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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - EPIMAIZE (Understading the Maize Epigenome and its Role in Development)

Teaser

Summary

Phenotypes are determined by both genetic (i.e. DNA sequence-dependent) and epigenetic factors (i.e. reversible, DNA sequence-independent modifications of chromatin). Sequence-based information is essential to define phenotypic outcome, but depends on the establishment of a proper chromatin environment (the epigenome), which determines the transcriptional competence of genes. Given their role in gene expression, epigenetic factors are of great interest for fundamental as well as translational research, and understanding the interplay between genetic and epigenetic factors is an important, open question in plant biology.
Owing to its relative simplicity as an experimental model, Arabidopsis thaliana has thus far provided the bulk of our current understanding of epigenetic regulations in plants. It is however a rather unusual plant species with a small genome size and a low proportion of repetitive elements, in sharp contrast to the genome of most crops. In addition, altering the epigenome in crops, particularly in maize, has profound developmental consequences. By contrast, Arabidopsis is highly resilient to epigenomic instability.

Reproductive development is of course central to plant breeding, yield, and food security. In contrast to animal models, however, where the relation between epigenetics and reproduction is a major theme of research, our understanding of the mechanisms controlling epigenome dynamics during plant reproduction remains rudimentary. However, published data indicate that, at many crop species, alterations of key enzymes controlling chromatin states results in altered reproductive development, including sterility, but also intriguing phenomena such as clear tendencies for clonal reproduction through seeds (a process known as apomixis in plants), or formation of unreduced gametes, both of which represent potentially very useful tools in plant breeding.

In this proposal, we proposed a dedicated effort to elucidate the role of chromatin regulation on reproduction in a crop species, using maize as our primary experimental model. Maize is a essential crop in both the US, where the host lab is located, and Europe. It is also an exceptional model to address the interplay between genetic and epigenetic factors, owing to a long history of epigenetic research, collections of mutants affecting epigenetic determinants, and, as far as this project is concerned, an exceptional cytology. The project relies on a unique collection of (mostly unpublished) maize mutants affecting chromatin factors acting during reproduction, and on the strong experience of the participants in both the construction of epigenetic maps, and the analysis of plant reproductive development. We hope to harness this expertise to assess the functional importance of key epigenetic pathways in shaping maize reproductive outcomes, and derive novel tools for plant breeding, as described below.

Work performed

As part of the project, we have generated a collection of maize mutants affecting key effectors of epigenetic regulatory pathways in maize. Genetic analyses, where we measured segregation distortions for the different mutants, indicates that most have strong effects on reproduction, affecting either the definition of the germ line, meiosis, or embryogenesis. Particularly striking, we identified mutant capable of generating significant proportions of unreduced male and female gametes, but also parthenogenetic embryos at a low frequency. These preliminary data confirmed the functional importance of the epigenome for maize reproduction, and suggest that many important exciting discoveries will be generated by further characterizing these genes. Particular attention will be paid to mutants resulting in either unreduced gametes, ectopic embryogenesis, or altered recombination rates, as described below.

A key aspect of the first year was its training component. I used this first year to gain considerable new expertise in the field of bioinformatics applied to epigenomics data. In particular, I learned with some of the top experts in the field the art of constructing proper whole-genome single based resolution methylomes, from library construction to data analysis in the context of a large genome such as maize. Similar training covered the analysis of small-RNA libraries, and Chromatin immuprecipitation experiments. In addition, I took advantage of the Advanced Microscopy Core Facility at Cold Spring Harbor to train in hyper-resolution microscopy.

Finally, this year was also an opportunity both to get trained in key technologies by the host lab, and to contribute to some of the project in the host lab, training some of the junior scientists in specific aspects of plant reproductive development, when useful to their projects. A key component of the one-year period at Cold Spring Harbor Laboratory was indeed the acquisition of specific novels skills. From that perspective, we view the results as a real success. Specifically, Daniel Grimanelli acquired up-to-date knowledge in three distinct areas:
1- Proper methodologies for the construction of high quality samples for Next Generation Sequencing related to Genome Wide Bisulfite Sequencing, Chromatin Immunoprecipitation, and RNA sequencing.
2- Analysis and interpretation of large scale genomic and epigenomic data. This was an essential component of the training, particularly with respect to the analysis of data for a complex genome such as maize. The Martienssen lab, which has extensive experience from that perspective, was thus the perfect place for such training.
3- Methodologies for hyper-resolution microscopy.
All three objective were achieved by a mix of formal training and hands-on practicing.

Final results

We have identified rather unusual phenotypic responses associated with the disruption of specific regulators of epigenetic states, belonging to the ARGONAUTE and DNA methyltransferase families of chromatin regulators. The most stinking phenotypes correspond to meiotic defects, ectopic germ cell formation, or heterochronic development of parthenogenetic embryos. We initiated the development of epigenome maps (so far mostly describing DNA methylation status) for the most interesting mutants. Cytological analyses are also underway to understand the germ cell-specific alterations induced by the disruptions.