A protein scaffold essential for K+ transport and stomatal control

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

Abstract

This proposal builds on the discovery in this laboratory of an unusual SNARE protein complex that is essential for K+ channel regulation in Arabidopsis, and of our recent finding of its impact on K+ uptake by the roots and plant growth. Because the SNARE, SYP121, is already known to have roles in pathogen defence and cell volume responses meidated by the plant hormone abscisic acid (ABA), these findings offer the first substantive evidence that physically links the protein complex to actions in abiotic stress, mineral nutrition, water use efficiency (defined as the amount of dry matter produced per unit of water transpired through stomata) and cell expansion in the plant. 'Closing the circle' of K+ channel control, its contributions to K+ assimilation, water flux and cell volume control in the growing plant is expected to yield substantive insights in future efforts towards 'hardening' and development of water-effcient crops. In general, eukaryotic cells engage SNARE proteins as part of a well-defined mechanism for traffic of membrane vesicles, proteins and soluble cargo between compartments within the cell. Work from this laboratory first identified genes encoding two plasma membrane SNAREs from tobacco and Arabidopsis, and demonstrated their association with the ABA signalling cascade leading stomata to close and reduce water loss from the plant. Much is known about these cellular signals as well as the mechanisms they engage in regulating transmembrane ion flux. By contrast, we have little knowledge of the mechanisms coordinating ion transport with cellular volume and membrane surface in guard cells. Indeed, how plant cells regulate transport of osmotically-active solutes (especially of K+) in parallel with cell volume, whether reversible as in guard cells or during irreversible expansive growth, remains a matter of considerable debate. Knowing how transport is coordinated with changes in cell surface area will be of great fundament importance to understanding cell volume control in plants generally, and is likely to have practical relevance in our ability to manipulate the growth and homeostasis of plants, for example in altering stomatal controls to improve water use efficiency of crops. Our working hypothesis is that SNARE-K+ channel interaction serves as a molecular governor, analogous to the mechanical invention of James Watt, to coordinate channel-mediated uptake of the osmotically-active K+ ion with cell expansion We want to explore the molecular mechanics of the interaction, its implications for such a governor model in cell volume control and, equally, we want to examine its potential for applications in agriculture. Our immediate goals are (1) to explore the dynamics of the protein partners and their interactions, (2) to analyse their impact on ion transport and its coordination with membrane traffic, and (3) to assess their importance for stomatal function and water use by plants under stress.

Technical Summary

This proposal builds on our discovery of an unusual SNARE protein complex that is essential for K+ channel regulation in Arabidopsis, and of our recent finding of its impact on K+ uptake and plant growth. In general, eukaryotic cells engage SNARE proteins as part of a well-defined mechanism for traffic of membrane vesicles, proteins and soluble cargo between compartments within the cell. Our discovery of a SNARE-K+ channel complex in plants, and evidence of its unusual role in controlling K+ uptake as well as facilitating cell expansion, provides the very first opportunity to explore how such interactions regulate transport of osmotically-active solutes in parallel with cell expansion in plants. Knowing how transport is coordinated with changes in cell surface area will be of great importance to understanding cell volume control in plants generally, and is likely to have practical relevance in our ability to manipulate the growth and homeostasis, for example in altering stomatal controls to improve water use efficiency of crops. Our working hypothesis is that SNARE-K+ channel interaction serves as a molecular 'governor' to coordinate channel-mediated uptake of the osmotically-active K+ ion with cell expansion. The immediate goals are to analyse the molecular basis for the interaction, its implications for cell volume control and, equally, to examine its potential for applications in agriculture. These studies will make use of site-directed mutagenesis, multi-component protein interaction analysis, electrophysiology and single-cell imaging technologies to identify the protein motifs essential for SNARE-K+ channel interaction and to characterise their role(s) in K+ transport control in parallel with changes in cell volume.

Planned Impact

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant cell biology community. The research should stimulate thinking about these topics and help facilitate a paradigm shift in approach. These studies will also extend our recent development of a novel 'bridge' assay for high-throughput molecular interaction analysis. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for the analysis of multicomponent systems. The research will feed into higher education training programmes through research training at the postgraduate and postdoctoral levels. Finally it will help guide future efforts in applications to agricultural/industrial systems. MRB has established links with industrial/technology transfer partners (Agrisera, Dualsystems, Plant Bioscience) and research institutes (SCRI and JIC) 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 Plan.

Publications

10 25 50

publication icon
Blatt M (2014) Focus on Water in Plant Physiology

publication icon
Blatt M (2014) The conceptual approach to quantitative modeling of guard cells in Plant Signaling & Behavior

publication icon
Blatt MR (2018) Plant Physiology Launches Associate Features Editors. in Plant physiology

publication icon
Blatt MR (2015) Vigilante Science. in Plant physiology

publication icon
Blatt MR (2017) Small Pores with a Big Impact. in Plant physiology

publication icon
Blatt MR (2014) Applications of fluorescent marker proteins in plant cell biology. in Methods in molecular biology (Clifton, N.J.)

publication icon
Blatt MR (2018) New Faces behind the Scenes. in Plant physiology

 
Description Work on this project built on a long-standing interest in understanding how plants control turgor and cell volume. On the basis of these studies, we can now show that direct physical interactions between a subset of ion channels (for solute uptake and turgor) and a conserved group of vesicle-trafficking proteins are key to mutual control of the turgor and cell expansion. A key finding of this work is the direct coupling of vesicle traffic with ion channels to confer a coupling to solute uptake and growth.
Exploitation Route The work completely rewrites the textbooks on how vesicle traffic is controlled in eukaryotes, demonstrating that plants have a unique capacity to coordinate traffic with solute uptake. The knowledge gained should, in the long run, contribute to our understanding and application of plant growth.
Sectors Agriculture, Food and Drink,Education,Environment,Other

URL http://www.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