Analysing GORK clustering for enhanced 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 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. The bulk of water used in agriculture passes through the stomatal pore. Thus stomata represent an important target for 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 speed of stomatal response can be enhanced.

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. Guard cells harbour ion channel proteins to facilitate cation flux for stomatal movement. Uniquely, the opening (or gating) of one class of plant ion channels is also sensitive to external K+ concentration. These channels are found in guard cells of tobacco, Vicia and Arabidopsis, in the latter encoded solely by the GORK gene. Increasing K+ outside moderates channel opening in parallel with the equilibrium voltage for K+ and affects whole-cell conductance. These channels are the main pathway for K+ efflux during stomatal closure, but their K+-sensitivity constrains K+ flux capacity, notably at higher external K+. Estimates based on recent modelling suggests that stomatal closure could be accelerated 3-fold with only a moderate increase in the flux capacity of these channels.

The K+-sensitivity of the GORK channel is a property of the channel protein itself, which should facilitate manipulating K+ efflux capacity to accelerate stomatal closure. My laboratory has uncovered evidence that the K+-dependence of GORK gating is associated with its assembly in clusters. These assemblies require the the so-called 'voltage-sensor domains' (VSDs) of GORK to interact with one another. Movement of the VSDs is known to couple voltage to channel gating, so it is likely that interaction between VSDs provides a mechanism for cooperative self-regulation. I propose now to complete the analysis of GORK VSD interaction and the consequences for channel control and for stomatal movements. Regardless of the mechanism, it is clear that these discoveries offer the means to explore a unique and fundamental property of this class of K+ channels in plants and to manipulate channel activity, potentially enhancing the kinetics of stomatal closure and water use by the plant.

Technical Summary

Gating of outward-rectifying K+ channels in plants, including the GORK channel of Arabidopsis guard cells, is uniquely sensitivity to extracellular K+. This sensitivity restricts substantially channel activity and K+ efflux during stomatal closure. Work from my laboratory has shown that the K+-dependence of gating is linked to GORK clustering. We have resolved one of the two, critical binding sites located on the voltage-sensor domain (VSD) of GORK and have confirmed that the second binding site at the N-terminus of the VSD. This project will resolve the second of the binding sites, and will utilize this knowledge to control clustering and modify gating. GORK is the primary K+ channel responsible for K+ efflux in the Arabidopsis guard cell, and makes this channel a important target for manipulations directed to accelerating stomatal closure and improving water use by the plant. Thus we will use knowledge of the binding sites to explore the potential for enhancing GORK activity.

Experiments will use in vivo and in vitro screens for binding and functional analysis. We will draw on the toolchest of vectors and expression methods developed in my laboratory for transient and stable transformations and mutant complementations. The work will be supported by structural modelling. Electrophysiological and related methods will be used quantify transport in vivo and after heterologous expression. We will assess stomatal responses to relate the effects of channel mutation on stomatal kinetics and their association with transpiration and carbon assimilation. We will use these data to parameterise physiological models and to test systems analysis 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 targeted manipulations, much as our previous efforts provided hitherto unexpected insights through the mutational analysis.

Planned Impact

This proposal is for research developing new concepts at the core of ideas emerging within the international transport, plant, and systems biology communities. The research will stimulate thinking around fundamental ion channel biophysics and membrane transport, plant growth and water relations, and it should strengthen biological and 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 and targeted methods 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.

Publications

10 25 50
 
Description This work has led us to demonstrate, for the first time in any eukaryotic cell, the direct connection between ion channel clustering and ion sensing that affects channel activity. These findings will rewrite the textbooks on how ion channel gating is regulated in eukaryotic cells. The outcomes will also give us a practical handle with which to manipulate guard cell behaviour and stomatal control of photosynthesis. Furthermore, we can show that the connection of clustering with channel gating can be used to enhance stomatal regulation and improve the water use of plants
Exploitation Route The work is not yet at a point that could be readily exploited commercially, but it has promise and we are pursuing patent protection for the IPR.
Sectors Agriculture, Food and Drink,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