Receptor-like kinase palmitoylation: resolving a crucial feature of plant cell signalling
Lead Research Organisation:
University of Dundee
Department Name: School of Life Sciences
Abstract
Regulating plant perception - the role of protein palmitoylation
Plants are unable to move away from danger and must therefore be able to detect any potential threat or dangerous change in the environment and adapt accordingly to survive. Plants are also multicellular organisms with complex body plans composed of many specialized tissue and cell types. Organising and maintaining their structure requires tightly regulated communication between the individual cells making up the plant. Many of these developmental and environmental signals are detected by a group of proteins called receptor-like kinases (RLKs). RLKs are the largest group of proteins in plants, accounting for 2.5% of all the genes in the model organism Arabidopsis thaliana. RLKs detect signals from outside of the cell such as pathogens, hormones, changes in the integrity and structure of the cell wall or patterning and developmental signals produced by the plant itself. These signals are transmitted across the cell membrane to start intracellular responses such as altering gene activity, changing cell division and differentiation, modifying the cell wall or production of chemicals to fight off pathogens. Understanding how RLK signaling is regulated is therefore critical to all aspects of plant biology.
Each RLK detects a different signal and in response to signals bind to specific protein partners to start the appropriate signaling processes. When RLKs interact they alter each other through a process known as post-translational modification. Post-translational modification usually involves the addition or removal of small molecules, such as phosphate groups, to specific amino acids in a proteins structure to alter its activity, change which proteins it can interact with, promote its degradation or change where in the cell the protein is found. Two post-translational modifications of RLKs are fairly well understood; phosphorylation and ubiquitination. Phosphorylation of RLKs occurs after signal perception and interaction of the RLK with its signaling partner, and generally causes activation of signaling. Ubiquitination usually results in the RLK being degraded to stop further signaling.
I have recently discovered an entirely novel post-translation modification of RLKs that is essential for their correct signaling. This modification is called palmitoylation. I have found that unstimulated RLKs are palmitoylated but when they detect a relevant signal their palmitoylation state rapidly decreases. We currently don't know why RLKs are de-palmitoylated or when it happens in relation to other events in RLK signaling. This proposal seeks to answer these questions and characterize the role of this novel and important factor controlling plants primary method of responding to extracellular signals. We will also identify the enzymes required to de-palmitoylate RLKs to understand how this process is regulated.
This greater knowledge of RLK function will help us to understand how plants regulate responses to threats, environmental problems and how they regulate their development. This information can be used in crop breeding and development programs to introduce or improve desired traits in plants and provide more insight into how plants will respond to particular circumstances.
Interestingly plant RLKs are related to the Toll-like and interleukin receptors used by mammalian immune systems to detect pathogens, activate and enhance immunity and regulate inflammation. I have recently shown that these receptors are regulated in the same way as plant RLKs meaning that the information from my plant work can be able to help understand immune system regulation in humans.
Plants are unable to move away from danger and must therefore be able to detect any potential threat or dangerous change in the environment and adapt accordingly to survive. Plants are also multicellular organisms with complex body plans composed of many specialized tissue and cell types. Organising and maintaining their structure requires tightly regulated communication between the individual cells making up the plant. Many of these developmental and environmental signals are detected by a group of proteins called receptor-like kinases (RLKs). RLKs are the largest group of proteins in plants, accounting for 2.5% of all the genes in the model organism Arabidopsis thaliana. RLKs detect signals from outside of the cell such as pathogens, hormones, changes in the integrity and structure of the cell wall or patterning and developmental signals produced by the plant itself. These signals are transmitted across the cell membrane to start intracellular responses such as altering gene activity, changing cell division and differentiation, modifying the cell wall or production of chemicals to fight off pathogens. Understanding how RLK signaling is regulated is therefore critical to all aspects of plant biology.
