Developing a synthetic approach to manipulating guard cell membrane transport and stomatal control

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Stomata are pores that provide for gaseous exchange across the impermeable cuticle of plant leaves. They open and close to balance the requirement for CO2 entry for photosynthesis against the need to reduce the transpiration of water vapour and prevent leaf drying. Stomatal movements are driven by solute transport - and consequent uptake/loss of water - across the cell membrane of the guard cells which surround the stomatal pore. Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years: globally, agricultural water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is projected to double again before 2030. Thus stomata represent an important target for breeders interested in manipulating crop performance. Significantly, stomatal responses are often delayed in the face of environmental fluctuations, especially of light. Improving water use efficiency (=amount of carbon fixed in photosynthesis/amount of water transpired) should be possible, without a cost to carbon assimilated in photosynthesis, if the stomatal response to light are enhanced. However, the complexity of guard cell transport and its coupling to gas exchange and transpiration has presented a formidable barrier to manipulations so that efforts at genetic improvements have generally proven constrained.

Synthetic methods offer one approach to physiologically enhancing stomatal function. Furthermore, in combination with quantitative systems analysis they present an opportunity to gain fundamental insights into the coordination of transport in the homeostasis of a plant cell system. I developed previously the OnGuard software for quantitative dynamic modelling of the guard cell. OnGuard models build explicitly on the wealth of molecular, biophysical and kinetic knowledge for guard cell transport and metabolism; they accommodate stomata of different plant species, over the full range of conditions studied in the laboratory to date; and they have been shown to incorporate the real predictive power needed to guide experiments at the cellular and physiological levels that start with molecular manipulations in silico. As the next major step, I wish to establish directed synthetic strategies for design of stomatal function, based on this deep knowledge of stomatal guard cells, and on developments in work with light-driven ion pumps and channels.

I propose now to use the OnGuard software to model and explore the most effective approaches to enhancing stomatal kinetics. In parallel, my laboratory will develop a synthetic approach to manipulate guard cell transport with so-called optogenetic tools - light-driven pumps and channels - using these to test and validate the model predictions. Most important, the combined modelling and experimental approaches will connect the molecular components with physiological properties of stomata. They will reinforce an understanding of guard cell transport and will enable novel explorations of water use and its balance with photosynthetic carbon assimilation. These studies will target expression of selected optogenetic tools based on OnGuard model predictions. The knowledge gained will aid in refining these models and will allow exploration of the kinetic and homeostatic coordination between the serial membranes of the plasma membrane and tonoplast. This coordination is known to be fundamental to the control of stomatal aperture, but its mechanism remains unknown. Finally, the studies will serve to establish the potential for improving the efficiency of water use by plants drawing on synthetic methods to reduce the mismatch in dynamic environmental responses between stomata and photosynthesis.

Technical Summary

These studies will utilise targeted expression of light-driven ion pumps and channels - including bacteriorhodopsins, halorhodopsins and channelrhodopsins - to probe the coordination of transport between the plasma membrane and tonoplast of stomatal guard cells. They will build on the OnGuard software, previously developed in this laboratory, that has proven effective in uncovering a hitherto unrecognised homeostatic connection in K+ and Cl- channel regulation. We will test, by in silico modelling and experimental validation, the hypothesis that transport coordination between these serial membranes is emergent from the interactions implicit in the sharing of transported substrates/products. Additionally, we will explore the potential for synthetic manipulation to accelerate stomatal kinetics for improved water use efficiency. The experimental work will focus on Arabidopsis for which we have a substantial molecular toolchest. The modelling and experimental methods proposed are independent, but their combination gives added value to both. We will build on methods at the research forefront in so-called optogenetics that have enabled substantial advances in understanding of neural networks. Electrophysiological and related methods will be used quantify transport at the cellular and subcellular levels, and will assess stomatal responses, especially to light, to relate the consequences of targeted pump and channel expression on stomatal kinetics and their association with transpiration and carbon assimilation. We will use these data to parameterise models and to test the micro-macro link of the models in predicting enhanced stomatal behaviour and its consequences for water use efficiency. Thus, I fully expect new and exciting insights into guard cell transport and its coordination through synthetic manipulations, much as our previous modelling efforts provided hitherto unexpected insights through the mutatiional analysis.

Planned Impact

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant, systems and synthetic biology communities. The research will stimulate thinking around strategies for systems modelling and synthetic applications, especially in relation to membrane transport, plant growth, development and pathology, and it should strengthen in silico methodologies for approaching crop engineering. Thus, the research is expected to benefit fundamental researchers and, in the longer-term agriculture and industry, through conceptual developments as well as synthetic approaches to improving plant water use efficiency and productivity. The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. The applicant has established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Pathways.
 
Description This is work has demonstrataed our ability to manipulate transport by introducing novel transport processes borrowed from algae and viruses in order to enhance stomatal function. Efforts to incorporate bacterial pumps proved challenging and this work was shelved. By contrast, we have been successful in introducing a synthetic, light-driven ion channel. This work demonstrates that optogenetic manipulation of transport in a plant cell can be used to alter physiological processes with significant gains both in water use efficiency and in photosynthetic carbon fixation and biomass gain by the plant. The first set of results were published in Science in 2019 and additional results are anticipated in a follow-on study over the next 2 years.
Exploitation Route Broad applications building on our proof of principle in a plant system.
Sectors Agriculture, Food and Drink,Education,Energy,Environment,Other

URL http://psrg.org.uk
 
Description The work has led us to engage Plant Biosciences Ltd in filing a patent application related to the IPR of this project
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Environment,Other
Impact Types Economic

 
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