Engineering the GORK K+ channel to enhance stomatal kinetics

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

Abstract

Stomata are pores that open and close to balance the requirement for CO2 entry to the leaf for photosynthesis against the need to reduce water loss via transpiration and prevent leaf drying. Stomata are 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 droughts of 2010-12 and 2018 cost UK farmers alone an estimated £1.2B and worldwide costs year-by-year are estimated in the hundreds of billions of pounds over the past five years. Thus stomata are an important target in efforts to improve crop performance, especially in the face of global climate change. Stomatal opening and closing are driven by solute and water transport of the guard cells which surround the stomatal pore. Our deep knowledge of these processes has made the guard cell one of the best-known plant cell models and gives real substance to prospects for engineering stomata to improve water use by crops.

In the natural environment light fluctuates, for example as clouds pass over. The stomata of most plants respond to light by opening the stomatal pore to increase CO2 access for photosynthesis, and they reduce the pore aperture when the light intensity drops and the demand for CO2 by photosynthesis declines. Photosynthesis generally tracks light fluctuations, but stomata are much slower to respond. The slower response of stomata can limit gas exchange and reduce carbon assimilation by photosynthesis when light intensity rises and lead to transpiration without corresponding assimilation when light intensity drops quickly. We and others have reasoned that assimilation, and consequently crop yields, could be enhanced concurrent with an decrease in water use by plants if the rates of stomatal movements could be better matched to variations in photosynthetic demand.

Recently, we found that accelerating ion flux in stomatal guard cells by introducing a synthetic, light-activated K+ channel, BLINK1, was sufficient to increase the biomass and reduce the associated water use by 2-fold in the model plant Arabidopsis. Furthermore, we have demonstrated that analogous gains are possible by altering the intrinsic controls on the activity of a K+ channel that occurs naturally in stomata and other plant cells. These findings demonstrate the potential of accelerating stomata as a strategy to enhance crop gains while conserving water and a second strategy based on the properties of a channel native to stomata.

We propose here an interlinked effort, combining our knowledge of native K+ channel regulation and of optogenetics in two distinct but related strategies. We will engineer native K+ channels for gains in water use efficiency and biomass yield and we will combine our knowledge of these channels with optogenetics to bring channel regulation under direct control by light. As a proof-of-principle, we will use Arabidopsis as a model that harbours K+ channels with orthologues in many crops. Additionally, we expect to develop and validate a new set of optogenetic tools and strategies based around modifications to the interactions of a known optogenetic photoswitch that will be widely applicable in plants. These aims dovetail with our longer-term interests in developing optogenetic approaches to bioengineering that integrate within processes native to the plant.

Technical Summary

We propose a concerted appraisal of two approaches to manipulating stomatal kinetics and its relevance to biomass gain and water use. Our aims are (1) to examine the potential for engineering the widely-distributed and naturally-occurring GORK K+ channel for these purposes through exploration of domains identified with inter-channel interactions in channel gating, and (2) to use knowledge of these domains in developing and applying new optogenetic strategies in vivo to accelerate stomatal kinetics for biomass and water use efficiency gains. A part of this work will develop and validate a new set of optogenetic tools based around separable components of the LOV2-J(alpha) photoswitch that are common to plants. We will build on our recent success with the light-activated K+ channel BLINK1 in Arabidopsis as a guide.

Both the practical and fundamental challenges will take advantage of targeted optogenetic expression within the leaf epidermis and, additionally, on the proven capacity for adjusting the gating controls of GORK to facilitate guard cell K+ flux and stomatal movements. Experiments will follow methodologies similar to those we have used successfully to date in molecular biology, protein-interaction and electrophysiological analysis, and in gas exchange and biomass studies. We will use extant knowledge of photoswitch variants and preliminary evidence for their deployment as separable components in developing and validating new optogenetic tools. In vivo, we will target expression to assess, in response to light, the impact and dynamic range of responses in stomatal guard cells that enhance their coupling to photosynthesis. We expect these studies to expand our fundamental understanding of stomatal mechanics, to establish a native K+ channel as a bona fide target for future efforts towards crop improvement, and to establish a new set of optogenetics tools with wide applicability in controlling protein-protein interactions.

Planned Impact

This proposal is for a synergy of fundamental research, albeit with practical implications in the longer term. The research builds on a core of ideas at the centre of the international plant photosynthesis and stomatal biology communities. It will stimulate thinking around strategies for enhancing crop yields and reducing agricultural water consumption, and it should inform methodologies for approaching crop engineering. In its essence, the research will yield basic insights into the physiology of stomatal guard cells as well as developing new methods in their engineering. Impacts will include the construction of a range of modified channel forms, both those without and with optogenetic control, and a new set of conceptual strategies for modifying membrane transporters native to the cell. In the long term, the research is expected to benefit practical research as well as informing agriculture and industry through the introduction of new technologies relevant to plant productivity and water use efficiency. The research will build capacity through higher education training programmes at the postgraduate and postdoctoral levels, yielding highly-trained researchers with broad expertise across molecular, cellular and whole-plant biology. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. The research will help guide future efforts in applications to agricultural/industrial systems, and the applicants have established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Details of these, and additional impacts will be found in the Case for Support and in the attached Impact Pathways.
 
Description This is a placeholder. The grant is only just started.
Exploitation Route nothing as yet
Sectors Agriculture, Food and Drink,Other

 
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 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 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
 
Description JIC 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with colleagues at the JIC on projects relating to environmental adaptation and improved crop yields
Collaborator Contribution Researchers at the JIC undertake development of crop models with specified genetic modifications for testing and analysis
Impact This is an ongoing collaboration. Outputs are still in development
Start Year 2020
 
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