Each RLK detects a different signal and in response to signals bind to specific protein partners to start the appropriate signaling processes. When RLKs interact they alter each other through a process known as post-translational modification. Post-translational modification usually involves the addition or removal of small molecules, such as phosphate groups, to specific amino acids in a proteins structure to alter its activity, change which proteins it can interact with, promote its degradation or change where in the cell the protein is found. Two post-translational modifications of RLKs are fairly well understood; phosphorylation and ubiquitination. Phosphorylation of RLKs occurs after signal perception and interaction of the RLK with its signaling partner, and generally causes activation of signaling. Ubiquitination usually results in the RLK being degraded to stop further signaling.
I have recently discovered an entirely novel post-translation modification of RLKs that is essential for their correct signaling. This modification is called palmitoylation. I have found that unstimulated RLKs are palmitoylated but when they detect a relevant signal their palmitoylation state rapidly decreases. We currently don't know why RLKs are de-palmitoylated or when it happens in relation to other events in RLK signaling. This proposal seeks to answer these questions and characterize the role of this novel and important factor controlling plants primary method of responding to extracellular signals. We will also identify the enzymes required to de-palmitoylate RLKs to understand how this process is regulated.
This greater knowledge of RLK function will help us to understand how plants regulate responses to threats, environmental problems and how they regulate their development. This information can be used in crop breeding and development programs to introduce or improve desired traits in plants and provide more insight into how plants will respond to particular circumstances.
Interestingly plant RLKs are related to the Toll-like and interleukin receptors used by mammalian immune systems to detect pathogens, activate and enhance immunity and regulate inflammation. I have recently shown that these receptors are regulated in the same way as plant RLKs meaning that the information from my plant work can be able to help understand immune system regulation in humans.
Technical Summary
Receptor-like kinases (RLKs) are the main method of extracellular perception in plants and their signaling is highly regulated through post-translational modification (PTM). I recently discovered a new PTM essential for regulating RLK function - palmitoylation. RLK palmitoylation rapidly decreases upon ligand perception and suppression of depalmitoylation impairs signaling. In this proposal we aim to identify how palmitoylation regulates RLK function, define what is required for de-palmitoylation to occur and isolate the enzymes that de-palmitoylate RLKs.
Due to the fundamental nature, technical requirements and resources needed this project will use Arabidopsis. However, as RLK signaling is a universal plant process this work will be directly applicable to all plants. We will use the FLS2-flg22 model RLK receptor-ligand pair to examine RLK de-palmitoylation in detail in response to stimulus. The effects of palmitoylation on FLS2 protein-protein interactions will be assessed by co-IP and FLIM-FRET. The timings and requirements of FLS2 de-palmitoylation will be determined by assessing changes in de-palmitoylation in various FLS2 and interacting partner mutant backgrounds. The interplay of FLS2 depalmitoylation with phosphorylation and ubiquitination will be directly assayed by mass spectrometry, western blotting and palmitoylation assays using palmitoylation and phosphorylation defective versions of FLS2 and ubiquitin ligase mutants. The enzymes responsible for depalmitoylation have been cloned and specific members acting on FLS2 will be identified through transient assays in Arabidopsis protoplasts. These results will be confirmed using Arabidopsis knock-out mutants in the appropriate genes. All of these data will be backed up by in-vivo assessment of the effects of de-palmitoylation on defined responses to flg22. This will provide an exhaustive examination of a novel regulatory mechanism affecting a fundamental and conserved aspect of plant perception.
Due to the fundamental nature, technical requirements and resources needed this project will use Arabidopsis. However, as RLK signaling is a universal plant process this work will be directly applicable to all plants. We will use the FLS2-flg22 model RLK receptor-ligand pair to examine RLK de-palmitoylation in detail in response to stimulus. The effects of palmitoylation on FLS2 protein-protein interactions will be assessed by co-IP and FLIM-FRET. The timings and requirements of FLS2 de-palmitoylation will be determined by assessing changes in de-palmitoylation in various FLS2 and interacting partner mutant backgrounds. The interplay of FLS2 depalmitoylation with phosphorylation and ubiquitination will be directly assayed by mass spectrometry, western blotting and palmitoylation assays using palmitoylation and phosphorylation defective versions of FLS2 and ubiquitin ligase mutants. The enzymes responsible for depalmitoylation have been cloned and specific members acting on FLS2 will be identified through transient assays in Arabidopsis protoplasts. These results will be confirmed using Arabidopsis knock-out mutants in the appropriate genes. All of these data will be backed up by in-vivo assessment of the effects of de-palmitoylation on defined responses to flg22. This will provide an exhaustive examination of a novel regulatory mechanism affecting a fundamental and conserved aspect of plant perception.
