ABSTRACT
Methylation of cytosines plays an important role in epigenetic regulation of gene expression. Several methods exist to determine the methylation status of DNA. Here, we describe a rapid and cost-effective method called Chop-qPCR to determine dynamic changes in the DNA methylation patterns, as they occur for instance in response to environmental stresses.
Subject(s)
DNA Methylation/drug effects , DNA Methylation/genetics , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Salicylic Acid/pharmacology , Epigenesis, Genetic/drug effects , Epigenesis, Genetic/genetics , Polymerase Chain ReactionABSTRACT
MicroRNAs (miRNAs) regulate plant development by post-transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri-miRNAs) to miRNA duplexes, which associate with AGO1. Additional proteins act in concert with DCL1 (e.g. HYL1 and SERRATE) or AGO1 to facilitate efficient and precise pri-miRNA processing and miRNA loading, respectively. In this study, we show that the accumulation of plant microRNAs depends on RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1), a scaffold protein that is found in all higher eukaryotes. miRNA levels are reduced in rack1 mutants, and our data suggest that RACK1 affects the microRNA pathway via several distinct mechanisms involving direct interactions with known microRNA factors: RACK1 ensures the accumulation and processing of some pri-miRNAs, directly interacts with SERRATE and is part of an AGO1 complex. As a result, mutations in RACK1 lead to over-accumulation of miRNA target mRNAs, which are important for ABA responses and phyllotaxy, for example. In conclusion, our study identified complex functioning of RACK1 proteins in the Arabidopsis miRNA pathway; these proteins are important for miRNA production and therefore plant development.
