BOBASPIS · Integrated approaches to bioprocess optimization of bioactive substances production with plant in vitro systems

Final Activity Report Summary - BOBASPIS (Integrated approaches to bioprocess optimisation of bioactive substances production with plant in vitro systems)

1. Main objectives Plant cell walls are gaining increasing interest due to their importance in nature and emerging industrial applications. However, our current state of knowledge about its fundamental biology is far from adequate. Today we are still left with the decade-old questions of how the intricate biosynthesis is achieved and what exact roles the wall components play in host-microbe interactions. By employing novel approaches, this research proposal aimed at untangling the complex glycobiology of plant cells. Specific goals are the following: i) to elucidate the spatial and functional regulation of the wall-synthesising enzymes; ii) to explore the roles of the wall polysaccharides in host-microbe interactions. The results would advance our knowledge in cell wall biosynthesis that may help shape the stage for future agricultural industrial application of plant biomass, e.g. for increased production of food and bioenergy. 2. Work performed and scientific accomplishments Bioimaging of biosynthesis The previous studies have elucidated the global co-transcriptional regulation of the cell wall biosynthetic enzymes. In the post-genome era, the regulation at the protein levels, namely, protein-to-protein interactions and sub-cellular localisations is beginning to be addressed. Therefore, there was the goal of establishing a way to investigate the functional regulation of the cell wall biosynthetic enzymes in the cellular context. To this end, highly efficient molecular cloning and bioimaging tools were implemented in the research group. By using these tools, dozens of putative cell wall biosynthetic enzymes have been fused expressed with a variety of fluorescent proteins and their expressions in planta were observed by using confocal scanning laser microscopy. The main results were the following: - Cell wall biosynthetic enzymes show distinct sub-cellular localisation, e.g. ER and Golgi. Furthermore, they appear to show distinct sub-Golgi localisation. These results may suggest that the cell wall biosynthetic enzymes are spatially organised within Golgi. - By combining biochemical and bioimaging analyses, we showed that the Golgi-localisation of a glycosyltransferase involved in homogalacturonan biosynthesis was strictly dependent upon the presence of its homolog. By using bifluorescent molecular complementation, we were further able to show that the two proteins interact inside Golgi. These results provided the first evidence of protein complex formation involved in pectin biosynthesis and provided a biological explanation for the observed protein complex formation. Conclusion: the implementation of bioimaging method has led to new insights into protein complex formation and spatial organisation of cell wall biosynthesis. - Host-microbe interaction via cell wall. Pectin is typically considered as a gelling material or 'space-filler' within the cellulose-hemicellulose cross-linking network of the cell wall. Except for the elicitor function of oligogalacturonides released upon pathogen attack, roles of pectin in interaction with microbes are not well known. I have investigated the pathogen susceptibility of two pectic mutants defective in arabinan and xylogalacturonan biosynthesis. To this end, I first implemented the patho-assays of the necrotic fungal pathogen Botrytis cinerea and the biotrophic bacterial pathogen Pseudomonas syringae in the research group. The main results are the following: - Arabinan is involved in interaction with B. cinerea, but not with P. Syringae. - Xylogalacturonan may be involved in interaction with P. syringae but not with B. cinerea. Conclusion: we have identified for the first time to our best knowledge that arabinan is likely to play role in pathogen resistance. The results may indicate more wide roles of pectic polymers in host-microbe interaction than previously expected.

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