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.
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.
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.
People |
ORCID iD |
Michael Blatt (Principal Investigator) |
Publications
Hecker A
(2015)
Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies.
in Plant physiology
Zhang B
(2015)
The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane.
in The Plant cell
Grefen C
(2015)
A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.
in Nature plants
Papanatsiou M
(2015)
Hydrogen sulfide regulates inward-rectifying K+ channels in conjunction with stomatal closure.
in Plant physiology
Grefen Christopher
(2015)
A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion (vol 1, 15108, 2015)
in NATURE PLANTS
Papanatsiou M
(2016)
Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir.
in Plant physiology
Minguet-Parramona C
(2016)
An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells.
in Plant physiology
Vialet-Chabrand S
(2016)
Modelling water use efficiency in a dynamic environment: An example using Arabidopsis thaliana.
in Plant science : an international journal of experimental plant biology
Chen ZH
(2016)
Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis.
in The New phytologist
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 |
Description | Work under this project has been used to develop and manipulate the 'clustering' domain of this channel and its response to extracellular potassium. We are now engaged with Plant Bioscience Ltd in developing the IPR in order to accelerate its contribution to stomatal movements and enhance gas exchange and photosynthesis while also conserving water. |
First Year Of Impact | 2021 |
Sector | Agriculture, Food and Drink |
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 |