Controlling pluripotency and differentiation in plant cells: the KNOX-TCP regulatory module

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


Development in multicellular organisms involves the differentiation of specific cell-, tissue- and organ-types from progenitor stem cell populations. In plants such as Arabidopsis thaliana, a commonly used model organism used in the study of plant developmental biology, pluripotent stem cells are located in a pool of undifferentiated cells termed the shoot apical meristem (SAM), which gives rise to lateral organs such as leaves. Leaf cells subsequently undergo differentiation associated with their specialised functions, such as photosynthesis and gas exchange.
Numerous genes have been identified that are involved in controlling the formation and sustained function of the SAM. Some genes act to confer a state of pluripotency - that is to say, an undifferentiated state with the ability to form any type of cell - while others promote differentiation into specialised cell types. Most of the genes currently known to regulate SAM function encode a type of protein called a transcription factor (TF). TFs are proteins that regulate the expression of other so-called 'target' genes by activating or repressing their expression - specifically by controlling the transcription of a gene from DNA into RNA, which is subsequently translated into a functional protein. Of the many different types of TF that have been shown to be involved in SAM regulation, the KNOTTED1-like homeobox (KNOX) gene SHOOT MERISTEMLESS (STM) encodes a TF that is absolutely critical for the specification and maintenance of pluripotent cell fate in the SAM.
My recent work has shown that STM represses the expression of a different type of TF belonging to the TCP family (TCP4), which is involved in promoting differentiation in leaves. Interestingly, TCP TFs have previously been shown to repress the expression of STM. Hence, I have revealed a mutually antagonistic relationship between these two important classes of TF: STM promotes pluripotency and represses TCP expression in the SAM, thereby preventing meristem cell differentiation, while TCP represses STM expression and promotes differentiation in developing leaves. Loss of STM function or elevated levels of TCP causes the cells of the SAM to differentiate and cease activity. Conversely, loss of TCP mimics the effect of elevated STM expression, such as inhibited leaf differentiation. Given these similarities, one might expect that STM and TCPs regulate, in an antagonistic manner, a common set of target genes that lead to cells adopting pluripotent or differentiated fates.
The control of pluripotency and differentiation is the central theme of this research project. Specifically, this project seeks to understand how cell fate decisions are made in the SAM by studying the interplay between STM and TCP, identifying TCP-regulated target genes and comparing these with those previously identified for STM. Using fluorescent 'reporters' for these two genes, changes in STM and TCP expression in the living SAM will be monitored when levels of TCP or STM levels are artificially increased or decreased respectively, revealing in which cells their expression changes and the how quickly this occurs. Having previously identified the STM target genes that promote pluripotency, characterisation of TCP target genes will enable the identification of target genes that promote differentiation. Comparative analyses will reveal which genes are competitively regulated by STM and TCP. These would likely represent key players for the adoption of pluripotent or differentiated cell fates and would represent a considerable advance in our understanding of the control of pluripotency and differentiation in plant development.

Technical Summary

In Arabidopsis, pluripotent stem cells in the shoot apical meristem (SAM) generate lateral organs such as leaves in which cells differentiate to acquire specialised functions. Transcription factors (TFs) such as the KNOX protein SHOOT MERISTEMLESS (STM) promote pluripotency in the SAM, while TFs such as class-2 TCPs induce leaf cell differentiation. My recent work showed that STM represses the expression of class-2 TCPs including TCP4. TCPs are known to repress the expression of KNOX genes including STM, hence this revealed a mutually antagonistic regulatory module where STM promotes pluripotency and represses TCP expression and hence differentiation in the SAM, while TCP represses STM expression and promotes differentiation in leaves. Supporting this, loss of STM function or ectopic TCP activity causes SAM differentiation, while loss of TCP partially mimics elevated STM expression phenotypes, suggesting that STM and TCPs antagonistically regulate common target genes that control cell fate. This project seeks to understand how cell fate is controlled in the SAM by investigating the STM-TCP4 regulatory module using live imaging and transcriptomics - specifically by studying the interplay between STM and TCP4, identifying TCP4-regulated target genes and comparing these with STM targets previously identified in my lab. To understand the spatial and temporal dynamics of the STM-TCP regulatory module, changes in STM and TCP4 expression in the SAM will be analysed using fluorescent reporters when levels of TCP4 or STM are altered by inducible expression, providing parametric data for modelling. TCP4 targets will be identified using inducible upregulation of TCP4 followed by RNA seq to identify TCP4-responsive genes combined with ChIP to identify TCP4 DNA binding sites. Comparison with STM targets will reveal genes that are competitively regulated by both STM and TCP4, comprising key factors in pluripotent or differentiated cell fate acquisition.

