Comparative cell-specific profiling to understand the molecular basis of nodulation

Lead Research Organisation: University of Warwick
Department Name: Warwick HRI

Abstract

Plants must acquire the elemental nutrient nitrogen from their surrounding environment and its availability is often a major limitation to plant growth. To try and cope with this, all plants form lateral roots that explore the soil and increase the surface area on which to take up nitrogen. In addition legumes (peas and beans) have a unique strategy to deal with nitrogen limitation. They enter symbiotic interactions with soil bacteria that are able to fix an atmospheric form of nitrogen that plants cannot take up, and convert it into a useable form. Understanding more about how this happens could allow us to develop nodulation outside legume species, a discovery that would have significant effects on agriculture, the environment and nutrition. For example it would enable farmers to expand the range of crops that they could grow on their land without requiring the use of expensive nitrate fertilisers. Lower fertiliser use would in turn benefit the environment since the fossil-fuel rich process required for its manufacture would be diminished. Adding the ability to fix nitrogen to a commercial non-legume crop, for example wheat, might also increase plant nitrogen content and therefore the nutritive value of such improved crops for consumption. Since there are similarities in the way that lateral roots and nodules form on plant roots it is thought that the plan for 'building' a nodule in legumes comes from a lateral root 'blueprint' that exists in all plants. Despite detailed study of lateral roots and nodules little is known of what this 'blueprint' looks like. The answer might come from the fact that both lateral roots and nodules develop from single types of cells in the root - because of this specificity the important factors that link them have not yet been uncovered. We now have the state-of-the-art technology that will allows us to make the detailed analyses required for such study. We will compare legume vs. non-legume responses to nitrogen and responses during nodulation in single cells using these novel techniques to address how nodulation evolved in legumes.

Technical Summary

The ability to nodulate arose several times in legumes and it is thought that a pre-existing lateral root 'blueprint' present in all plants was co-opted for nodulation. Although regulators of nodulation and lateral root development have been characterised, molecular components underlying the proposed co-option have not been uncovered. A likely reason for this is that previous studies of nodulation have been at the whole-root level, and critically both lateral roots and nodules originate from single cell types. In order to identify shared programs of gene regulation the ideal method would therefore be to separate signals from different cells. Recent developments that combine Fluorescently Activated Cell Sorting (FACS) with dynamic treatments to profile environmental responses of individual cell types make a new developmental comparison of the two organs possible. In this systems biology proposal we will compare genomic responses in single cell types of the root between the legume Medicago truncatula and the non-legume Arabidopsis thaliana. The responses will be the effects of nitrogen treatment to induce lateral root development, and Nod-factor treatment to induce nodulation in Medicago. We will measure these responses over a high resolution timecourse and combine the expression data with promoter motif identification and orthology assignment into causative network models. This bioinformatic analysis will enable us to narrow down to gene responses that are shared between different species, different treatments and different cell types, and thus implicate specific genes in co-option. We will then test these genes for predicted effects on lateral root development and nodulation in reverse genetic studies. The best candidates will be characterised for effects on nodulation in Medicago at a completely novel level by profiling single cell types with FACS in inducible gene expression lines. Together this work will shed new light on the evolution of nodulation in legumes.

Planned Impact

Plants must acquire the elemental nutrient nitrogen from their surrounding environment and its availability is often a major limitation to plant growth. To overcome this limitation farmers apply nitrogen at a high concentration to their crop plants through the application of fertilisers. The production of nitrate fertiliser is a highly energy demanding process and currently accounts for approximately 2% of the worlds energy usage. The primary energy source for fertiliser production is fossil fuels and as such fertilisers not only account for a significant cost in food production, but also almost 50% of carbon dioxide emissions from agriculture. As energy prices rise the cost of food production increases, mostly due to the link between energy usage and fertiliser production. Reducing agricultural reliance on inorganic fertilisers will dramatically enhance sustainable and affordable food production. This is crucial if we are going to meet the increasing demands for food from an expanding global population. The major natural contributor to biologically available nitrogen in terrestrial systems are nitrogen fixing bacteria in symbiosis with plants. This symbiosis is restricted to a subset of plant species, including legumes such as peas and beans, but absent in many of our major crop plants such as wheat, rice and maize. There is no 'quick fix' to the global nitrogen challenge and biological nitrogen fixation likely holds the key to solving this problem. Our work will use state-of-the-art experimental technologies and analytical methods to uncover how the symbiosis is controlled in individual cells of the legume root and uncover information about how nodulation evolved by comparison to a non-legume root. We need this detailed understanding of the mechanisms that control this symbiosis if we are to use this process to tackle the nitrogen problem.

