PIN proteins and architectural change in plants.

Lead Research Organisation: University of Bristol
Department Name: Biological Sciences

Abstract

A key challenge in biology is to understand how body parts with complex shapes and specialized functions arise during development. In plants, the overall shape reflects the pattern of branching, the pattern of leaf initiation and the relative growth of leaves initiated from the tip. These traits impact strongly on plant productivity because they affect light interception in photosynthesis. Because many crop species are grasses that have little branching, the shape of leaves and their arrangement around the stem are particularly important. For these reasons, understanding the basic mechanisms that regulate leaf arrangements and growth is of considerable interest to scientists.

To study this problem, we are working on a moss (Physcomitrella patens), which like many plants, has leaves that are arranged in a spiral pattern around the stem. Physcomitrella has many advantages as a model for leaf development. Mosses are an evolutionary ancient group in which most gene families functioning in other plants are represented by fewer family members, making it simpler to pinpoint gene function. The plants are very small and the shoot apex is made up of a single cell. Leaves initiate as a single cell and are a single cell layer thick. This means that we have been able to develop a technique for filming shoot initiation, leaf initiation and leaf development microscopically, and can generate quantitative information about how cell division and growth contribute to overall plant shape.

Analysis and interpretation of this data is difficult without computational input, so we are working with computer scientists to identify key contributors to shape. Such computational analyses have so far abstracted leaf development to the tissue scale, or focussed on a specific aspect of development to minimise computer processing constraints. Because moss leaves have few cells, we have been able to generate a cellular model of leaf development that has made specific predictions about the contribution of cell division and growth to final leaf shape.

A plant hormone, auxin, plays a primary role in modulating leaf initiation patterns and leaf shape in plants like tomato and Arabidopsis as it regulates decisions about cell identity and growth. The regulated distribution of auxin is a key aspect of its activity, and transport is effected by carrier proteins belonging to a small gene family. Current approaches for evaluating how the carrier proteins effect the auxin distribution and impact on overall shape are limited by the contribution of multiple gene family members to transport. Monitoring the auxin distribution in flowering plant leaves has also been challenging as it can only be achieved indirectly by monitoring changes in the activity of auxin responsive genes, and flowering plant leaves have a complex tissue composition that limits tissue penetration in microscopy. The anatomical and genetic simplicity of the moss shoot again brings an advantage.

A further advantage in using moss to understand how plant shape is attained is that moss shoots have an independent evolutionary origin to most shoot systems. This means that if the mechanisms regulating shape are shared with other better studied groups like flowering plants, they are likely to be universal regulators of plant shape. The knowledge that we genenerate will therefore be very broadly applicable.

Technical Summary

We aim to understand the basic processes that regulate shoot development and leaf shape, and how they have changed in plant evolution. To this end, we have developed a moss, Physcomitrella, as a model system for shoot development. As moss shoots are gametophytic, and mosses and flowering plants diverged around 430 million years ago, similarities in form arise by convergence. This creates the opportunity to identify mechanisms that are fundamental requirements of shoot development.

Auxin and its transport by PIN proteins are primary regulators of leaf initiation, leaflet initiation, margin growth and vein insertion in generating flowering plant shape, and auxin transport has conserved roles in the vascular plants. Tissue complexity and genetic redundancy make it hard to dissect the contribution of auxin and its transporters to these aspects of leaf development in flowering plants, and basal vascular plants are not amenable to genetics. Our aim is to use the simple leaves of moss to identify links between auxin, growth and shape that would be hard to pull out in flowering plants.

We previously used live-imaging and clonal analysis to determine how cell division and growth contribute to moss leaf development. We instigated a collaboration with computer scientists working in the Prusinkiewicz group (Calgary) to synthesise our data in a computational model of leaf development. This has suggested that the growth rate distribution is a key determinant of shape. As auxin modulates growth, a corollary is that the auxin distribution, and auxin transport, are important regulators of shape.

The aims of this proposal are to:
1. test the function of Physcomitrella PIN homologues.
2. determine the growth rate distribution in leaves.
3. generate a reporter to determine the auxin distribution in moss leaves.
4. identify links between the auxin distribution, auxin transport, the growth rate distribution and shape using genetics and pharmacology.

Planned Impact

Who will benefit from this research and how?

