The role of the E2F transcription factors in regulating stem cell functions during Arabidopsis root development

Lead Research Organisation: Royal Holloway, University of London
Department Name: Biological Sciences

Abstract

Plants form organs, grow and develop throughout their lifetime, which can be over a thousand years in the case of some trees. For that purpose they maintain pluripotent somatic stem cells within the meristems, local pools of mitotically active cells. In roots these stem cells surround the mitotically less active organising centre, called the quiescent centre (QC) and together they form a stem cell niche. The quiescent centre is specified by combinatorial action of two gene sets. The PLETHORA genes are transcribed in response to the phytohormone, auxin accumulation and are required to position QC basal in roots while an independent developmental pathway of the genes named after the mutants, SCARECROW (SCR) and SHORT-ROOT (SHR), sets up the radial position for QC. Although we are beginning to understand the patterning in meristems, we do not know the mechanisms that keep cells undifferentiated around the region of QC and what sets up the field of differentiation as cells leave the meristematic zone. Auxin has long been known to be essential for cell division in culture and to be involved in a wide range of developmental processes including the maintenance and initiation of organs that depend on the regulation of the balance between cell division and differentiation. Channels are carefully positioned and regulated to allow auxin to get in and out from cells and to determine the directionality of auxin flow from cell to cell that leads to the formation of highly dynamic fields of auxin gradients. The idea is, that cells are able to distinguish these different auxin concentrations and respond to them differently, for instance by growth through elongation or by cell division, but the exact mechanism is not known. We know however, that different auxin concentrations are sensed by a protein known as TIR1, which is able to operate sophisticated switches on large sets of genes that rely on lifting repressor molecules by auxin-regulated protein degradation. Recently we have discovered two transcriptional regulators in cell division with antagonistic roles, one called E2Fb promotes cell proliferation and co-ordinates it with cell growth; while the other, called E2Fc, blocks cell division, and promote cells to attain specific functions, termed cell differentiation. We were surprised to find that the abundance of these proteins are oppositely regulated by auxin, E2Fb being stabilised while E2Fc is destabilised. We formulated a working hypothesis that auxin concentrations are converted into opposing concentration gradients of these positive and negative cell cycle regulators and constitute the switch between decisions to divide or elongate and differentiate. E2Fs are kept under control by the pocket protein called retinoblastoma (RB), because it was discovered in animals to cause uncontrolled tumour growth in the eye. It was recently discovered that in Arabidopsis an RB related protein is essential to maintain the stem cell niche in roots in response to the developmental regulator SCARECROW. Thus, E2Fs provide a converging point for developmental regulators and auxin to determine stem cell functions. We propose to test this model genetically by observing the phenotypes of mutants in E2F genes in combination with the RB related gene and mutants in regulators of auxin production or transport. We also plan to visualise the E2F protein distribution in relation to auxin gradients. Because E2Fs operate by controlling large number of genes, it is vital that we identify the genes E2Fs bind to and determine how these genes are regulated. Our work should uncover how auxin regulates cell division, and thus plant growth.

Technical Summary

The Arabidopsis root provide a model where proliferation and cell differentiation follows a strict pattern through well established asymmetric divisions of stem cells producing ordered cell files that is easily recognisable in a phase contrast microscope or by available developmental markers. We will characterise mutants for the E2F genes for root growth, stem cell organisation, asymmetric cell divisions, cell file organisation and differentiation of cells as they leave the meristem zone. We found that auxin alters the balance between the two opposing E2Fs, E2Fb and E2Fc. If indeed E2Fs are important to decode auxin concentrations to cell division and differentiation than mutants in these genes should have predictable alterations in auxin responses. We will study this in mutants of E2F genes by external or internal modifications of auxin distribution. Regulators for patterning root meristem organisation have been uncovered, and we will investigate how this is interfaced with the regulation of cell division through the E2F-RB pathway. Functional diversification among E2Fs and a key to the mechanism of how they act rely on their ability to bind to distinct promoters. We propose the use of chromatin immunopreciptitation (ChIP) combined with microarray hybridisation (ChIP-on chip) to find target genes for the E2Fs, and use datasets of gene expression profiles obtained from plants with altered E2F pathways and available datasets of expression profiles during root development to uncover the complex regulatory network how cell division and differentiation is coordinated during root development.

