Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 39 end of a gene coding for S-adenosyl-L-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

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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.

Transcript profiling was used to look for genes that differ in expression between the SAH hydrolase deficient and hypomethylated hog1-1 mutant and the parental (HOG1) line. This analysis identified a subset of gene transcripts that were up-regulated in hog1-1 plants. The majority of these transcripts were from genes located in the pericentromeric heterochromatin. About a third of the genes are annotated as transposons or having transposon homology. Subsequent experiments using Northern blots, RT-PCR and real-time RT-PCR confirmed the up-regulation of 19 of the genes and identified a set of molecular probes for genes that are up-regulated in the hog1-1 background. Six (of six genes tested) of the hog1-1 up-regulated genes are also up-regulated in the hypomethylated ddm1 mutant, three in the hypomethylated met1 mutant and three in the dcl3 mutant. The results suggest that the hypomethylation in the mutant lines may have a causal role in the up-regulation of these transcripts.

SummaryPlant development is critically dependent on the interactions between clonally unrelated cell layers. The crosstalk between layers can be addressed by studies of cell autonomy. Cell autonomy is a property of genetic mosaics composed of cells of differing genotypes. Broadly, if the phenotype of a mutant tissue reflects only its genotype and is unaffected by the presence of wild-type tissue, the trait is cell-autonomous. Conversely, if the phenotype of a mutant tissue reflects that of wild-type tissue in the mosaic, the trait is non-autonomous. Here we report a novel, versatile and robust method for studies of cell autonomy in Arabidopsis. Cell autonomy (CAUT) lines consist of a collection of homozygous stocks, each containing one of 76 mapped T-DNA inserts, each of which corrects the yellow ch-42 mutant to green (CH-42) by complementation. This has the effect of translocating the colour marker to 76 new locations around the genome. X-irradiation of heterozygous CAUT line seeds results in yellow sectors, with loss of the CH-42 transgene and adjacent wild-type genes. This property can be used to remove the wild-type copy of developmental genes in appropriate heterozygotes, resulting in yellow (ch-42) sectors that are hemizygous for the trait of interest. Such sectors can provide insight into cell autonomy. Experiments using the ap1, ap3, ag and clv1 mutants show that CAUT lines are useful in the study of cell autonomy.