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Genetics of homology‐dependent gene silencing in Arabidopsis ; a role for methylation
Author(s) -
Davies Gareth J.,
Sheikh Mazhar A.,
Ratcliffe Oliver J.,
Coupland George,
Furner Ian J.
Publication year - 1997
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1997.12040791.x
Subject(s) - gene silencing , biology , genetics , chalcone synthase , gene , mutant , dna methylation , methylation , locus (genetics) , arabidopsis , wild type , gene expression , microbiology and biotechnology
Summary Ninety‐eight independent transformed (T 1 ) Arabidopsis plants were generated, containing additional copies of the chalcone synthase ( CHS ) gene. Three T 2 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 F 1 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 F 1 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.