Publications

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Carter AD (2013) The use of fluorescence-activated cell sorting in studying plant development and environmental responses. in The International journal of developmental biology

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Grønlund JT (2012) Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting. in Journal of visualized experiments : JoVE

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Rosas U (2013) Integration of responses within and across Arabidopsis natural accessions uncovers loci controlling root systems architecture. in Proceedings of the National Academy of Sciences of the United States of America

 
Description (1) We have discovered that plant gene families are much more specialised than first thought. Typically we say that members of the same family of genes are redundant and can compensate for each other. However, we found, using high-resolution profiling of gene expression over time in specific cell types, that different members of the same gene family have vastly divergent expression patterns. This is even more true when we compare gene expression for genes that are shared (orthologs) between species.

(2) We discovered in the nitrogen-fixing legume Medicago a new control over the way that plants balance production of nitrogen-fixing nodules, with lateral branching roots. This seems to be controlled by some of the genes that regulate nodule levels (auto regulation of nodulation).


(3) Co-ordination of cell type responses is a key aspect of multicellularity. In many organisms it is known that interactions between environmental conditions, controlled by regulatory transcription factors and mediated by hormone patterning control organ development. However, we know much less about the cross-talk between cell types that underlies control of whole organ (e.g. plant root) phenotypes. To address this we need genetic tools that perturb cross-talk, and a way to gain high resolution data on how cell type communication controls whole organism responses. We have done both of these by analyzing the function of a SCARECROW-LIKE transcription factor at the cell type level. The results have broad significance since they identify how plant physiology is tuned to the environment and we identify a potentially conserved nutrient-hormone-development mechanism between non-legumes and nitrogen-fixing legumes.
Exploitation Route This can help drive synthetic biology approaches to develop nitrogen fixation in new species, and we are developing some of these ourselves. In 2017 our work received very good coverage.
Sectors Agriculture, Food and Drink,Environment

URL https://plantae.org/space-time-continuum-of-gene-expression-in-lateral-root-development/
 
Description BBSRC responsive mode
Amount £196,000 (GBP)
Funding ID BB/J001872/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2011 
End 06/2014
 
Description Capturing microbial co-symbiosis to sustain plant productivity
Amount £429,620 (GBP)
Funding ID BB/P002145/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2016 
End 11/2019
 
Description EU FP7
Amount € 349,000 (EUR)
Funding ID 289365 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2012 
End 12/2015
 
Title Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting 
Description This is a method to isolate single cell types of leaves. The method was published in 2012 in the Journal of Visualized Experiments 68: e4214. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact From this the use of cell sorting in Warwick has increased, and now we are one of the leaders in this technology use for plant cells, as evidenced by the participation in our cell sorting workshop: New Phytologist Workshop: Developing interdisciplinary omics tools for organelle and cell type-specific analysis of plants The aim of this tightly focused meeting is to objectively assess the feasibility of using proteomics in conjunction with Fluorescence Activated Cell Sorting (FACS), a tool to isolate single cell types, organelles and vesicles. We hope this meeting will help establish benchmarks and guidelines for FACS preparation for proteomics. Participants will present their current work, debate the value and application they see in cell-type specific proteomics, and discuss how to overcome challenges presented by the approach. The group will jointly prepare and analyse samples during the meeting using protocols and materials shared by delegates. There are 10 places available on this highly focused workshop that will run from 12:00 on 13th to 14:00 14th November at the University of Warwick. Delegates will have to pay for their own travel to the University of Warwick, but accommodation for 13th November, all meals and sample analysis will be covered by support from The New Phytologist Trust and ThermoScientific. Limited funds are available to support attendance of early career researchers. The organising committee is Charis Cook, Lorenzo Frigerio, Miriam Gifford, Alex Jones, Vardis Ntoukakis, Patrick Schafer (all University of Warwick) and Imogen Sparkes (University of Exeter). 
URL http://www.jove.com/video/4214/cell-specific-analysis-arabidopsis-leaves-using-fluorescence
 
