Cell Wall Metabolism · Functional analysis of osmo-sensitive signalling processes regulating the metabolic response to environmental stress

Functional analysis of osmo-sensitive signalling processes regulating the metabolic response to environmental stress

As the world population approaches ten billion, issues like climate change, scarcity of oil and limited availability of land and water are challenging the planet's capacity to produce enough food for everyone. According to World Bank, food production must increase by at least 50% over the next 30 years in order to meet the demands of the growing population and its rising affluence. In addition, green transportation fuels produced from cellulosic biomass are becoming more and more important as sustainable alternative to fossil fuels. Plant cell walls represent the first line of defence against environmental stress while representing the bulk of the biomass used for bioenergy production. Their composition and structure influences both efficiency of bioenergy production and resistance of food crops to biotic and abiotic stresses. Despite the fact that cell walls have been extensively studied, our understanding of the mechanisms regulating cell wall formation and maintenance is still very limited. In the last few years a cell wall integrity mechanism has been discovered in plants. The mechanism monitors the state of the wall and initiates changes in cell wall structure and composition to maintain functional integrity. This research project contributes to the dissection of this mechanism by analysing the cross-talk between cell wall stress perception and primary metabolism. Two main aims addressed with this project are: characterisation of the role of sugar signalling in cell wall damage response (CWD) and identification of proteins mediating the response to CWD. In this study, the pre-emergence herbicide isoxaben was used to cause CWD through cellulose biosynthesis inhibition (CBI) in Arabidopsis seedlings, and the response was studied using two approaches: FRET-based nanosensor technology and phospho-proteomics experiments. The first approach, FRET-based nanosensor technology was applied to investigate the function of hexoses during CWD response, by monitoring changes in soluble sugars with subcellular resolution in the 7 days old Arabidopsis plants. Those plants are crosses between Arabidopsis mutants (tonoplast monosaccharide transporter1-2-3 (tmt1-2-3), jasmonic acid resistant1 (jar1), mechanosensitive channel of small conductance-like (msl), murus4 (mur4), de-etiolated1 (det1) and de-etiolated3 (det3)) and plant lines expressing nanosensors specific for glucose and sucrose with different sensitivities targeted to the cytoplasm (Glu 3.2mM; Glu 600μM; Glu 2μM; Glu 170nM; Suc 90μM) and apoplast (Glu 3.2mM; Glu 2μM; Glu 170nM). Before performing FRET analysis, crosses between nanosensors and mutant plants (triple:tmt-1-2-3; quintuple: msl4-5-6-9-10; jar1; mur4; det1 and det3) were genotyped. PCR analysis of the genotype of analysed crosses resulted in the detection of homozygous mutant seedlings expressing cytosolic FRET nanosensors in rdr6-11 background, which eliminates silencing and results in high fluorescence levels. Analysis of crosses between mutants and apoplastic nanosensors resulted in plants in rdr6-11 background but lacking fluorescence. Therefore, those plants were excluded from further analysis. In order to monitor distribution of soluble sugars in vivo in FRET, epidermal cells of the root elongation zone of 7 days old Arabidopsis seedlings were tested. Tested mutant plants were growing on solidified hydroponic media supplemented with sugar to ensure high levels of nanosensor expression. In the next step, seedlings were transferred onto media lacking sugar for 2-3 days prior imaging. To test the cytosolic sensor activity, seedlings (from plates supplemented with/or lacking sugar) were perfused with media containing 600nM isoxaben. Obtained results suggest that isoxaben treatment causes changes in FRET (CFP/YFP) ratio, which differ depending on the seedlings analysed. Seedlings growing on media supplemented with sugar (tested for: FLIP-Glu600µM; FLIP-Glu2µM; FLIP-Suc90µM; FLIP-jar1 x Glu600µM; FLIP-tmt1-2-3 x Suc90µM; FLIP-tmt1-2 x Glu600µM) seemed to respond to isoxaben with a decrease in FRET ratio, while those on sugar starvation (tested for: FLIP-Glu600µM; FLIP-jar1 x Glu600µM) showed slight increase in the ratio. FRET analysis of crosses between msl mutants and cytosolic nanosensors specific for glucose 600µM and 2µM growing on media supplemented with sugar gave two opposite responses. In case of FLIP-msl9-10 x Glu600µM a slight increase in FRET ratio was observed, similar to that seen with seedlings growing in the absence of sugar. Interestingly, FLIP-msl4-5-9-10 x Glu2µM showed after isoxaben treatment a gradual decrease in FRET ratio, not observed before. Those opposite responses to isoxaben treatment could be caused by different combination of msl mutations. To sum up, the results obtained in this study seem to suggest that isoxaben treatments have an effect on FRET ratio of analysed plants. However, in order to draw further conclusions, it is first necessary to reproduce the preliminary results and then explore alternative possibilities in FRET analysis. The second approach involved phospho-proteomics experiments to identify and functionally characterise novel proteins implicated in cell wall related processes. For this part of the project liquid cultures of Arabidopsis Col-0 and ixr1-1 seedlings (to ensure specificity) treated with mock, isoxaben, 5% PEG (osmotic support) and isoxaben+5% PEG were taken after 15 and 60 minutes of treatment. In order to prepare samples for LC-MS analysis, first soluble and membrane-associated proteins were extracted from plant material, followed by in-solution tryptic protein digest and phosphopeptide enrichment. Due to the fact that the purchase of new LC-MS equipment at Imperial College London (planned for 2012) was delayed in time, obtained peptides were sent for analysis to the Proteomics Facility at the Norwegian University of Science and Technology (NTNU) in Trondheim (Norway), where project supervisor Dr Hamann accepted an Associate Professor position. However, the LC-MS analysis was not performed as anticipated due to the withdrawal of the project supervisor. Thus, MC fellowship was terminated after 14 months duration of the project and did not generate more results. If continued, the outcome of this research project could generate the foundation for understanding a novel, turgor-based regulatory mechanism that apparently coordinates cellulose biosynthesis with primary metabolism and photosynthesis. Primary metabolism and photosynthesis in turn give rise to the carbohydrates forming the building blocks of plant cell walls/lignocellulosic biomass. Therefore, this research could help to develop the underpinning science to support novel strategies for biomass quality and quantity improvement. Applications resulting from this research could increase the efficiency of bioenergy production and resistance of food crops to environmental stresses.

Data: CORDIS, © European Union