Planned Impact
To meet projected global food requirements in 2050 the Food and Agriculture Organisation (FAO) of the United Nations estimates that food production needs to increase by 70% overall (and by 100 % in developing countries). Pests/disease and lack of water are both major causes of crop loss (up to 65% in developing countries) and provide a substantial barrier to food security. With climate change increasing the geographical and temporal range of pathogens, breakdown of existing plant resistance traits, emerging pathogen insensitivity to chemical control and EU directives reducing the number and strength of chemical management strategies available, breeding plants with durable resistance is essential for sustainable food security. At the same time crops need to cope with a wider range of climatic conditions and agriculture needs to increase yield while reducing water consumption. Understanding the fundamental mechanisms of how plants perceive and regulate response to a changing environment is therefore required to breed or manipulate crops to deliver the desired improvements. The proposed research will be exploited as detailed in the Pathways to Impact and is expected to benefit the following areas.
1. Breeders, biotechnology and industry - This work seeks to characterize an entirely new mechanism regulating plant perception of the external environment, hormones and pathogens. This work will provide greater understanding of mechanisms at work during plant perception of stimuli and allow for greater subtlety and accuracy in manipulating desired traits such as yield, pathogen resistance or water use efficiency and reducing unwanted effects. This work has the potential to maximize land use, reduce losses pre- and post-harvest and reduce uncertainty in food production.
2. Consumers - Improved food production efficiency thereby reducing food costs and reducing the use of potentially harmful chemical control measures.
3. Global and UK economic competitiveness - Crops showing increased adaptability to environmental change, resistance to pathogens or improved yield will prove profitable to breeders and biotech companies. Jobs will be created to implement any novel advantageous mechanisms found. Reduced expenditure on disease control and irrigation or increased yield per unit area of land will lead to greater profit margins for growers while ensuring costs are kept low for consumers.
4. Environment - By reducing chemical control use ecological diversity can be maintained or improved and will prevent contamination of watercourses, reduce buildup of chemicals in the soil and reduce greenhouse gas emissions from chemical production, transport and application. Improved water use will reduce soil salination thereby increasing sustainability and will provide more fresh water for human consumption thereby improving health.
5. Policy makers - This work will help keep policy makers informed of progress towards safeguarding food supplies against current and emerging pathogens and environmental change.
6. Research Staff - Staff on the project will be trained in public speaking, presentation preparation, presenting data and information to expert and lay audiences, analytical processes, accurate record keeping and collaborative work. These are widely transferable skills applicable to all employment sectors.
This work is therefore aligned to the "Sustainably enhancing agricultural production" strategic priority.
1. Breeders, biotechnology and industry - This work seeks to characterize an entirely new mechanism regulating plant perception of the external environment, hormones and pathogens. This work will provide greater understanding of mechanisms at work during plant perception of stimuli and allow for greater subtlety and accuracy in manipulating desired traits such as yield, pathogen resistance or water use efficiency and reducing unwanted effects. This work has the potential to maximize land use, reduce losses pre- and post-harvest and reduce uncertainty in food production.
2. Consumers - Improved food production efficiency thereby reducing food costs and reducing the use of potentially harmful chemical control measures.
3. Global and UK economic competitiveness - Crops showing increased adaptability to environmental change, resistance to pathogens or improved yield will prove profitable to breeders and biotech companies. Jobs will be created to implement any novel advantageous mechanisms found. Reduced expenditure on disease control and irrigation or increased yield per unit area of land will lead to greater profit margins for growers while ensuring costs are kept low for consumers.
4. Environment - By reducing chemical control use ecological diversity can be maintained or improved and will prevent contamination of watercourses, reduce buildup of chemicals in the soil and reduce greenhouse gas emissions from chemical production, transport and application. Improved water use will reduce soil salination thereby increasing sustainability and will provide more fresh water for human consumption thereby improving health.
