Work in this project used an Arabidopsis thaliana seedling model system, where CWI is impaired either by chemical inhibition of cell wall synthesis, or enzymatic degradation of cell walls. We used this model system to screen for central components of the plant CWI maintenance mechanism, using both forward and reverse genetics approaches. In a forward genetics approach we screened a collection of chemically mutagenized Arabidopsis seedlings for a combination of pathogen defence and CWI signalling phenotypes. Mutants were first selected based on strong pathogen resistance and then treated with the chemical isoxaben, which inhibits biosynthesis of the main load bearing cell wall polymer, cellulose. Mutant lines with increased resistance and altered isoxaben response have been isolated. In parallel, a comprehensive reverse genetics analysis was conducted, where mutants for more than 30 candidate genes, hypothesized to be required for CWI signalling, were investigated using standardized conditions and readouts. Quantitative data for phytohormone accumulation, root lignification and root growth inhibition were collected and integrated by phenotypic clustering. This dataset revealed relative contributions of candidate genes to the mechanism and highlighted that specific signals seem to be perceived at the cell wall / plasma membrane interface upon cell wall damage. Further experiments suggested that mechanical sensing is of major importance for cell wall damage detection, whereas sensing of cell wall fragments did not play an important role in our system.
The phenotypic clustering showed that genes required for pattern-triggered immunity are inhibiting cell wall damage signalling. In addition, RNA-Sequencing of isoxaben-treated seedlings indicated that a danger-associated molecular pattern response is triggered through plant elicitor peptides (Peps). Indeed further experiments showed that Pep precursor genes are transcriptionally induced and the corresponding proteins secreted into the apoplast. To understand the function of Pep signalling during CWI impairment, seedlings exhibiting or lacking functional Pep receptors at the plasma membrane were co-treated with synthetic Peps and isoxaben. These experiments showed that Pep detection leads to reduced isoxaben-dependent phytohormone accumulation. As Pep signalling is also induced during pathogen-associated molecular pattern-triggered immunity, where it acts as defence signalling amplifier, our data reveal a mechanism potentially fine-tuning immune signalling and CWI signalling.
In order to investigate the role of metabolic signalling for CWI signalling, we analysed isoxaben responses in seedlings affected in trehalose-6-phospate metabolism (T6P), glycerol-3-phosphate metabolism (G3P) and nitrogen metabolism, respectively. Altered T6P or G3P metabolism affected isoxaben responses, but no clear correlation with metabolite abundance could be found. Instead, loss of nitrate reductase function strongly repressed isoxaben effects on hormone accumulation, lignification and cell cycle gene expression. The data suggest that alterations in T6P and G3P content influence the extent of isoxaben responses, while nitric oxide signalling through nitrate reductase activity might be essential for CWI maintenance.
Hyperosmotic conditions reduce the magnitude of cell wall damage responses. We show that cellulose biosynthesis inhibition and enzymatic degradation cause different structural cell wall damage, but similar osmo-sensitive responses. Several plasma membrane-localized proteins are known to be required for detection of osmotic stress. However, none of the osmosensors tested in our experiments was required for osmotic suppression. Current evidence suggests that mechanical signalling is of major importance for this osmo-sensitive mechanism.
Data obtained during the project period have been presented at several international conferences in Norway, Austria, the United Kingdom, Finland and Germany.
