An invariant aspartic acid in the DNA glycosylase domain of DEMETER is necessary for transcriptional activation of the imprinted MEDEA gene

Abstract

Helix-hairpin-helix DNA glycosylases are typically small proteins that initiate repair of DNA by excising damaged or mispaired bases. An invariant aspartic acid in the active site is involved in catalyzing the excision reaction. Replacement of this critical residue with an asparagine severely reduces catalytic activity but preserves enzyme stability and structure. The Arabidopsis DEMETER (DME) gene encodes a large 1,729-aa polypeptide with a 200-aa DNA glycosylase domain. DME is expressed primarily in the central cell of the female gametophyte. DME activates maternal allele expression of the imprinted MEDEA (MEA) gene in the central cell and is required for seed viability. We mutated the invariant aspartic acid at position 1304 in DME to asparagine (D1304N) to determine whether the catalytic activity of the DNA glycosylase domain is required for DME function in vivo. Transgenes expressing wild-type DME in the central cell rescue seed abortion caused by a mutation in the endogenous DME gene and activate maternal MEA:GFP transcription. However, transgenes expressing the D1304N mutant DME do not rescue seed abortion or activate maternal MEA:GFP transcription. Whereas ectopic expression of the wild-type DME polypeptide in pollen is sufficient to activate ectopic paternal MEA and MEA:GUS expression, equivalent expression of the D1304N mutant DME in pollen failed to do so. These results show that the conserved aspartic acid residue is necessary for DME to function in vivo and suggest that an active DNA glycosylase domain, normally associated with DNA repair, promotes gene transcription that is essential for gene imprinting.

Fig. 1.

Effect of the D1304N mutation on seed viability. Siliques in A-D were dissected and photographed 14 days after self-pollination. (Scale bars = 0.5 mm.) Arrows indicate aborted seeds. (A) Wild-type silique. (B) Heterozygous DME/dme-2 silique. (C) Silique is heterozygous DME/dme-2 and hemizygous for a DME:DME-4 transgene. (D) Silique is heterozygous DME/dme-2 and hemizygous for a DME:DME(D1304N)-3 transgene.

Fig. 2.

Effect of the D1304N mutation on transmission of the maternal mutant dme-2 allele. No transgene, heterozygous DME/dme-2 plant was pollinated with wild-type pollen and no F₁ progeny with the dme-2 allele were detected (790 checked); DME:DME(D1304N), plant heterozygous DME/dme-2 and hemizygous for a DME:DME(D1304N)-3 transgene was pollinated with wild-type pollen, and three F₁ progeny with the dme-2 allele and DME:DME(D1304N)-3 transgene were detected (379 checked); DME:DME, plant heterozygous DME/dme-2 and hemizygous for a DME:DME-4 transgene was pollinated with wild-type pollen and 36 F₁ progeny with the dme-2 allele and the DME:DME-4 transgene were detected (97 checked).

Fig. 3.

Effect of the D1304N mutation on maternal MEA:GFP transcription in the central cell. Fluorescence micrographs of ovules harvested from stage 12 flowers (46) are shown. GFP and chlorophyll fluorescence were converted to green and red, respectively. Arrows point to central cells. (Scale bars = 0.04 mm.) (A) Ovules from a wild-type flower that is homozygous for a MEA:GFP transgene. (B) Ovules from a flower that is heterozygous DME/dme-2 and homozygous for a MEA:GFP transgene. (C) Ovules from a flower that is heterozygous DME/dme-2, hemizygous for a DME:DME-4 transgene, and homozygous for a MEA:GFP transgene. (D) Ovules from a flower that is heterozygous DME/dme-2, hemizygous for a DME:DME(D1304N)-3 transgene, and homozygous for a MEA:GFP transgene.

Fig. 4.

Effect of the D1304N mutation on ectopic paternal MEA allele expression. CaMV:DME-4 and CaMV:DME-5 represent two independently isolated transgenic lines that ectopically express the wild-type DME cDNA (8). CaMV:DME(D1304N)-1 and CaMV:DME(D1304N)-3 represent two independently isolated transgenic lines that ectopically express the mutant D1304N form of DME. Total RNA was isolated from pollen harvested from open flowers, and the approximate level of MEA and DME RNA was determined by semiquantitative RT-PCR. (A) CaMV:DME transgenes activate paternal MEA allele gene expression in pollen. (B) CaMV:DME(D1304N) transgenes do not activate paternal MEA allele gene expression in pollen.

Fig. 5.

Effect of the D1304N mutation on ectopic paternal MEA:GUS gene transcription. Light micrographs were taken 12 h after staining for GUS activity. (A) Stamen is hemizygous for a MEA:GUS transgene. (B) Stamen is hemizygous for a CaMV:DME-4 transgene and hemizygous for a MEA:GUS transgene. (C) Stamen is hemizygous for a CaMV:DME(D1304N)-1 transgene and hemizygous for a MEA:GUS transgene. (Scale bars = 0.005 mm.)