5. Policy makers - This work will help keep policy makers informed of progress towards safeguarding food supplies against current and emerging pathogens and environmental change.
6. Research Staff - Staff on the project will be trained in public speaking, presentation preparation, presenting data and information to expert and lay audiences, analytical processes, accurate record keeping and collaborative work. These are widely transferable skills applicable to all employment sectors.
This work is therefore aligned to the "Sustainably enhancing agricultural production" strategic priority.
Publications
Hurst CH
(2015)
Current perspective on protein S-acylation in plants: more than just a fatty anchor?
in Journal of experimental botany
Hemsley PA
(2015)
The importance of lipid modified proteins in plants.
in The New phytologist
Kumar M
(2016)
S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.
in Science (New York, N.Y.)
Hemsley PA
(2017)
An outlook on protein S-acylation in plants: what are the next steps?
in Journal of experimental botany
Turnbull D
(2017)
Fats and function: protein lipid modifications in plant cell signalling.
in Current opinion in plant biology
Hurst CH
(2017)
Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.
in BioTechniques
Hurst CH
(2018)
Variable Effects of C-Terminal Fusions on FLS2 Function: Not All Epitope Tags Are Created Equal.
in Plant physiology
Cowan GH
(2018)
Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.
in Plant physiology
Turnbull D
(2019)
AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity.
in Plant physiology
Hurst CH
(2019)
Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods.
in Methods in molecular biology (Clifton, N.J.)
Hurst CH
(2019)
Juxta-membrane S-acylation of plant receptor-like kinases is likely fortuitous and does not necessarily impact upon function.
in Scientific reports
Hurst CH
(2023)
S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signaling.
in Current biology : CB
Description | We have found a new mechanism by which plants regulate their responses to changes in their environment. This process involves the rapid addition of fatty acids to activated receptors and controls their ability to activate specific signalling pathways. Failure to perform this results in reduced signalling through the pathway. This therefore provides potential for regulating signal strength outputs of receptors and could be important for introducing novel phenotypes such as dwarfing to plants through GM means. This is a unique ability of this modification and will therefore be of greater interest for crop manipulation than other modifications where blocking results in total loss of function. This has opened up a novel research area and we are currently trying to capitalise on our findings. As part of this work we determined that the original sites of modification proposed for study resulted from evolutionary chance and, although modified, were not required for function in our study protein. Subsequent work identified receptors where these sites appear to be functional, evolutionary conserved and contributed to evolution of new receptor mechanisms. These data were published as Hurst et al 2019 Sci Rep. Additional work is now being performed to determine the role of these modifications. We also found that a common approach to studying proteins in cells, addition of tags that can be recognised by antibodies or used to visualise movement in cells, disrupts receptor function. This necessitated redesign and recloning of most of our constructs and called into question many published works on the function of these proteins. These data were published as Hurst et al 2018 Plant Phys. In the process of this work we have improved the overall assay method and this method has seen uptake by the wider eukaryotic research communities. This method also allows these assays to be performed in a single tube, medium-throughput manner for the first time, greatly enhancing speed and scalability of the assay. |
Exploitation Route | The main findings on changes in S-acylation state and reduction in signalling magnitude have potential use in breeding or GM manipulation of plant signalling mechanisms to generate novel phenotypes such as dwarfing or altered response to hormones. I am currently discussing suitable receptor associated traits with early stage barley and potato breeders to investigate this further. Planned BBSRC grant to investigate this. The findings on tagging receptor like kinases (Hurst et al 2018) have already caused a change in how the field approaches it's work, hopefully leading to a correction of the literature and better data in the future. Our improved S-acylation assay has been adopted widely by the field and we have had reports of medium throughput small molecule screening programs using it to assess S-acylation changes. |
Sectors | Agriculture, Food and Drink |
Description | BBSRC responsive mode |
Amount | £414,203 (GBP) |
Funding ID | BB/P007902/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2017 |
End | 06/2020 |
Description | Control of dynamic palmitoylation: Identification of de-palmitoylating enzymes and their substrates in plants |
Amount | £367,875 (GBP) |
Funding ID | BB/P007902/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2017 |
