Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1)

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles

Genetic Rearrangements Can Modify Chromatin Features at Epialleles

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana. Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality