A Complexity Science Approach to Plant Tissue Regeneration

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

Tissue regeneration in multi-cellular organisms is a topic of great interest both from a basic scientific point of view and from an applied one. In the attempt to shed new light on the fundamental nature of the mechanisms underlying tissue regeneration, this project intends to test experimentally a quite technical but intriguing hypothesis on the way cellular divisions are organized during regeneration.

The experimental case studied is that of a plant's root that completely regenerates its tip when this is excised by mechanical means, and a multi-disciplinary approach combining live imaging and advanced statistical analysis is followed.
From the experimental point of view, the main challenge is to develop a type of microscope that allows very frequent observations of the internal organisation of a regenerating root, without of course killing the root. The idea is to be able to collect data about when and where cell divisions occur during the tissue re-growth and reorganization, and this can be achieved through a non-invasive technique that detects fluorescent proteins in the sample. The trick is to genetically engineer a fluorescent protein that is expressed in a cell only at the moment of its division, and this has been done, already.

The initial part of this project is dedicated to the optimisation of a microscope - starting from existing prototypes - specifically redesigned to image for many days a regenerating root, quite often and at cellular resolution. Time-lapse sequences will be then systematically collected with this instrument, to capture in great details the regeneration of the root tip.
The more advanced part of the project is aimed at the statistical analysis of such data, to test the hypothesis that the dynamics of cellular divisions guiding root regeneration shares common features with a wide class of complex phenomena found in physics and biology, as different from each other as sand piles, earthquakes, flying birds or neural networks. This class is sometimes called Self-Organizing Critical (SOC) systems, and is characterized by the ideas that (i) global patterns emerge simply from interactions among all the system's elements and without the need of a central director (self-organization) and that (ii) the system exhibits a spontaneous convergence towards a special (critical) state. At the critical state the system is said to be scale-free, which means that there is no typical length or duration in the underlying dynamics. Moreover, at the critical state some statistical distributions that describe the system's dynamics take a universal shape (power law distributions). It is a bit technical, but it turns out that these statistical distributions can be measured from the data collected with the microscope used in this project, and therefore it becomes possible to test whether a regenerating root behaves like a SOC system or not. We will then be able to test more in details the causal link between cellular interactions and global tissue behavior by developing a mathematical model designed to give a simplified representation of all the key characteristics of the system. This will be based on previous experience gained by applied mathematicians in the field of complexity science, where SOC and similar phenomena are studied in great details.
Finally, it is understood that any major improvement of our understanding on how a root can regenerate its tip after a major injury opens the possibility to enhance growth and resistance of plants of economic interests in harsh conditions. Moreover, any new insight in natural mechanisms leading to tissue repair and regeneration is of major interest for the biomedical and biotech community.

Technical Summary

The goal of this multi-disciplinary project is to study the phenomenon of tissue regeneration from the novel point of view of complex systems, focusing on an original quantitative analysis of the cell division dynamics. The working hypothesis is that the process of regeneration can be described as the emergent property of a complex dynamical system, and that the underlying statistics of cell division events carries crucial information on the fundamental mechanisms driving the process. One intriguing possibility is that such dynamics could exhibit some degree of scale-free behaviour as would be indicated by (approximate) power-law distributions, suggesting a parallel with a very broad category of natural phenomena which operates at or near a state of self-organised criticality. Such a result would have a wide impact, well beyond developmental biology.

The approach proposed is multi-disciplinary in its essence, generating novel experimental data within developmental biology and analyzing it with mathematical tools adopted from current studies in complex systems. The methods will include novel high spatial and temporal-resolution live imaging, statistical analysis of response distributions in time series of cell division events, and mathematical modelling of effective cell-cell interactions leading to intermittent activity.

In synthesis, we propose to look for classic signs of scale-free behaviour in the temporal distribution of cell divisions during root tip regeneration. A scale-free dynamics, one where no characteristic scale length or duration can be identified, is a classic hallmark of criticality observed, at least to some degree, in a variety of phenomena ranging from earthquake events to brain neuronal activity. The main question here addressed is whether this notion can be extended to complex phenomena in developmental biology.

