RNA-directed DNA methylation in plant development

Plant development is very plastic since plants cannot escape adverse conditions, they tailor their shape and physiology to the environment. A spectacular manifestation of this strategy is post-embryonic organogenesis. On different individuals, organs can be and are formed at different positions, depending on need. This ability implies the existence of a robust system for reprogramming and securing the identity of a subset of cells, giving rise to the organs. Plant hormones such as auxin, are vitally important for initiating the reprogramming process and for defining its position. Hormones or external cues may, however not be sufficient for automatically triggering gene expression changes. Physical characteristics of the coding DNA, namely the presence of methyl groups at its cytosine residues, can drastically affect its on/off status, expression rate or responsiveness to stimuli. An interesting question is whether plant hormones influence gene expression, at least partly, by interfering with the methylation status of target genes or it is rather the opposite and it is the methylation that defines what a hormone can reach. We have an excellent tool at our disposal for studying these crucial questions. We found a mutation where a single amino acid exchange in a subunit (NRPE5) of the DNA polymerase complex Pol V results in both aberrant hormone-dependent development and in altered DNA methylation. We will analyze global gene expression and in parallel, gene methylation status in detail, using a next generation sequencing (NGS) approach. Our data will shed light on the nature of causality between hormone action and DNA methylation. Furthermore, the new mutant (termed freak show, fks) displays a series of unique and severe visible phenotypes. Characterization of these traits and identification of the genomic locations causing them will enable us to describe methylation-controlled developmental processes in more detail. Combining fks with mutants perturbed in various sub-pathways of the DNA methylation process will help us further analyzing the role of NRPE5 in establishing methylation and its potential interplay with methylation maintenance. Comparison of fks with other known NRPE5 alleles suggests that the mutation did not turn the Pol V complex inactive but it rather caused a shift in its functionality. Whether this shift is due to a change in target selection, specificity, affinity, efficiency, the usage of alternative subunits or something else, is an exciting question. We will start discriminating between these scenarios using biochemical tools: protein interaction assays with other polymerase subunits will provide the first clues. Characterizing and using the nrpe5fks mutant allele in subsequent research will provide us with unprecedented information for the mechanistic understanding of Pol V action, DNA methylation establishment and its hormone dependence in higher plants.

For a complete and proper life cycle, it is not enough to have all the genes required for every function, but information must be available about whether, where and how a gene is allowed to work. Part of this information is encoded in the form of DNA methylation, a chemical modification at selected cytosine residues throughout the chromosomes. Having such a modification can interfere with the ability of proteins to interact with DNA, affecting a genes functionality. Depending on location and density of this modification, genes can selectively be kept on or off as well as their activity can be kept within the acceptable range. In higher plants, cytosine DNA methylation is mainly deposited by a process called RNA-directed DNA methylation (RdDM), while during DNA replication (e.g. when cells divide and plants grow), DNA methylation is copied over to the new DNA mostly by different mechanisms, the most important one being the so called MET1 pathway. Currently, textbooks say that the core of RdDM and the MET1 pathway hardly have anything to do with each other, they do not interact and their components are distinct. Generally, plants with disturbed RdDM look surprisingly healthy, while without a proper MET1 machinery, plants do not develop normally, and frequently cannot finish their life cycle. In our research, we worked with a novel mutant called freak show (fks) of Arabidopsis, a tiny weed, the laboratory mouse of plant research. In fks, an important component of RdDM does not look exactly as it should. Strikingly, instead of showing the molecular characteristic of RdDM mutants and being otherwise healthy, fks looks like a plant without a fully functional MET1 pathway, both macroscopically and at the molecular level. Using genetic and biochemical tools we confirmed that this strange behavior is caused by the modified RdDM component. Our results suggest an unanticipated mechanistic overlap between the two major DNA methylation mechanisms. The abnormal RdDM component fks clearly interferes with the efficiency of DNA methylation through MET1, challenging the current dogma of independent DNA methylation processes in plants. Whether this is a physiologically significant, direct or indirect effect, is still an open question, and requires further research work. However, an important step towards a better understanding the mechanism of DNA methylation had been made.