Systems analysis of guard cell oscillatory mechanics in stomatal dynamics

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

Stomata in the epidermis of plant leaves play a vital role in regulating CO2 exchange for photosynthesis while minimising transpirational water loss between the inner leaf air space and the atmosphere. Guard cells surrounding the stomata take up inorganic solutes and water, increasing in volume to open the stomatal pore when CO2 in the leaf is depleted; and they lose solutes and water, decreasing in volume to close the stomatal pore and conserve leaf water under stress, in the dark and when CO2 is high. We know a great deal about the mechanisms that drive stomatal movements between the extremes in pore aperture. By contrast, our knowledge is remarkably poor of the mechanisms that give rise to the dynamic continuum of apertures normally observed in the field, much less how such fine-tuning is regulated. This gap in knowledge can be seen, for example, in the focus of past efforts in quantitative modelling. Stomatal characteristics underpin models for transpiration and plant water use efficiency that have proven successful in reproducing and predicting transpirational behaviours at the plant and community levels. However all of these models reflect a 'top-down' approach and consider guard cell mechanics as a 'black box', subsuming these processes within a few empirical parameters, hydraulic pathways and conductances. There are very few models that have been developed 'bottom-up' from the properties of the guard cells themselves, despite the wealth of knowledge we have for guard cell transport and signalling, and none that are sufficiently generalised to be widely applicable in predicting stomatal behaviour. A further complication is that much of our knowledge at the cellular level is based on in vitro studies with guard cells in epidermal peels or isolated as protoplasts. We need to bridge these gaps in our knowledge and to understand how stomata compensate dynamically in the face of real environmental challenges. Studies over the past 15 years have yielded several important clues to the mechanisms behind stomatal dynamics. The clues point to oscillations of the guard cell membrane between two quasi-stable states that control and balance osmotic fluxes. This postulate finds support in well-documented observations that stomatal apertures also oscillate and can be driven experimentally under defined conditions. Indeed, such a 'time-averaging' mechanism has already been predicted from a systems analysis of guard cell ion transport, albeit using a mathematical model with significant parameter limitations. These several lines of evidence need now to be drawn together and subject to rigorous experimental testing in order to address a number of key issues. We need to know whether more comprehensive mathematical models for guard cell transport / incorporating, for example, known regulatory properties for the major ion transporters / are able to return the full range of observed behaviours in aperture and voltage, and to predict novel ones. We want to know how these behaviours are underpinned by the dynamics of guard cell ion fluxes and osmotic contents. Finally, we want to test whether experimental manipulations of the relevant guard cell parameters can be shown to yield well-defined and predictable changes in stomatal behaviour. We propose here to develop this line of enquiry jointly through systems kinetic modelling to derive quantitative and testable predictions and through experimental analysis and validation. Our knowledge of guard cell transport and homeostasis is now sufficiently well-developed to make an approach of this kind a readily achievable goal. We fully expect answers to the questions we pose to yield new and exciting insights into the behaviour of stomata and to open entirely new dimensions to practical applications in agriculture and crop development.

Technical Summary

Stomata of plant leaves play a vital role in regulating CO2 exchange for photosynthesis while minimising transpirational water loss to the atmosphere.We know a great deal about stomatal movements between the extremes in pore aperture and about the mechanisms that drive these movements at the level of the guard cell in vitro in epidermal peels and protoplasts. By contrast, our knowledge is remarkably poor of the mechanisms that give rise to the dynamic continuum of apertures normally observed in vivo in the leaf. This project seeks to bridge these gaps in knowledge to understand how stomata achieve a dynamic range of apertures in the face of environmental challenge. We propose a systems modelling approach in conjunction with experimental analysis of the oscillatory behaviour of guard cells in epidermal peels and in situ in the leaf. The project will follow two lines of enquiry. (1) We will develop a software platform for quantitative mathematical modelling of guard cell membrane transport and homeostasis. We will draw on the wealth of detailed kinetic information for guard cells to fully-constrain parameters and generate models with true predictive power and experimentally-testable outputs. (2) We will utilise imaging, electrophysiology and related techniques to quantify the oscillatory behaviours of guard cells, both to bridge the gap between in vitro preparations and stomata function in the leaf, and to dissect the underlying mechanics and function(s) of these oscillations. These studies will also serve as a test-bed for experimental analysis of model predictions and for model validation. We anticipate the results to yield new and exciting insights into the behaviour of guard cells and stomata / much as previous modelling efforts have provided hitherto unexpected (indeed, counter-intuitive) insights into cellular homeostasis and human disease / and we anticipate the work to open entirely new dimensions in applications to agriculture and crop development.

