Periodic Reporting for period 1 - miR156evo (The role of the miR156-SPL genetic network during land plant evolution: a comparative analysis of sporophyte development in divergent plant lineages)
Periodo di rendicontazione: 2021-11-01 al 2023-10-31
Plants first colonised land ~470–515 million years ago. Prior to this the Earth’s terrestrial surface was an extremely inhospitable place, largely devoid of life. The arrival of plants on land from aquatic algal ancestors changed both the Earth’s surface and atmosphere, enabling a huge explosion in terrestrial life. The expansion of land plants initiated the development of soils on a previously barren landscape and led to atmospheric oxygen concentrations that could support animal life. The ‘greening’ of Earth changed the course of evolution and was essential to the emergence of vertebrate animals including humans. Understanding the nature of early land plants is thus fundamental to understanding our own evolutionary history.
The land plants we live among and depend on today look very different to the ancestral green alga that first colonised the land. Green algae have basic body plans that are usually restricted to two-dimensions with limited apical growth (i.e. directional proliferative growth facilitated by the self-renewing activity of undifferentiated stem cells). Furthermore, the diploid stage of the life cycle is brief with meiosis occurring immediately following fertilisation. On the other hand, land plants grow in complex 3D structures with extensive apical growth and prolonged diploid development prior to meiosis. What, then, were the critical innovations that led to the establishment of plants on land? With this fundamental question in mind, the aim of this project was to elucidate the ancestral role of a key genetic network in land plants.
To do this we carried out a comparative analysis of gene function in a vascular plant (the flowering plant Arabidopsis) and a bryophyte (the moss Physcomitrium), which arose following an ancient divergence in land plant evolution. Sustained apical growth is a defining feature of vascular plants and the establishment of elaborate shoot systems with delayed reproductive development enabled their extraordinary evolutionary success. In contrast, mosses undergo more transient apical growth prior to rapid reproductive development. In Arabidopsis, a well-studied genetic network regulates apical growth and promotes developmental transitions. However, the role of this module in plant evolution remains to be determined.
Excitingly, early results suggest that this network has an ancestral role in regulating developmental transitions in plants and that the appearance of the network shortly after plants colonised the land was a critical event in plant evolution.
The land plants we live among and depend on today look very different to the ancestral green alga that first colonised the land. Green algae have basic body plans that are usually restricted to two-dimensions with limited apical growth (i.e. directional proliferative growth facilitated by the self-renewing activity of undifferentiated stem cells). Furthermore, the diploid stage of the life cycle is brief with meiosis occurring immediately following fertilisation. On the other hand, land plants grow in complex 3D structures with extensive apical growth and prolonged diploid development prior to meiosis. What, then, were the critical innovations that led to the establishment of plants on land? With this fundamental question in mind, the aim of this project was to elucidate the ancestral role of a key genetic network in land plants.
To do this we carried out a comparative analysis of gene function in a vascular plant (the flowering plant Arabidopsis) and a bryophyte (the moss Physcomitrium), which arose following an ancient divergence in land plant evolution. Sustained apical growth is a defining feature of vascular plants and the establishment of elaborate shoot systems with delayed reproductive development enabled their extraordinary evolutionary success. In contrast, mosses undergo more transient apical growth prior to rapid reproductive development. In Arabidopsis, a well-studied genetic network regulates apical growth and promotes developmental transitions. However, the role of this module in plant evolution remains to be determined.
Excitingly, early results suggest that this network has an ancestral role in regulating developmental transitions in plants and that the appearance of the network shortly after plants colonised the land was a critical event in plant evolution.
The key objective of the project was to characterise the function of the miR156-SPL genetic network in the moss Physcomitrium patens. There are three genes that encode miR156 (MIR156A/B/C) in Physcomitrium and three miR156-targeted SPL genes (SBP3/6/13). To account for this redundancy, and to provide a detailed analysis of the genetic network, we took a multi-layered approach that required the generation of a suite of genetic tools. The design of these tools focused on the resolution of three questions: 1) What are the miR156 and SPL gene expression patterns during moss development? 2) Is SPL activity necessary for moss development? 3) What are the consequences of de-regulation by miR156 on moss development? The initial cloning steps required for these experiments, and the subsequent moss transformation process, are technically demanding and time consuming. However, we have generated all the vectors necessary for transformation (16) and have successfully generated most of the transgenic moss lines required for project success (13/16).
The work has progressed furthest using plants in which either miR156, or a target analog that sequesters miR156 and therefore decreases its activity (named MIM156), is overexpressed. The initial characterisation of these lines has been very encouraging. The manipulation of miR156 appears to regulate Physcomitrium plant shape, leafy shoot initiation and length, leaf shape and reproductive development (as indicated by the accompanying figure). This would indicate that miR156 function emerged in the earliest land plants and its role is not restricted to the vascular plant lineage. Despite the cessation of the Fellowship, we plan to complete this exciting project and anticipate the publication of our findings within the next 12-18 months.
The work has progressed furthest using plants in which either miR156, or a target analog that sequesters miR156 and therefore decreases its activity (named MIM156), is overexpressed. The initial characterisation of these lines has been very encouraging. The manipulation of miR156 appears to regulate Physcomitrium plant shape, leafy shoot initiation and length, leaf shape and reproductive development (as indicated by the accompanying figure). This would indicate that miR156 function emerged in the earliest land plants and its role is not restricted to the vascular plant lineage. Despite the cessation of the Fellowship, we plan to complete this exciting project and anticipate the publication of our findings within the next 12-18 months.
We have generated multiple moss transgenic lines that were not previously available: miR156 or target mimic MIM156 constitutive or inducible overexpressors, CRISPR-Cas9 mediated knockouts of three moss genes and reporter lines that indicate protein activity in both a miR156-sensitive and miR156-insensitive manner. The generation of these lines was an essential step in our aim to investigate the early evolution of land plants. These fundamental tools will be made freely available to the plant genetics community where they will be of significant interest to other researchers. Having produced the lines necessary for our investigations, we will now use them to carry out detailed analyses of the role of miR156 during moss development. The results of these studies will provide critical insights into the establishment of plants on land – an event that changed the world.