End | 06/2020 |
Description | Delivering the impossible - novel fatty acid delivery reagents to enable in vivo research and discovery |
Amount | £357,280 (GBP) |
Funding ID | BB/W000261/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2022 |
End | 05/2025 |
Title | Diels-alder scavenging of maleimides from aqueous solutions |
Description | Allow for precipitation free processing of samples during acyl-switch S-acylation assays |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | Invited book chapter in methods in molecular biology, invitation to biochemical society conference |
URL | https://www.biotechniques.com/BiotechniquesJournal/2017/February/Maleimide-scavenging-enhances-deter... |
Description | Turner |
Organisation | University of Manchester |
Department | School of Biological Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Assessment of S-acylation in a variety of cellulose synthase mutant plant lines. Experimental guidance. |
Collaborator Contribution | Generation of cellulose synthase mutant plant lines. |
Impact | Kumar M, Wightman R, Atanassov I, Gupta A, Hurst CH, Hemsley PA, Turner S. S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization. Science. 2016 Jul 8;353(6295):166-9. doi: 10.1126/science.aaf4009. |
Start Year | 2013 |
Description | Gatsby Masterclasses |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Gatsby plant science master class - 30 minute lectures delivered on key aspect of plant science research and future applications, with a 30 minute practical and open discussion. |
Year(s) Of Engagement Activity | 2017,2018 |
Description | Gatsby Plant Science Summer School |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Undergraduate students |
Results and Impact | Led discussion groups for undergraduate students interested in plant sciences and for secondary chool science teachers. Topics for discussion were 3 lectures given by prominant plant scientists and discussion led to list of questions to ask the presenter. Students reported that they were likely to engage more with plant sciences at University more in future while teachers contacted me afterwards to ask for clarification of more advanced ideas and how to incorporate them into their teaching. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.gatsby.org.uk/plant-science/programmes/gatsby-plant-science-summer-school |
Description | Plant Power |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Plant Power is an annual event that takes place at the University of Dundee Botanic Garden where various different groups and organisations participate with plant related activities/shows. A science strand is delivered by researchers from the Division of Plant Sciences at the University of Dundee and the James Hutton Institute. They presented different interactive hands-on activities related to their respective groups research to the visitors. These activities are either brand new or have been developed over a number of years at various events. The aim is to allow the public to learn about the research taking place locally and why this research is important. Various modes were used to communicate the research as shown by the diversity of activities e.g. use of games (pin the plant & botany trail); craft activities (chromosome modelling & lino printing); science experiments (raspberry DNA extraction); art (animating science). My research was represented in this program of work by discussions on plant adaptations to biotic and abiotic stress. Approximately 970 people came to the Botanic Garden for the event. They are generally family groups with young children (below 10 years of age). Feedback from the public indicated that they enjoyed all the activities. Researchers fed back the questions and statements from the public asked while interacting with them. Examples include people not realising that DNA is everywhere in a plant (DNA extraction activity); asking questions about how experiments are conducted with plants and the differences between plant and animal cells (chromosome modelling). Legacy: Follow on plans are for the activities developed for Plant Power to become formal borrow boxes. An overall Plant Science box aligned with the Curriculum for Excellence and investigating formally sharing activities via publications would be a subsequent step. |
Year(s) Of Engagement Activity | 2016,2017,2018,2019 |
Description | Plant Science Gatsby Lectures 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Speakers from the division of Plant Sciences delivered four lectures to secondary pupils and teachers on the topics of climate change and pharming. Lectures lasted roughly 40 minutes and were followed by hands-on activities and the chance for pupils to speak to scientists and postgraduate students about the topics. Students were very positive about the experience, the hands-on activities in particular, and shared that they had learned new information that was pertinent to the curriculum. Following the lectures two of the schools expressed interest in working with Life Sciences on further projects, and a collaborative project around sustainability and lab research will begin at the end of February 2020 with them. |
Year(s) Of Engagement Activity | 2019 |