Publications

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Armengaud P (2009) EZ-Rhizo: integrated software for the fast and accurate measurement of root system architecture. in The Plant journal : for cell and molecular biology

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Bassham DC (2008) SNAREs: cogs and coordinators in signaling and development. in Plant physiology

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Blatt MR (2013) The conceptual approach to quantitative modeling of guard cells. in Plant signaling & behavior

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Caesar K (2011) A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana. in The Plant journal : for cell and molecular biology

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Chen Z (2009) Systems analysis of membrane transport and homeostasis in stomatal guard cells in Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology

 
Description This project yielded a detailed and quantitative modelling platform for stomatal biology that not only reproduces existing knowledge of stomatal mechanics but also has produced a large number of unexpected, often counterintuitive, predictions about how stomata work. A number of these predictions have been followed up over the past 8 years, all showing the remarkable accuracy of the modelling platform. This knowledge has enabled us to expand on the platform to the whole plant in the field, effectively providing the essential link between molecular function and whole-plant transpiration that is needed to guide experiments to engineer stomatal behaviour in the intact plant.
Exploitation Route The modelling platform is now freely available at the above web site for users to explore their own questions about how stomata function. It has obvious applications in 'reverse engineering' of stomatal function to improve water use efficiency in crops. It also offers a superb tool with which to train the next generation of researchers in how cells function.
Sectors Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education,Energy,Environment,Other

URL http://psrg.org.uk
 
Title 2in1 vector systems 
Description Synthetic biology vectors for transient and stable transformation with quantitative visual reporting on cell-by-cell basis 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Computer software tool for quantitative measurement and analysis of root growth/development 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software for electrophysiology and imaging data aquisition and analysis 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Multicistronic vector systems 
Description Synthetic biology vector systems for transient and stable transformation for expressing multiple, tagged proteins and for quantitative analysis of membrane traffic and transport 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title OnGuard 
Description Systems biology software for quantitative modelling of cellular transport and homeostasis 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SUS vector systems 
Description Synthetic biological vector systems for protein-protein interaction screening 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title Software tools for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Software for quantitative trait analysis and acquisition for root growth/development 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software package for electrophysiology and imaging data acquisition and analysis 
Type Of Material Data handling & control 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title OnGuard 
Description Quantitative systems biology modelling of cellular transport and homeostasis 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SDM-assist 
Description Software for molecular primer design that enables introduction of silent markers for molecular cloning 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Description PBL 
Organisation Plant Bioscience Limited Technology
Country United Kingdom 
Sector Private 
PI Contribution IPR on ABA receptor technology and ABA signalling
Collaborator Contribution Funding related to IPR on ABA receptor technology and ABA signalling
Impact Multiple outcomes in publications and industrial contacts
 
Description PSG 
Organisation POSCO - South Korea
Country Korea, Republic of 
Sector Private 
PI Contribution Base support for meetings and exchange of materials
Collaborator Contribution Base support for meetings and exchange of materials
Impact Base support for meetings and exchange of materials
 
Title Software tools and packages for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Technology Software 
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Description International online services 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Prof. Blatt and members of his laboratory have contributed to various media events over the years, including online interview contributions (e.g. People behind the Science, a US-based media program)
Year(s) Of Engagement Activity Pre-2006,2006,2008,2011,2015,2016,2017,2018
 
Description Invited presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I regularly speak to audiences, from small groups (5-20) to large audiences (>1000) in a variety of settings. In addition to teaching and extramural activities associated with the university, I also speak on invitation to national and international groups a number of times each year and in a variety of settings, academic as well as public. I also reach audiences through short video presentations mounted on the web, these primarily via my laboratory website and the ASPB websites. Anyone reading this entry is welcome to visit these sites to learn more.

The impacts arising from my presentations are varied. For example, a common consequence of my speaking in academic settings is to attract potential researchers to visit my laboratory and, frequently, to interest potential collaborators and students/postdocs to my research group. At scientific meetings, my talks often attract interest also from researchers interested in the various tools and materials that my research has produced, including the various vector systems and software packages that I
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Schools and displays 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact As these were multiple events, this question is not informative or useful. Participants varied from numbers in the tens to several thousands

Extensive training of participating laboratory members as well as broad scope reach to schools and communities, in the case of the GCC science days to the west of Scotland and in the case of the IFPD activities to audiences within and outside the UK
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk