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SugarOsmoSignalling Report Summary

Project ID: 657835
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - SugarOsmoSignalling (Analysis of sugar- and osmo-signalling mechanisms in cell wall integrity maintenance)

Reporting period: 2016-03-16 to 2018-03-15

Summary of the context and overall objectives of the project

Plant cell walls represent the largest source of renewable biomass and significant scientific and economic efforts are aimed at modifying cell wall composition in a controlled manner to facilitate bioenergy production. In addition, cell walls form a defence barrier against pathogen infection, making them also attractive targets for crop protection. However, cell walls are not invariant, but dynamically adapt to different growth and stress conditions to ensure survival and adaptation of the plant to the environment. Recently, it has become obvious that they also adapt to targeted changes and neutralize them, hampering optimization of food crop performance and facilitation of bioenergy production from plants in a sustainable manner. The mechanism enabling this adaption, called the plant cell wall integrity (CWI) maintenance mechanism, appears to be functionally conserved across the plant kingdom and can be also found in other organisms, such as yeast.
In this project, we investigated the mode of action of the plant CWI maintenance in the flowering plant Arabidopsis thaliana, which is frequently used as green test tube to facilitate targeted follow up studies in crop species. We were interested in understanding how plants detect cell wall damage and how the initial signal is passed on to activate downstream responses. Previously it was shown that carbohydrate metabolism is strongly affected by cell wall damage. Here we aimed to elucidate how metabolic signalling could be involved in the CWI maintenance mechanism. Since cell wall damage occurs frequently during pathogen infections, another objective of our work was to elucidate the coordination of pathogen defence with CWI maintenance. Finally, we aimed to increase our knowledge on the influence of osmotic pressure on cell wall damage responses.
In summary, we identified novel components required for osmo-sensitive CWI maintenance signalling and successfully gained insights into the mechanism coordinating pathogen defence with CWI maintenance. This increased understanding of the regulatory mechanisms will facilitate generation of novel crop varieties, which allow food and bioenergy production in a more sustainable manner.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has resulted in several major advances. We have defined a core group of signalling components required for plant CWI signalling. Interestingly, for a subset of them homologues have been identified in food crops like rice and maize where loss of function alleles affect growth and give resistance against pathogens. This indicates the direct relevance and potential the results produced here have for improving crop performance. Simultaneously, the mode of action of the mechanism itself has been further elaborated and we have managed to develop a model explaining how the activity of the CWI maintenance mechanism is integrated with plant immune signalling. This provides insights into the processes enabling plants to adapt their responses and growth to environmental change. The mechanism enables them to either respond to abiotic or biotic-stress derived challenges in a highly specific manner increasing their chances of survival. This novel knowledge has obvious application potential, since it will enable the scientific community to solve problems arising from plant plasticity in a knowledge-driven manner.

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