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.

Project details

Project name: RNA-directed DNA methylation in plant development
Project reference: P29988-BBL
Funding Scheme: FWF Stand-Alone Project
Contribution: EUR 351.799,89
Funding provided by the Federal Province of Lower Austria and the “Nationalstiftung für Forschung, Technologie und Entwicklung”Contract type: ERC-SG - ERC Starting Grant
Duration: From 2017-07-01 to 2020-06-30
Project leader: Prof. Jiří Friml