Academic beneficiaries
The scientific community will benefit from the outputs and training provided by this research as described in the 'Academic Beneficiaries' section.

Beneficiaries in secondary education
This work is underpinned by a strong grounding in comparative biology, and comparative techniques like phylogeny. We think that phylogeny provides a strong a strong unifying theme for teaching in evolution, genetics, population genetics, health and disease and biodiversity. We are working with the Astra Zeneca Teachers' Trust and Science And Plants for Schools (SAPS) based at the Cambridge Botanic Garden to strengthen understanding and use of phylogeny in secondary teaching. So far this has resulted in the development of a web resource that showcases the use of phylogeny in medicinal plant bio-prospecting in a local 6th form college.

Public sector
The Cambridge Botanic Garden offers an exciting opportunity to showcase the evolutionary aspects of our work in the context of a living collection that manifests the diversity of plant life. We are working with education and outreach officers at the Botanic Garden and SAPS to use the living collection to effectively communicate key ideas relating to the radiation of plants on land. For instance, we are developing interpretation for the 'Life before flowers: a green world greenhouse display', and we will use this as a starting point to weave a storyline about plant evolution as a trail through the rest of the garden. We will work with local artists in the 'fascination of plants' day held at the Botanic Garden to invite visitors to look closely at fossils and basal lineages of land plants in the context of how changes in plant shape have happened through time. We think that this will challenge public views of plant shape in a way that could be constructive in relation to engineering change in crops.

Private sector
We are collaborating with researchers in the Biochemistry Department to exploit moss in generating green energy in bio-photovoltaic fuel cells. A design prototype, the 'moss table' in which many cells act in parallel, has arisen from a collaborative project between biologists and engineers to educate the public about the challenges of generating renewable energy and has been widely exhibited within the UK (http://www.robaid.com/bionics/moss-table-employs-biophotovoltaic-technology-to-generate-power.htm). The electrical output of fuel cells in the 'moss table' is currently low, but a long term aim is to make small self-powering organic-synthetic hybrid objects for use in daily life. We are working with Dr. Bombelli to improve the efficiency and reproducibility of moss fuel cells using our moss growth expertise. We are also exploring possibilities for making self-sustaining, sealed objects powered by moss by building new prototypes, and will explore routes for commercialisation in collaboration with Dr. Bea Schlarb-Ridley, the Cambridge Enterprise Officer, who has an extensive network of private sector contacts.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/L002248/1 02/09/2013 30/06/2015 £320,184
BB/L002248/2 Transfer BB/L002248/1 01/07/2015 31/08/2016 £109,630
 
Description This grant aimed to look at the role of PIN auxin transporters in a moss, in which polar auxin transport was previously thought not to regulate leafy shoot development. We have found that Moss PIN proteins are similar to those that regulate polar auxin transport in Arabidopsis, and have determined the functions of two.
We built on this finding to show that moss PINs do not regulate branching patterns in moss gametophytes and we postulated an alternative mechanism for auxin transport.
We have made promoter reporter and protein reporter lines for all 4 moss PINs, and are making multiple knockouts.
I am senior author or sole author on publications relating to work on this grant:
Dennis et al (2019). Journal of Bryology 41: 314.
Coudert et al. (2017). New Phytologist 215: 840.
Harrison (2017). New Phytologist 215: 545.
Harrison (2017). Phil. Trans. R. Soc B 372: 20150490.
Coudert et al. (2015). eLIFE 4.
Harrison (2015). Trends in Plant Science 20: 468.
Bennett et al. (2014). Current Biology 24: 2776.
Bennett et al. (2014). Molecular Biology and Evolution 31: 2042.
Colleagues working on this grant have also contributed to several further publications from my lab including:
Whitewoods et al. (2018). Current Biology 28: 1.
Coudert et al. (2019). Current Biology 28: 2743.
Exploitation Route This award moved over with me to Bristol from cambridge and is the same grant as 2248/1
Please see info there
Sectors Agriculture, Food and Drink,Other

 
Description Bristol Centre for Agricultural Innovation funding
Amount £44,000 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 01/2023
 
Description Leverhulme Project Grant
Amount £192,806 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 01/2019 
 
Title IPTOE line generated 
Description A cytokinin overprducer line was generated 
Type Of Material Biological samples 
Provided To Others? No  
Impact Transferable to other projects 
 
