructed and expressed in transgenic tobacco plants. The expression of the SbHRGP3-GUS gene was not induced by wounding alone in transgenic tobacco plants; sucrose was also required. Expression was specific to phloem tissues and cambium cells of leaves and stems. In transgenic tobacco seedlings, SbHRGP3-GUS gene expression was activated by the maturation of the primary root and then inactivated; however, reactivation was specifically at the epidermis of the zone from which the lateral root was to be initiated. Its reactivation occurred just before the lateral root initiation. These results indicate that the SbHRGP3 gene in different tissues responds to different signals.

The catalytic subunit of Arabidopsis DNA polymerase α ensures stable maintenance of histone modification

Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase α in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance.