A novel pathway of cell cycle activation in root formative divisions

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences


We all learn at school that reproduction is a fundamental property of life, and the same is true of the cells from which organisms are built. All cells arise from a parent cell by division. Usually a cell divides to make two cells of the same type, increasing the population size. We refer to this as a "proliferative division", and is easy to think about in a tumour where there are many identical cells. However, when a complex organism is developing, for example as an embryo grows from a single initial fertilised egg cell, new cell types have to be produced. These arise from a special kind of division, known as a "formative division". In this case one daughter cell (or sometimes both daughter cells), are different from their parent cell and have a new identity. This process of formative division is also essential in the maintenance and function of stem cells- these are cells in the body that can undergo formative divisions to generate another stem cell and a new cell of different identity.

We know a lot about the molecules that regulate the processes of proliferative cell divisions, in part because of their importance in cancer. The molecular and cellular aspects of the process of cell division are known as the "cell cycle". However we know very little about what different mechanisms operate in the cell cycle of formative divisions or indeed even if there are different mechanisms. We have been studying a particular type of formative division in the root of a plant called Arabidopsis. We study plants because the cells do not move around, and the identity of a cell is easy to establish because it is determined by its position in the root. We use Arabidopsis because it grows rapidly, there is a great wealth of earlier studies to draw on, and there are a lot of resources that make the research fast and relatively cheaper. The roots are also thin and transparent so we can study living roots using a confocal microscope that allows us to visualise the action of proteins and genes as cells divide. The root consist of concentric layers of cells, each layer with a different identity, wrapped around a central core that conducts water and nutrients.

The particular division we have been studying involves the formation of two of these layers, the cortex and endodermis from a single layer of root ground tissue. The endodermis is a crucial tissue because it forms an impermeable layer controlling the movement of water and ions into the central conducting tissue. Without the endodermis the root cannot grow and function properly. This is exactly what happens in mutants of a gene called SHORT-ROOT. In this mutant, the formative divisions do not take place. In collaboration with a leading US group, we showed in a paper published last year in the journal "Nature" that SHORT-ROOT directly controls expression of a cell cycle regulating gene called cyclin D6, which is only switched on in cells carrying out the formative division. If cyclin D6 is missing, the formative division is not properly controlled. Cyclins work together with a partner protein called a cyclin-dependent kinase. This has now been identified, and mutants in this gene also have a defect in the formative division, confirming it is also involved. These two proteins do not normally work together, so we believe that we have identified a new mechanism by which the cell cycle is switched on in formative divisions, which also involves a third candidate we have identified. In this project, we will analyse this new mechanism in detail. We think it involves three feedback loops that all activate each other, so to understand them, we will use mathematical modelling to predict the effect of making changes to the system and test these predictions. We will carry out the work in collaboration with world-leading groups in the US, Holland and the UK, bringing exceptional expertise to bear on this problem.

Technical Summary

Cell divisions fall into two categories. Proliferative divisions increase the number of cells of a given type, but formative divisions result in the production of two cells with new identities. Whilst we know a lot about control of the cell cycle in general, we know very little about the mechanisms that underpin these different types of division, or indeed whether there are differences in the cell cycle mechanisms that operate. These processes are easy to study in plants due to the relationship between spatial position and cell identity.

Here we present evidence of new cell cycle regulatory mechanisms in formative divisions of the Arabidopsis root ground tissue and propose to analyse and model this novel mechanism for cell cycle control. These formative divisions that create the cortex and endodermis depend on regulation by the SHORTROOT (SHR)/ SCARECROW (SCR) transcription factors. We propose here that the novel cell cycle activation involves three interlocking positive feedback loops converging on phosphoryation of the RETINOBLASTOMA-protein homologue RBR via novel cyclin-CDK complexes. We have previously shown will Philip Benfey's group that a unique D-type cyclin CYCD6;1 acts in the formative division downstream of SHR (Nature 466:128-32; 2010) and controls the spatio-temporal pattern of division. Further work by the Scheres group has shown that RBR itself also binds and regulates SCR, limiting its activity and forming the first positive feedback loop. The applicant's lab has also identified a phenotype associated with the CDK bound by CYCD6;1 and a further candidate cyclin also involved. The project proposes to analyse these regulatory mechanisms combining in vitro and in vivo approaches with mathematical modelling of the system allowing predictions to be made and tested. The work will be in collaboration with the Scheres, Benfey and Bennett groups, three of the world-leaders in studying root development.

Planned Impact

This project will define a new pathway controlling cell division that operates specifically when new cell types are being formed. This new knowledge will have a broad impact across the biological sciences, improving our understanding of how biological processes operate in general and how pattern is generated during the development of organisms. It will also show for the first time that different types of regulatory mechanism operate in different types of cell division.
Immediate beneficiaries will be academic biologists worldwide who seek to understand how cell division is controlled and how complex organisms develop, broad questions of fundamental and general interest. The significance of the work is therefore likely to generate further interest in the media and wider public. Precursor work led to publicity and media interviews for the applicant illustrating its wider impact. The general public will therefore benefit through an enrichment of their understanding of how biological processes function. Education may well benefit since this mechanism is likely to become included in undergraduate textbooks and courses as an illustration of formative divisions.

In the longer term, business and industry will benefit from an increased understanding of the processes that are important in generating biological form. Such knowledge will be required in order to realise long term goals of engineering crops for improved yield, biomass and resistance to stress (for example through improvements in root structure or architecture). This type of knowledge will help to underpin the knowledge based bioeconomy (KBBE), goals for both UK government and the European Commission. Furthermore, this project places the UK at the centre of a collaboration involving top European and US labs, enhancing UK global prestige and scientific standing and indirectly contributing to the UK position as a leading country for research and development (R&D) and hence helping to attract associated investment.

The project will involve the researchers in state-of-the-art techniques, leading to highly skilled and trained individuals. Since the project integrates both biology and systems modeling approaches, the biologists will benefit from practical knowledge of how mathematics can be used in biological research and the mathematician in understanding practical applications of modeling. These types of integrated skill sets acquired by the researchers are essential for a highly trained and flexible workforce that will be required to deliver the KBBE and contributing to future economic development and associated societal benefits. Such individuals also enhance the skill and knowledge base available to academic and business research and further contribute to the UK's attractiveness for outside investment in R&D.


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Description The coordination of plant cell division and expansion controls plant morphogenesis, development, and growth. Cyclin-dependent kinases (CDKs) are not only key regulators of cell division but also play an important role in cell differentiation. In plants, CDK activity is modulated by the binding of INHIBITOR OF CDK/KIP-RELATED PROTEIN (ICK/KRP). Previously, ICK2/KRP2 has been shown to mediate auxin responses in lateral root initiation. Here are analysed the roles of all ICK/KRP genes in root growth. Analysis of ick/krp null-mutants revealed that only ick3/krp5 was affected in primary root growth. ICK3/KRP5 is strongly expressed in the root apical meristem (RAM), with lower expression in the expansion zone. ick3/krp5 roots grow more slowly than wildtype controls, and this results not from reduction of division in the proliferative region of the RAM but rather reduced expansion as cells exit the meristem. This leads to shorter final cell lengths in different tissues of the ick3/krp5 mutant root, particularly the epidermal non-hair cells, and this reduction in cell size correlates with reduced endoreduplication. Loss of ICK3/KRP5 also leads to delayed germination and in the mature embryo ICK3/KRP5 is specifically expressed in the transition zone between root and hypocotyl. Cells in the transition zone were smaller in the ick3/krp5 mutant, despite the absence of endoreduplication in the embryo suggesting a direct effect of ICK3/KRP5 on cell growth. We concluded that ICK3/KRP5 is a positive regulator of both cell growth and endoreduplication.

Seeding establishment following seed germination requires activation of the root meristem for primary root growth. We investigated the hormonal and genetic regulation of root meristem activation during Arabidopsis seed germination. In optimal conditions, radicle cell divisions occur only after the completion of germination and require de novo GA synthesis. When the completion of germination is blocked by ABA, radicle elongation and cell divisions occurred in these non-germinating seeds. Conversely under GA-limiting conditions, ABA-insensitive mutants complete germination in the absence of radicle meristem activation and growth. Radicle meristem activation and extension can therefore occur independently of completion of the developmental transition of germination. The cell cycle regulator KRP6 partially represses GA-dependent activation of the cell cycle. Germination of krp6 mutant seeds occurs more rapidly, is slightly insensitive to ABA in dose-response assays, but also hypersensitive to the GA synthesis inhibitor PAC. These conflicting phenotypes suggest the cell cycle uncouples GA and ABA responses in germinating Arabidopsis seeds, and that KRP6 acts downstream of GA to inhibit mitotic cell cycle activation during germination.

We have uncovered mechanisms linking root growth and control of the cell cycle in formative divisions in the root. This part of the work was in collaboration with Prof Ben Scheres in Wageningen. These are divisions of stem cells that give rise to a new cell type.In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.
Exploitation Route Primarily of academic interest.
Sectors Agriculture, Food and Drink

Description Size Matters: A systems approach to understanding cell size control in a developing multicellular tissue
Amount £421,568 (GBP)
Funding ID BB/S003584/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 01/2022
Title Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids 
Description BACKGROUND: A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance. RESULTS: Here, we introduce 'Phytotracker', a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers. CONCLUSION: We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research. 
Type Of Material Data handling & control 
Year Produced 2012 
Provided To Others? Yes  
Impact Currently 8329 Accesses and 4 Citations (March 2020). 
URL http://sourceforge.net/projects/Phytotracker