RNA splicing control of meristem identity, cell division and differentiation in Arabidopsis.

Plants and animals control their development, from single cell to complex multicellular organism, by regulating the activities of thousands of genes. Each gene produces an RNA molecule, and at one time it was thought that one RNA molecule contains the information to produce one protein. It has been discovered in recent years that complex organisms can in fact splice sections of a given RNA molecule, to generate alternative versions of the final, mature RNA, and this in turn means that a greater diversity of proteins can be produced from a single gene. This production of a range of spliced versions of RNAs is called alternative splicing, and contributes to the fine level of control to regulate the activities of cells. The mechanism and role of alternative splicing in specific developmental processes is still, however, at a very early stage. We have identified a plant gene, termed MDF, for which we have new information indicating a novel role in regulating RNA splicing to regulate cell identitity and cell division activity in the growing regions of plants, the apical meristems. The project aims to understand which RNAs are spliced by MDF, and how these target RNAs contribute to the complex network of genes regulating the activity of meristems.

We previously identified the Arabidopsis gene MERISTEM-DEFECTIVE (MDF) as being expressed in the basal domain of the developing embryo and in the root and shoot apical meristems. Loss-of-function mutants reveals an essential role in maintaining cell identity and cell division activity in the embryo and seedling meristems. MDF encodes an SR family protein, and we hypothesized it has a possible role in splicing, based on homology with human hSART1 and yeast snu66 splicing factors. RNA-seq in two independent mdf mutants and an MDF transgenic overexpressor is consistent with this role, revealing a range of splicing defects in the RNA population. MDF expression is resticted to embryonic and seedling meristems, and represents a potential novel tissue-specific global regulator of meristem identity genes via splicing control. The proposed project aims to characterize the global effects of MDF activity on the RNA population, to characterize the roles of likely MDF targets in the Arabidopsis root meritem, and to define the functional links between MDF and its putative targets. We will use a range of techniques (bioinformatics, gene expression analysis, mutant characterization, advanced bioimaging) to address these questions. The project will lead to a new understanding of the molecular mechanisms of alternative splicing in plants, and the identification of new components in the gene-signalling regulatory network in the Arabidopsis root meristem.

Who will benefit?
The project will be of value to:
1) staff trained on the project;
2) academic research scientists - molecular biologists interested in understanding the mechanisms of RNA splicing, and developmental biologists interested the control of stem cell identity and meristem function, especially how this is regulated by alternative splicing;
3) in the longer term, an improved understanding of the genetic basis root meristem function has the potential to provide breeders with the molecular tools to identify genes in crops, such as identified by GWAS, that contribute to root architecture.

How will they benefit?
Understanding the mechanisms of both alternative splicing in eukaryotes, and meristem function in plants, represent two important areas of basic science. The project will provide new information on both, and our findings will therefore directly impact the broader biological research community. Dissemination of our methods and findings through publications and conference presentations will ensure that a global audience can benefit from our work.

For the PDRA (Dr. Helen Thompson), the award to funding would allow her to capitalise on the excellent work she has carried out to date on her (fixed term) Daphne Jackson Fellowship, a scheme that aims to help back into research those who have taken career breaks. Helen's aim is to improve her skills and expertise in modern molecular biology and genetics, to maximise her opportunity for a future career in science, whether in academia or industry. Many of the skills learned will be transferrable also to careers outside biology, should that be a choice considered by Helen.

Plant breeders increasingly rely on molecular tools to improve the efficiency of breeding, and fundamental research in model systems such as Arabidopsis greatly facilitates gene identification and functional analysis. This can inform the interpretation of genome-wide association studies, for example, such as (in the case of this project) may be associated with improved root traits (i.e. associated with the control of the root meristem). The root meristem responds to environmental conditions to regulate the architecture of the root system, and is a target for breeders who aim to develop crops with, for example, shallow root systems (e.g. to capture poorly-mobile phosphate fertilisers) or deeper root systems (to capture water and more mobile nitrogen). Clearly this project is not directly targeted at these breeding objectives, but gene identification can be expected to inform crop molecular breeding at some point in the future.

The data generated will be made publicly available, to support new hypothesis-driven research in the wider scientific community.