Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus

Abstract

Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.

Evolutionary history of Methyltransferase 1 genes in hexaploid wheat

“…Homoeologous group 2 were expressed in most tissues at nearly all developmental stages, named according to the Zadoks (Z) scale [35], but with highest expression levels at Z30 in the stem and Z32 in the spike. MET1 expression levels in other species peak in proliferating cells such as in meristems and in reproductive organs [27, 28, 36]. In wheat we observed TaMET1 expression at early developmental stages especially during early stem extension (Z30-Z32) when wheat is switching from the vegetative to reproductive phase.…”

“…However possible roles for the homoeologous group 7 (MET1-a lineage) is challenged by recent data collected in rice indicating that the main MET1 function is ensured by Met1b and not Met1a . Indeed, RNAi against Met1a does not significantly affect plant development while a met1b null mutant is lethal [28, 53]. …”

“…MET1 is a gene of particular importance for genome maintenance in many organisms, which we hypothesize will be a crucial component of epigenetic mechanisms controlling transposable elements that in wheat make up to 80% of the genome. To date MET1 gene function have been described in several plant species including Arabidopsis [26], maize [27], rice [28] and brassica [29] but not in wheat. We identified MET1 genes in hexaploid wheat ( TaMET1 ).…”

Enrichment of Triticum aestivum gene annotations using ortholog cliques and gene ontologies in other plants

“…The gene is located in the nucleus and is involved in methyltransferase activity in flowering, circadian rhythm and photoperiodism. Other methyltransferase genes were isolated and characterized in monocots such as maize [ 43 ] and rice [ 44 ]. In wheat, five homologous cDNA sequences were connected with methyltransferase activity [ 45 ] and their expression patterns were studied.…”

Epigenetic regulation of rice flowering and reproduction

“…DRM2 is required for de novo cytosine methylation in both symmetric and asymmetric sequence contexts, which is guided to the target region by RNA-directed DNA methylation (RdDM) pathway ( Cao and Jacobsen, 2002 ; Law and Jacobsen, 2010 ; Stroud et al, 2014 ). While Arabidopsis contains only one MET1 gene, rice has two MET1 genes, MET1a (also named OsMET1-1 ) and MET1b/OsMET1-2 ( Teerawanichpan et al, 2004 ; Yamauchi et al, 2008 ). The transcripts of MET1b accumulate more abundantly than those of MET1a in all of the examined rice tissues, indicating that MET1b may play a more important role in maintaining DNA methylation ( Yamauchi et al, 2008 ).…”