title: Genetics of homology-dependent gene silencing in Arabidopsis; a role for methylation.
abstract: Ninety-eight independent transformed (T1) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T2 generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology-dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self-fertilization of plants hemizygous for the B and C-inserts suggested that these CHS-silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C-insert homozygotes was reduced to undetectable levels. Outcrosses of B- and C-insert homozygotes to wild-type plants resulted in F1 plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild-type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F1 plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C-insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild-type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C-insert-induced silencing and retained methylation and partial silencing after the silencing T-DNA was lost.
authors.affiliation: Department of Genetics, University of Cambridge, UK.
authors.forename: G J
authors.initials: GJ
authors.lastname: Davies
authors.forename: M A
authors.initials: MA
authors.lastname: Sheikh
authors.forename: O J
authors.initials: OJ
authors.lastname: Ratcliffe
authors.forename: G
authors.initials: G
authors.lastname: Coupland
authors.forename: I J
authors.initials: IJ
authors.lastname: Furner
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000872:Anthocyanins / Q000096:biosynthesis / Q000235:genetics; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000032:analysis; D018744:DNA, Plant / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000032:analysis; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant; D010641:Phenotype; D030821:Plants, Genetically Modified; D013395:Sucrose / Q000494:pharmacology
chemical_list: D000872:Anthocyanins; D004269:DNA, Bacterial; D018744:DNA, Plant; D004277:DNA, Single-Stranded; C036483:T-DNA; D013395:Sucrose; D000217:Acyltransferases; C020141:flavanone synthetase