Planned Impact

The proposed project takes fully advantage of a typical systems biology approach. It naturally falls within the Exploiting New Ways of Working theme identified by BBSRC as integral part of its strategic priority Systems Approaches to Biosciences. The project promotes a combination of mathematical modelling imported from complexity science and novel live imaging, bringing together integrative systems biology and quantitative developmental biology. The case study is plant root regeneration, but the actual questions addressed are much more general and refer to the essence of multi-cellularity itself.
Due to the multi-disciplinary nature of the proposed approach, this project is likely to impact more than one scientific community and group of interest.

- Academic communities.
The project intersects topics in developmental biology, regenerative biology and root biology, together with generating mathematical and computational tools. This makes it likely to impact the work of developmental biologists, plant biologists, bio-physicists and applied mathematicians interested in complexity and self-organization, and computer scientists involved in automated image processing.

-Training the next generation.
The PDRA involved in this project will receive a unique training in developing ad hoc imaging tools, in taking a quantitative approach to developmental biology - particularly in complex morphological dynamics - and in developing original mathematical models. The proposed line of work will induce a strong multidisciplinary and out-of-the-box attitude within the lab and the department, without any doubts a critical skill to reach new achievements in world-class bioscience.

- Translations to agronomy and other industrial applications.
Since the exploration proposed in this application is mainly grounded in basic science, any potential practical application must be viewed with a long-term perspective.
Among these, the topic of plant root regeneration is of clear relevance for the agronomy industry, interested in translating mechanisms regulating plant growth and robustness into enhanced crop cultivations. This proposal is based on the model organisms Arabidopsis, but it is well understood that basic knowledge gained in this system often translates into agricultural or horticultural applications involving economically relevant crop species such as rice and corn. More specifically, the project presented here has the potential to significantly advance our understanding of tissue repair in roots, a crucial organ for plants. This would open the possibility to improve root resistance to harsh soil conditions, a topic very much pursued in the agronomy industry.
Other industry-related fields that will be interested in applying the results emerging from this project are the new and rapidly expanding area soft robotics and the more established bio-inspired engineering. At the core of the proposed study is the desire to expose new fundamental mechanisms driving tissue self-repair. This topic is highly relevant for anybody interested in either enhancing existing organisms, or producing completely artificial systems taking inspiration by solutions already successful in nature. The potentials for impact are vast.

- General public
We will seek opportunities to engage the general public by promoting and discussing the notion of multidisciplinary approaches to gain understanding of natural phenomena. Any potential for contributing to the "knowledge economy" will be considered and pursued. In particular, expanding on the idea that cross-pollination between disciplines has the potential to deliver truly innovative solutions to old problems and inspiration for new applications. This project and its results will be presented to the public as a clear example of that strategy.
 
Description Optimising live light sheet microscopy on growing tissue, like plant roots. Developed and optimized automated tracking methods to follow growing root tip in microscope chamber.
3D temporal series of growing or regenerating Arabidopsis root tips expressing CYCB::GFP mitotic reporter. We also developed algorithms to track the growing root tip and to automatically detect and count cell divisions in the time-series. A statistical analysis of the spatial and temporal distribution of cell division events is providing a complete quantitative picture of the proliferation dynamics of the tissue.
Two manuscripts are still in preparation.
Exploitation Route Adopting some of our optimised methods to perform live imaging of plant root meristem for prolonged periods, at high spatial and temporal resolution. Some of the methods could in principle be adapted to other model systems. The data and interpretation of the cell division dynamics during regeneration could result in better understanding of morphological plasticity in plants and shape regulation.
Sectors Agriculture

Food and Drink

Education

 
Description BBSRC IAA
Amount £29,728 (GBP)
Funding ID BB/S506667/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 10/2019
 
Title LSFM 
Description The Light Sheet Fluorescence Microscopy (LSFM) imaging system has been fully developed and optimised, starting from the existing crude prototype. Both hardware (optics and hydroponic perfusion) and software (automatic live imaging and tracking) has been completed and tested. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact This was a crucial step in the project, and allow us to press on with systematic live imaging acquisition. It allows high spatial and temporal resolution live imaging of regenerating Arabidopsis root. 
 
