Molecular genetic approaches to study the roles of SHORTLEAF and SCARECROW in moss gametophytic development

Abstract

Physcomitrella patens or moss, of the phyla Bryophyta belong to an ancient lineage of plants, which colonised land around 400mya after diverging from the vascular plant lineage. Leading a haplo-diplontic life cycle, with a dominant haploid stage, this model organism has a very simple yet sturdy body plan. The dominant body plan in moss is the gametophyte, which bears structures similar to those borne by sporophytic vascular plants. Moss bears leaf like structures known as phyllids, (which lack a proper vasculature) and root like organs known as Rhizoids. A forward genetics approach allows for the creation of mutants which target random regions in the genome. One such event carried out in the lab lead to the generation of a shlf (shortleaf) mutant, which as the name suggests bore phyllids which were smaller in size. The gene region that was targeted in the forward genetics approach was characterised to a certain extent during my study. SHLF contains four repeats , with each repeat being highly similar, one of my targets was to understand the functionality of a single repeat. SHLF also has paralogs in the sister clade of Marchantiophyta. Another one of my objectives aimed at understanding the extent of conservation of the gene across the Bryophyta lineage. I also wanted to study the localisation of the protein generated by this gene by raising GFP tagged lines. Understanding the localisation might help us understand the potential function of the protein. Reverse genetics is another methodological approach to create mutants by specifically targeting a known region of the genome. Another such event targeting SCARECROW lead to the generation of a mutant bearing needle like leaves. This gene, in vascular plants is usually involved int patterning the development of the root vasculature and leaves to a certain extent. My study on this mutant included characterising the promoter activity by generation of fluorescent protein (tdTomato) tagged lines, followed by understanding the cell division pattern in the mutant phyllid. Based on the results, the localisation of the protein SHLF could be determined, the functionality of a single repeat of the gene was better understood and a partial conservation of the paralog of SHLF could be concluded.