Title Method for quantifying leaf shape 
Description Cell segmentation and analysis tool published in Dennis et al 2019 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? Yes  
Impact Too soon to say 
URL https://www.tandfonline.com/doi/full/10.1080/03736687.2019.1668109
 
Title Model of moss branching patterns 
Description We developed a computational model of branching patterns 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact The method will underpin work by Dr Yoan Coudert as he sets up his research group in Paris. He was awarded an ATIP futur avenir fellowship in 2015 
 
Title Quantification of moss branching patterns 
Description We developed a method for comparing branching patterns between species or mutants 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Publications with collaborating labs 
 
Description Cambridge Botanic Garden Outreach 
Organisation University of Cambridge
Department Cambridge University Botanic Garden
Country United Kingdom 
Sector Academic/University 
PI Contribution We are working towards building an evolution trail in the Garden.
Collaborator Contribution I designed a trail and hand out, freely available on my lab blog: http://harrisonlab.blogspot.co.uk.
Impact Evolution trail now incorporated in BG and first year teaching on 'Evolution and behaviour' at Cambridge University.
Start Year 2013
 
Description Computational modelling of moss leaf development 
Organisation University of Calgary
Country Canada 
Sector Academic/University 
PI Contribution We are contributing expertise in moss genetics, cellular resolution 4D imaging, image segmentation and analysis.
Collaborator Contribution Our partner is a leader in the plant computational modelling field, and the project is a collaboration to understand the dynamics of moss leaf development.
Impact We have not yet published our work.
Start Year 2007
 
Description Evolution of branching patterns in mosses 
Organisation Natural History Museum
Country United Kingdom 
Sector Public 
PI Contribution project design use of specimens data analysis paper writing
Collaborator Contribution access to specimens project design knowledge of plant groups
Impact There will be a paper, currently accepted with New Phytologist The project has primed a new group forming at the Natural history museum in Paris
Start Year 2013
 
Description Evolution of branching patterns in mosses 
Organisation Royal Botanic Garden Edinburgh (RBGE)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Project design data analysis paper writing
Collaborator Contribution knowledge of plant phylogeny and plant group project design
Impact Paper accepted in New Phytologist
Start Year 2014
 
Description CUBG trail files 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact request from Science and Plants for Schools for access to files for BG Trail so that it can be transferred to other gardens
Year(s) Of Engagement Activity 2015
URL http://harrisonlab.blogspot.co.uk/2016/01/source-files-for-bg-trail.html
 
Description CUBG trail launch 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact As an impact activity associated with my BBSRC grant 'PIN proteins and architectural diversification in plants' (BB/L00224811), I have worked with Dr Alison Murray (CUBG) and Prof Beverley Glover (CUBG and Plant Sciences Department, Cambridge University) to establish an evolution trail in the Cambridge University Botanic Garden.

The trail currently comprises a selection of plants chosen to represent key steps in plant evolution mapped out in the Garden with a handout to be used by undergraduates taking the 'evolution and behaviour' module that can be signed out from the Brookside entrance.

The trail handout can be downloaded here.

New interpretation boards to showcase the relevance of plants in the 'life before flowers' greenhouse have been designed and will be produced in 2016.

The trail conveys ideas about how plant life has changed over geological timescales from the time that tiny bryophyte-like plants first started to colonise land, through the establishment of the earliest vascular plant shooting systems to the radiation of today's dominant flowering plant flora.

The conquest of land by plants cooled the earth's climate, established the first soils, and provided the food and shelter that allowed animals to colonise land.

Recurring themes of plant interrelationships, plant adaptation to new environments and plants shaping their environment have great relevance to future challenges to humanity in the face of rapid population growth and climate change.
Year(s) Of Engagement Activity 2015
URL http://harrisonlab.blogspot.co.uk/2015/12/evolution-trail-goes-live-at-cubg.html
 
Description Ross Dennis collaboration with moss fuel cell group 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Develop a moss fuel cell for use in schools

A publication relating to a moss fuel cell for use in schools
Year(s) Of Engagement Activity 2012,2013,2014
URL http://www.cam.ac.uk/research/news/the-hidden-power-of-moss
 
Description Talk at Graduate School of Experimental Plant Sciences, Wageningen. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research visit and invited talk in Wageningen, discussions with potential collaborators
Year(s) Of Engagement Activity 2016