Publications

10 25 50
 
Title Artist in residence Kerry Lemon 
Description Drawing of plants with increased understanding how development shapes growth 
Type Of Art Artwork 
Year Produced 2014 
Impact Stimulating discussions with students. Media release. Planned exhibition. 
URL http://www.kerrylemon.co.uk/
 
Description The RETINOBLASTOMA RELATED (RBR) genes is an evolutionary conserved master regulator for controlling cell proliferation, organ growth and development both in animals and plants. It does so through interactions with transcription factors and provide a platform to build repressor complexes. In this grant we characterised how RBR regulates root growth through the E2F transcription factors. We have discoverred that RBR regulates growth by maintaing the proliferative capacity of meristems through E2FA and the exit from proliferation through E2FB. These two functions are critical to tune growth.
Exploitation Route RBR is a central hub to modulate plant growth. Engeneering connections to RBR has a great potential to increase yield in crops and match growth to environmental conditions. We have demonstarted that tuned expression of specific RBR-associated TFs can have growth benefits. Mapping signalling connections to RBR network components is another avenue with great promise.
Sectors Agriculture, Food and Drink

URL http://pure.rhul.ac.uk/portal/en/persons/laszlo-bogre(1bd7dd10-4a82-479b-ab69-ec13db21076d).html
 
Description The RBR-E2F transcriptional repressor complex is central to regulate plant growth and serves as an integrator of incomming signals. We developped methods to tune the pathway that led to increased growth potential of plants. This has been tested by Bayern Crop Science and considerred to incorporate this technology to their biotechnology method to increase yield in crops.
First Year Of Impact 2012
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Advisor for the Hungarian Academy of Sciences
Geographic Reach Asia 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
 
Description Advisory board Agricultural Biotechnology Centre
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
URL http://www.abc.hu/en/
 
Description Structuring Framework 7 calls
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
 
Description Inference of RBR network and dynamic RBR complexes during leaf development.
Amount € 319,888 (EUR)
Funding ID 330789 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 03/2013 
End 03/2015
 
Title Purification of protein complexes 
Description Use genomic tagged GFP lines for rapid purification of protein complexes and identification of protein complex components 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact Established collaborations and accepted manuscript in EMBO J in 2017 
 
Title mutant lines, antibodies, GFP-tagged lines 
Description Tools for lipid signalling kinases, MAPKs, E2F-RBR such as antibodies, mutant lines, GFP-tagged lines 
Type Of Material Cell line 
Provided To Others? Yes  
Impact shared research material facilitate research in other groups 
 
Description Dr Zoltan Magyar 
Organisation Hungarian Academy of Sciences (MTA)
Department Biological Research Centre (BRC)
Country Hungary 
Sector Academic/University 
PI Contribution Working on RBR-E2F, connecting translational regulation and cell cycle
Collaborator Contribution Providing antibodies and mutants in the RBR-E2F pathway
Impact research papers, collaboration with Bayern Crop Science
 
Description Patrick Giavalisco 
Organisation Max Planck Society
Department Max Planck Institute of Molecular Plant Physiology
Country Germany 
Sector Charity/Non Profit 
PI Contribution Connecting TOR-S6K to RBR
Collaborator Contribution TOR silenced lines
Impact Joined project proposal
Start Year 2010
 
Description Pavla Binarova 
Organisation Academy of Sciences of the Czech Republic
Country Czech Republic 
Sector Learned Society 
PI Contribution Analysing RBR phosphorylation and interaction with microtubules.
Collaborator Contribution Microtubules, cell biology
Impact research papers, joined projects
Start Year 2010
 
Description Scheres 
Organisation Utrecht University
Department Cancer, Stem Cells and Developmental Biology
Country Netherlands 
Sector Academic/University 
PI Contribution Functional characterisation of E2F transcription factors during root growth and development
Collaborator Contribution Methodologies and tools to study root development, collaboration on RBR phospho-mutants
Impact Modulating RBR-E2F levels to increase plant growth
 
Description Artist in Residence Kerry Lemon 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Stimulating discussions to bridge the gap between science and artistic thinking

Artistic drawing with understanding of plant development
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL http://www.kerrylemon.co.uk/
 
Description Open day 
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 Schools
Results and Impact students were engaged with experiments on plants and appreciate knowing more about plants for food security, climate change

Regular contact with teachers, regular school visits, talks
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
URL https://www.royalholloway.ac.uk/studyhere/opendays/home.aspx
 
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact increase awareness in plant research

Increased interest, motivation of school kids
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,