Cell wall feedback signalling in Arabidopsis

The cell wall surrounds plant cells like a rigid armour, limiting cell expansion while at the same time giving the plant body strength and resilience. The network of cellulose and other polysaccharides is one of the biggest investments of assimilated carbon in plants. It is also a key determinant of susceptibility to pathogens. We are beginning to understand how the different components are made, but we don't understand at all how plants sense the status of their wall, whether it is intact and whether there is the right balance of the different components. How is information on the integrity of the wall transmitted into the cell? What are the immediate responses to cell wall damage on the molecular level? And what happens to the normal growth and development of the plant if we interfere with this sensory pathway? Is there feedback control of the production of the different cell wall polysaccharides? How is cell wall sensing related to pathogen detection? Finding answers to these questions of outside-inside communication will help us to better understand the fundamental workings of a plant cell. For both plants and animals, the matrix of polysaccharides and proteins that surrounds all cells is a lot more than just glue to hold tissues together- it has a major influence on what the cells do and what they develop into as the organism grows. In animals, tumour cells that 'get out of touch' with this extracellular matrix form metastases. In yeast, with a cell wall more comparable to that of plants, the surveillance system for wall integrity is hard-wired to many developmental and stress response processes. We have much indirect evidence that this is also the case for plants, but no molecular details. I want to study in a cell culture what happens when cellulose production is blocked with a herbicide. In growing cells, this soon leads to a structural defect of the cell wall. I expect that most of the signalling response happens directly beneath the cell surface, and that much of it is based on taging proteins with phosphate -a universal signal to change a protein's activity. Getting a global view of which proteins become tagged will give me a broad understanding of what happens in the cell and how it compensates for the damage. After identifying the phosphate-tagged proteins that respond to cell wall damage, I want to use genetics to understand what the proteins do and how the wall surveillance system influences development, e.g. with plants that have one or several of the genes for these proteins deleted. I will also compare the cell wall damage response with the response to microbial signals (an ongoing project) to understand how the different sensory systems are linked. There is great interest in manipulating the composition of the cell wall, for example to enhance disease resistance, or to facilitate processing of plant material for biofuel and other industrial purposes. However, many desirable changes have a negative impact on plant growth. Frequently, this may not necessarily be because of changes in the physical stability of the wall, but because the different structure triggers an alarm. In the long term, interfering with this surveillance system to make the plants more tolerant to changes in wall structure would be a practical contribution to improving biomass crops.

How do plant cells monitor the status of their cell wall? How do they perceive structural changes induced by growth, or damage from pathogen attack or herbicides? I propose a phosphoproteomic approach for the discovery of proteins that participate in wall feedback signalling and/or execute the cellular responses. The discovery part of the project is a large-scale profiling of protein phosphorylation at the plasma membrane before and after cell wall disruption by isoxaben. Plasma membranes are isolated from control and treated suspension-cultured cells, inverted and digested with trypsin to yield peptides. Control- and up to three treated samples are labelled separately with iTRAQ reagents and then pooled. Phosphopeptides are isolated from this mixture by affinity chromatography, and then separated, quantified and identified by mass spectrometry. This approach that has been very successful for responses to microbial elicitors in the suspension culture. Reverse genetic analysis of the identified proteins will include cell wall characterisation of knockout mutants, study of growth and anatomical phenotypes, genetic interactions with known cell wall mutants, and the response to artificial cell wall damage. Finally, a more detailed structure-function analysis will be performed for proteins that emerge as potential key regulators. This includes the study of subcellular localisation, interacting proteins and the role of phosphorylation for their function. Another aim is to identify the upstream kinase(s) and to establish the network that connects the different phosphoproteins. The comparison of elicitor responses (previous work) and cell wall damage responses on the level of phosphorylation profiles will help to design experiments addressing cross talk between cell wall integrity and microbial defence pathways. Taking in more responses will help to reach an integrated view of plant stress signalling networks.