Receptor-like kinase palmitoylation: resolving a crucial feature of plant cell signalling

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.

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.

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.