Planned Impact

All terrestrial life on Earth ultimately depends on plant meristems - small populations of undifferentiated cells located at the growing tips of plants. Meristems generate plant organs such as leaves and flowers in addition to housing a pluripotent stem cell population that is maintained throughout the plant life cycle to permit indefinite growth. Meristems produce most of the autotrophic biomass in terrestrial ecosystems and are therefore of broad importance and interest not only to plant scientists and developmental biologists but to other stakeholders such as ecologists, agronomists, farmers and wildlife management organisations. This project will address a fundamental question in biology: how do cells in the meristem decide to whether differentiate into a new organ such as a leaf, or to remain as pluripotent stem cells? Understanding the mechanism behind this key cell fate decision is critical for a comprehensive understanding of meristem regulation, leading to multiple potential applications in the scientific, agronomic, educational and economic spheres.
This proposal will identify novel genes involved in the specification of pluripotency and differentiation in Arabidopsis, achieved by studying the target genes of two key developmental transcription factors. The most immediate beneficiaries are other academics working on the genetic regulation of plant development, though other stakeholders such as plant breeders will benefit considerably from this work. Notably, deliverables from this project could be used to engineer plant form and function, for example by altering the size of the meristem or by manipulating leaf size and shape to maximise crop yields. The potential applications in food security are especially pertinent given that most edible fruit and vegetable biomass is in the form of differentiated leaf or floral tissues. Supporting this the applicant is forming a collaboration with IRRI (Philippines) to investigate how these novel genes might be applied in rice engineering.
This work will have impact not only in science but in the agriculture and horticulture industries, education and training opportunities, job creation and economic prosperity. It will generate data that will lead to scientific advancement by expanding the current biological knowledge base, especially for plant science, feeding directly into the remit of sustainable agriculture and food security through the development of new products and processes (in this case, novel genes for GM manipulation). Additionally there will be a less direct but nonetheless important impact through enhancing education at all levels, particularly as the fundamental nature of this work means it could become included in text-books. Educational impact is especially important as it leads to training opportunities that develop skills and enhance employment potential. Importantly, the PDRA employed on this project will have extensive opportunities for training and skillset development.
The applicant will undertake the following engagement and communication activities to enhance project impact in addition to producing two peer-reviewed publications and conference proceedings: Firstly, the applicant will create a project-specific website that will describe the rationale, methodology and major findings of this project. This will be accessible by the public and other interested parties. Secondly, the applicant will create 3D printed models of the meristem using colour-coding to show the cells expressing the STM and TCP expression patterns, and how these change dynamically during organ formation. These will be used at events such as the Fascination of Plants Day to showcase UK plant science to the public. Finally, the project will generate high-impact publications, enhancing UK global prestige and scientific standing and indirectly contributing to the UK position as a leading country for research and development, thereby helping to attract overseas investment.


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Description DNA constructs created for generating transgenic lines. Transgenic lines created for TCP4 overexpression and TCP4 reporter gene constructs. TCP4 induction causes meristem termination via repression of STM. ChIP-seq performed to identify transcription factor binding sites in Arabidopsis genome - publication in preparation. Chromatin particle spectrum analysis performed and key differences observed in STM regulated genes when STM is overexpressed - publication in preparation. Role of miR319a in STM repression of TCP4 investigated. STM represses TCP4 directly via binding to STM promoter and indirectly by promoting miR319 expression. Comparative RNA-seq experiment conducted to identify TCP4 and STM target genes. ChiP-seq experiment for TCP4 conducted. Imaging of reporter lines not completed.
Exploitation Route Use to control plant meristem function and therefore can be used for crop engineering eg. increasing biomass yields
Sectors Agriculture, Food and Drink,Creative Economy,Education,Manufacturing, including Industrial Biotechology

Description Superscience saturday October 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact School pupils and general public attended the event, which focused on plant science
Year(s) Of Engagement Activity 2019