Analysis of membrane traffic in adaptive stress tolerance in plants

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

Abstract

Stress-mediated adaptation is a key factor in the ability of plants to cope under long-term or repeated environmental challenge, and is especially important for stomata to compensate for gas exchange during extended periods of water deficit. Stomata in the epidermis of plant leaves play a vital role in regulating CO2 exchange for photosynthesis while minimising transpirational water loss between the inner leaf air space and the atmosphere. Guard cells surrounding the stomata take up inorganic ions and water, increasing in volume to open the stomatal pore when CO2 in the leaf is depleted; and they lose ions, other solutes and water, decreasing in volume to close the stomatal pore and conserve leaf water under stress, in the dark and when CO2 is high. Arguably, stomata are at the centre of a crisis in water availability that is expected to unfold over the next 20-30 years. Globally, water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2040, driven mainly by irrigation and agriculture. Clearly, a full understanding of guard cells and stomatal regulation will have significant implications for future efforts to improve the water use efficiency of agricultural crops and, consequently, for society to face these future challenges. In fact, we know a great deal about the cellular and molecular physiology of guard cells, especially in relation to abscisic acid (ABA), salinity and drought that triggrer stomata to close, and the depth and breadth of this information has helped to elevate the guard cell system to that of one of the best understood plant cell models. However, virtually all of this knowledge centres on the short-term responsive behaviour of guard cells. By contrast, our knowledge is remarkably poor of the mechanisms by which guard cells adjust stomatal movements to adapt to longer-term environmental stress. Recently, this laboratory observed that ABA triggers the traffic to an internal compartment of one of the plasma membrane K+ channels mediating ion fluxes in guard cells. This response shows all the hallmarks essential for a function in adaptive modulation of the channel population. It complements a second discovery that the Arabidopsis mutant Atsyp121, which is defective in a major vesicle trafficking protein at the plasma membrane, selectively impairs the ability of stomata to 'remember' the recent history of stress and remain closed (so-called 'progammed' stomatal closure). These discoveries firmly tie membrane vesicle trafficking to [Ca2+]i signalling and adaption in guard cells, and suggest that K+ channel traffic between the plasma membrane and the internal compartment is an important mechanism for adaptation in these cells. I propose to address this idea directly using cellular and molecular tools now available in the laboratory. The outcome will greatly extend our understanding of ABA signalling and adaptation in plants, and should add fundamentally to our knowledge of the dynamics of membrane traffic and ion channel control in eukaryotic cells generally.

Technical Summary

Guard cells are able to adapt stomatal response to the past history of stress, as evinced by a number of well-defined behaviours such as so-called 'programmed' stomatal closure which reflects an ability to 'remember' the recent history of stress. Yet our knowledge is remarkably poor of the mechanisms that underlie this, and similar processes in plants and other eukaryotes. We recently discovered (i) that the water-stress hormone ABA triggers the traffic to an endosomal compartment of one of the plasma membrane K+ channels mediating ion fluxes in guard cells, and (ii) that the Arabidopsis mutant Atsyp121, which is defective in a major trafficking protein at the plasma membrane, impairs progammed stomatal closure. These discoveries firmly tie membrane traffic to [Ca2+]i signalling and adaption in guard cells. They lead to the postulate that K+ channel traffic is important to adapt the capacity for solute flux across the guard cell membrane to the recent history of stress. I propose to follow this line of enquiry to address three key questions underlying this postulate: (i) Is ABA-evoked channel traffic integrated with known ABA signalling pathways? (ii) Do the K+ channels recycle to the plasma membrane and is internalisation and/or recycling affected by the Atsyp121 mutant? Finally, (iii) Does the programmed closure stimulus and/or ABA evoke a traffic of other ion channel proteins associated with ABA signalling? The work will make use of a combination of confocal imaging and electrophysiological techniques, taking advantage of a toolchest of marker constructs and mutants now on hand in the laboratory, and will be supported by biochemical and cell fractionation studies. Various strategies will be used, including a combination of 'pulse-chase', localisation and kinetic analyses as well as in vivo epitope labelling methods.

Publications

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Bassham DC (2008) SNAREs: cogs and coordinators in signaling and development. in Plant physiology

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

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Blatt MR (2013) The conceptual approach to quantitative modeling of guard cells. in Plant signaling & behavior

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Caesar K (2011) A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana. in The Plant journal : for cell and molecular biology

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Chen Z (2009) Systems analysis of membrane transport and homeostasis in stomatal guard cells in Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology

 
Description Work on this project established a direct connection between membrane traffic and the ability of guard cells (and hence gas exchange for photosynthesis) to adapt to repeated stresses. In effect, the findings demonstrate a key role for membrane traffic in the 'memory' of the plant as it relates to the history of stress experience.
Exploitation Route This is purely fundamental research, but could help inform future efforts at improving crop performance in the field.
Sectors Agriculture, Food and Drink,Education,Other

 
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