Title Correcting for link loss in causal network inference caused by regulator interference 
Description There are a number of algorithms to infer causal regulatory networks from time series (gene expression) data. Here we analyse the phenomena of regulator interference, where regulators with similar dynamics mutually suppress both the probability of regulating a target and the associated link strength; for instance, interference between two identical strong regulators reduces link probabilities by ~50%. We constructed a robust method to define an interference-corrected causal network based on an analysis of the conditional link probabilities that recovers links lost through interference and validated our method against 'gold' standard data. The method is implemented in R and is publicly available as the NIACS package at http://www2.warwick.ac.uk/fac/sci/systemsbiology/research/software and was published in Bioinformatics in 2014 (PubMed 24947751) 
Type Of Material Data analysis technique 
Year Produced 2014 
Provided To Others? Yes  
Impact This method will enable other researchers to better analyse their own data. 
URL http://www2.warwick.ac.uk/fac/sci/systemsbiology/research/software
 
Description Collaboration to understand rhizoplane development 
Organisation James Hutton Institute
Country United Kingdom 
Sector Public 
PI Contribution This is a Warwick Collaborative Postgraduate Research Studentship (WCPRS) funded for a PhD student that started in Oct 2017 and is joint between Warwick and JHI. A student will be trained in cutting-edge methods by Dr. Gifford, Dr. Lionel Dupuy (JHI) and and Dr. Nicola Holden (JHI). Gifford training is in cell type analysis methods in roots.
Collaborator Contribution Following a recent Society for Experimental Biology scientific conference session organised by Dr. Gifford, and including a talk by Dr. Dupuy, a novel opportunity for collaboration on the mechanism of bacterial colonisation of plant surfaces arose. It stems from Dupuy's discovery that bacteria attach to very specific cells in a growing plant root cell. In order to discover the basis for this specificity, Gifford lab expertise in separating and analysing individual root cell types is essential. The ability of the Dupuy lab to both live-image and develop a mathematical model of bacterial surface colonisation is unique and distinct compared to the work of other microbial research groups at Warwick. This area of work is highly relevant to understanding both plant disease progression but also to development of biofilms - thus is relevant to both agriculture and industrial biotechnology.
Impact New student training in multi-disciplinary areas (plant science, microbiology, mathematical modelling). The work of the student will go towards new funding applications in future.
Start Year 2017
 
Description Extending Synthetic Biology collaborations between WISB and BSSB 
Organisation University of Sao Paulo
Department Biomass Systems and Synthetic Biology Center
Country Brazil 
Sector Academic/University 
PI Contribution This project has a range of activities under the overall aim of building a productive and mutually beneficial partnership with BSSB at USP. This is including visits of academics and exchanges of PDRAs, PhD students, and undergraduate students. The outcomes from such arrangements will include new grant funding, novel research opportunities, and expanded training opportunities. Specific activities are planned to strengthen the collaboration with USP in the area of Plant Synthetic Biology, and leverage additional research funding. My contribution has been on extending our work in cell type analysis collaborations since this is a particular expertise with work developed during this grant. Amongst other aspects a key contribution I have made was leading Warwick academics to carry out a grant-writing workshop in Sao Paulo (Nov 2016).
Collaborator Contribution There has been a very even spread of interactions between partners and collaborators in the UK, which we are now following up on in a visit by the Sao Paulo academics to Warwick (July 2017).
Impact - Participation of 12 Brazilian PhD students and postdocs at a Plant Synthetic Biology workshop in Warwick in March 2016 - First Grant writing workshop - USP in Nov 2016 Other activities are planned for 2017 including two research grant applications.
Start Year 2015
 
Description British Biology Olympiad 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I designed and ran the practical Botany training in the British Biology Olympiad. 16 pupils from across the UK came and became more engaged with botany, a subject they are rarely taught at school.
Year(s) Of Engagement Activity 2016
URL http://www.ukbiologycompetitions.org/british-biology-olympiad/2016/
 
Description Café Scientifique on 'Plants at Extremes' 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Invited Guest at the King's High School (Warwick) Café Scientifique on 'Plants at Extremes'. Leading discussion with KS4-5 pupils (GCSE and A-level) and their teachers.