Title Algorithms for cell division tracking 
Description With our collaborator Gunnar Pruessner, we developed new algorithms to image process the time-lapse data series we generated with the light sheet microscope applied on CYCB::GFP reporters. The algorithms enable us to automatically recognize and track in 3D cell division events, and to extract basic statistical properties of the temporal series. 
Type Of Material Computer model/algorithm 
Year Produced 2017 
Provided To Others? No  
Impact Novel analysis of the temporal series. Manuscript in preparation 
 
Title Time-lapse of CYCB::GFP roots 
Description Long-term (up to 7 days) time-lapse series of 3D stack of images taken of Arabidopsis root meristem growing or regenerating. This data has been obtained through our light sheet microscope to image transgenic roots expressing CYCB::GFP with or without their tip excised. It is a very rich database of cell division dynamics in plant tissue. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact A manuscript is in preparation. 
 
Description Gunnar Pruessner 
Organisation Imperial College London
Department Department of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing time-lapse images of Arabidopsis root regenerating, as collected with Light Sheet Microscope built with this grant. Specifically, we provided temporal series of cell division events, described by mitotic fluorescent reporter CYCB::GFP.
Collaborator Contribution One of Gunnar's students significantly contributed to the project by developing novel image processing algorithms to automatically extract and track in time and 3D cell division events from the raw images.
Impact Main outputs are algorithms to image process the temporal series of 3D images generated with our light sheet microscope. Multi-disciplinary: Life Science (Sena), Mathematics (Pruessner)
Start Year 2017
 
Description Henrik Jensen 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with Prof. Henrik J. Jensen and his group. We provide live images of the root regeneration process, and image processing.
Collaborator Contribution They help with data analysis (statistical analysis of time series) and mathematical modelling.
Impact No major output, yet. Multi-disciplinary: Biology, Physics, Mathematics
Start Year 2015
 
Title SPECIMEN MOUNTING DEVICE AND METHOD FOR LIVE MICROSCOPY 
Description A device for mounting a living (or otherwise shape-changing or moving) specimen in a chamber for microscopic examination, the device comprising: a first part for mounting the device in or on the chamber; an inlet port for receiving a flow of a liquid medium; and a second part attached to the first part, the second part being arranged to extend into the chamber in use and to form a substantially straight channel between the second part and one or more walls of the chamber, the channel being for constraining the specimen in use; wherein a first conduit extends from the inlet port, through at least part of the second part, to an outlet hole in the second part, for conveying the liquid medium into the chamber via the outlet hole. Also provided is an assembly comprising such a device mounted in a chamber, and a method of examining a specimen using such a device. 
IP Reference WO2017137779 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact None so far
 
Title Novel algorithm for tracking of intermittent events in 4D 
Description This method fills a gap in existing tracking techniques, to follow intermittent events (e.g. cell divisions) in challenging data-sets without constant reference points and in unregistered images. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2020 
Open Source License? Yes  
Impact Published and available. Too early to comment on general impact. 
URL https://plantmethods.biomedcentral.com/articles/10.1186/s13007-021-00723-8
 
Description International Innovation interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview with the magazine International Innovation, describing the Lab activity and in particular the BBSRC project on root regeneration.
I received several emails of appreciation, in response of this outreach exercise.
Year(s) Of Engagement Activity 2015
URL http://www.internationalinnovation.com/plant-regeneration/
 
Description Physics World interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact An interview with the magazine Physics World, from the Institute of Physics. A detailed description of this project
Year(s) Of Engagement Activity 2018
 
Description Poster, Induced Plant Development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Poster presented at the conference "Induced Plant Development", held at the Sainsbury Laboratory, in Cambridge, UK
Year(s) Of Engagement Activity 2016
 
Description Poster, Physics of Emergent Behaviour II 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Poster presented at the conference "Physics of Emergent Behaviour II", held at the Science Museum, in London, UK.
Year(s) Of Engagement Activity 2015
 
Description Talk at AAB conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Invited talk at the meeting "Mathematical Modelling in Plants", organized by the Association of Applied Biologists
Year(s) Of Engagement Activity 2018
 
Description Talk at the GARNet Workshop on Advances in Plant Imaging (Un. of Warwick) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Talk titled "Light-sheet microscopy for plant roots", where the light-sheet microscope we developed was described, together with methods.
Year(s) Of Engagement Activity 2019
URL https://garnetimaging19.weebly.com