75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

175 DEMETER DNA Glycosylase Regulates MEDEA Polycomb Gene Self-Imprinting and Seed
Viability by Allele-Specific Demethylation
Wenyan Xiao, Mary Gehring*, Jin Hoe Huh, Tzung-Fu Hsieh, Jon Penterman, Yeonhee Choi#, Robert Fischer
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm
development. Maternal mutant dme or mea alleles result in seed abortion. DEMETER (DME) encodes a large protein with
DNA glycosylase and nuclear localization domains. DNA glycosylases initiate the base-excision DNA repair pathway by
excising damaged or mispaired bases. An invariant aspartic acid in the active site is involved in catalyzing the excision
reaction. We mutated the invariant aspartic acid at position 1304 in DME to asparagine and found that the conserved aspartic
acid residue is necessary for DME to function in vivo and in vitro. DME is expressed primarily in the central cell of the
female gametophyte, the progenitor of the endosperm. MEDEA (MEA) is a Polycomb group gene that is imprinted in the
endosperm. The maternal allele is expressed and the paternal allele is silent. DME DNA glycosylase activates maternal MEA
allele expression in the central cell. We identified mutations that suppress dme seed abortion and found that they reside in
the METHYLTRANSFERASE1 (MET1) gene, which maintains cytosine methylation. DME and MET1 are antagonists
in the central cell. DME activates whereas MET1 suppresses maternal MEA:GFP allele expression. MET1 methylates
the maternal MEA allele whereas DME causes maternal-allele-specific hypomethylation at the MEA gene. DME excises
5-methylcytosine in vitro and in E. coli. We propose that excision of 5-methylcytosine by DME, followed by insertion of
cytosine by downstream enzymes in the base excision DNA repair pathway, results in DNA hypomethylation and expression
of the maternal MEA allele. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb
group proteins that are expressed from the maternal genome, including MEA, are required for 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 MEA allele. New approaches undertaken
to further understand mechanisms for DME-regulated gene imprinting and seed development will be discussed.
Present Address:
*Fred Hutchinson Cancer Research Center, Seattle, WA 98109
#Dept of Biological Sciences, Seoul National University, South Korea
176 VAJURA, an EF-2 Family Protein, Is Involved in Flower Development and
Gametophytogenesis
Noriyoshi Yagi 1 , Seiji Takeda 2 , Noritaka Matsumoto 1 , Kiyotaka Okada 1
1
Department of Botany, Graduate School of Science, Kyoto University, 2 Department of Cell and Developmental
Biology, John Innes Centre
In eukaryotes, pre-mRNA splicing is an essential process for gene expression. The splicing occurs in a large complex,
the spliceosome, which consist of five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4/U6, and U5) and
other non-snRNP splicing factors. The splicing machinery has been studied extensively using mammalian and yeast
cells. In Arabidopsis, one of multicellular organisms, recent studies identified many splicing factors, but little is known
about the role of pre-mRNA splicing in plant development.
The VAJURA (VAJ) gene encodes an EF-2 family protein, of which counterparts in human and yeast are one of the
components of U5 snRNP. It is known that, in plants, spliceosomal components localize in Cajal bodies and in nuclear
speckles. Transient expression assay using Arabidopsis protoplast revealed that VAJ:mRFP co-localized with SC35:GFP,
speckle marker. Sequence similarity and subcellular localization suggest that VAJ functions as a splicing factor.
To assess the role of VAJ during developmental process, we characterized vaj mutants. In vaj-1, a weak allele, sepals
and petals were narrower and longer than those of wild type. Although homozygous plants of vaj-1 were not lethal, in vaj-2
and vaj-3, T-DNA insertion lines, no homozygote was identified from self-pollinated heterozygous plants. The segregation
ratio of heterozygote and wild type indicated that vaj-2 and vaj-3 mutations affected gametophyte development. We are
now performing reciprocal crossing test and cytological studies of male and female gametophyte.
A VAJ-related gene, VAJURA-LIKE (VAL) was found in Arabidopsis. The val mutant showed no obvious phenotype.
EST of VAL was not found at database suggesting that VAL is not expressed or is expressed at very low level. To examine
VAL function genetically, we are generating VAJ/vaj val/val mutant. What kind of role VAJ and VAL play in plant
development will be discussed.