Outcomes: 3 A level students requested work experience and they did this in my group and Warwick University School of Life Sciences in January 2017.
Year(s) Of Engagement Activity 2016
 
Description Christmas Royal Institution-style public lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a 'Warwick Christmas lecture' - held at the University of Warwick Arts Centre as part of a series along the lines of the Royal Institution Christmas lectures. It was entitled 'Talk Amongst Your Cells', with games, activities and multimedia to talk to people about cells, their functions and how we can study them in the research lab. The audience was a lay audience of 570 people aged 8 and up (held in Dec 2014). Dr Gifford ran the show/lecture, with 15 PG and UG students from the department involved. The lecture was also videod.

After the session we talked to the public, finding them enthused about the research area and keen to learn more. We have since seen many of those people coming back for additional lectures/sessions.
Year(s) Of Engagement Activity 2014
URL http://www.warwickartscentre.co.uk/whats-on/2014/the-warwick-christmas-lectures/
 
Description Faces of plant cell biology 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Participation in publicising careers in plant science.

This has increased the understanding of plant research science opportunities.
Year(s) Of Engagement Activity 2012
URL http://www.plantcellbiology.com/2012/07/faces-of-plant-cell-biology-dr-miriam-gifford/
 
Description Gatsby Plant Sciences Summer School - tutor and careers panel member 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact I spoke at a careers event to showcase different careers within plant sciences. I was representing the 'academic' route on the panel. After a panel overview we split off to take student questions directly - these were vast with a lot of questions about career decisions and achieving a family-career balance.

After the talk students were increasingly enthused about staying in plant science and we now wait to see if this filters up to PhD level and beyond. The Gatsby foundation will track such numbers.
Year(s) Of Engagement Activity 2013
URL http://www.gatsbyplants.leeds.ac.uk/tree_ss_previous.html
 
Description International Biology Olympiad, Botany practical 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Over 260 young biologists from 68 countries came to the University of Warwick to take part in the 2017 International Biology Olympiad. This event takes place every year in different locations across the globe, sees pre-university students compete in a series of practical experiments and theoretical exams in what is the biggest biology competition in the world.
The competition, which spans a week in duration, took place from 23rd July to the 30th July, and - after a successful bid back in 2012 - was hosted by the UK at the University of Warwick's School of Life Sciences. Over a 6 month period Gifford developed, tested and finalised the Botany Practical Exam, a 2 hour long exam spanning plant evolution, development, physiology and anatomy. She then ran the practical at the IBO, leading a team of 20 PG/UG/staff helpers to prepare material, run and finally mark the exam. Impacts have included increased engagement with plant sciences by school and UG students as well as new material devised that will be used internationally. Another URL: https://exchanges.warwick.ac.uk/article/view/221
Year(s) Of Engagement Activity 2017
URL https://blog.rsb.org.uk/tag/ibo/
 
Description Sense about Science plant science panel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact As a member of the 'Sense about Science' plant science online Q&A panel I field questions relating to plant science from anyone, particularly the public. This helps to inform the public and stimulate debate.

After answering questions the questioners can come back for more information, or ask more questions - it is a great way to inform and we can track the impact of this.
Year(s) Of Engagement Activity 2013,2014
URL http://www.senseaboutscience.org/pages/plant-science-expert-panel.html#form
 
Description The Elizabeth Creak Charitable Trust Warwick Food Security Lecture 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Warwick University School of Life Sciences hosted a free public science evening on Food Security. This included talks (I gave one of these), demonstrations and talks to explain how Warwick research keeps our food secure via pest management, pollinators, and whole plant imaging.
Year(s) Of Engagement Activity 2017
URL http://www2.warwick.ac.uk/study/outreach/news/keeping_plant_disease
 
Description Warwick Crop Centre Open Day presentation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talk helped to explain to lay public what our research means for them, and what impacts could be realised. There was also good discussion of crop breeding and GM strategies.

Active engagement with local public could have contributed to the recent charitable trust award of £1,000,000 to Warwick from the Elizabeth Creak Trust (local farmer) for food security research chair and studentships.
Year(s) Of Engagement Activity 2012