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Identification and characterisation of plant Golgi biosynthetic multiprotein systems

Periodic Reporting for period 1 - GolgiBiosSys (Identification and characterisation of plant Golgi biosynthetic multiprotein systems)

Reporting period: 2016-05-01 to 2018-04-30

The crops of the future will need to provide sufficient food, feed and fuel necessary for the sustainment of the estimated 9 billion people by 2050. Over the coming decades the change in climate will impact all aspects of plant biology, as a result of increased atmospheric CO2, higher temperature and changing precipitation cycles. Knowing that 90% of all living biomass is plant derived, its stability and usability is a sizeable challenge for the future. The growth of the bioeconomy in such climate change scenario requires new knowledge in order to produce more resistant crops and to convert the plant biomass into useful products whilst maintaining ecosystem stability: new knowledge of the molecular biology of plants, especially on systems level. The application of molecular systems biology models on the interplay of mater and energy within the plant metabolism is essential to such knowledge. In particular, experimental investigations on the portion of the proteome involved in biomass production such as cellulose are necessary, both to advance our knowledge and to develop and inform further models of systems behavior.
I use Arabidopsis thaliana as a model organism with the main goal of understanding the proteomic organization of the Golgi apparatus. Probably the most significant functional feature of the plant Golgi apparatus is synthesis of extracellular polysaccharides. These polymers account for at least a third of all renewable organic carbon on the planet and are not only major components of many ecosystems, but also components of our food, fuel, building material, paper and fiber, as well as targets in the search for renewable energy. In terms of its biosynthetic aspects, previous studies have identified enzymes involved in the synthesis of diverse polysaccharides, with evidence that these enzymes are organized in protein complexes as molecular machineries. The organization, stoichiometry and composition of these protein complexes impact their properties as well as the efficiency of their function.
The overall research objective of this project was the application of state of the art quantitative mass spectrometry technologies for the proteomic measurements of plant Golgi protein complexes in order to understand the details of its biosynthetic machineries. More precisely, the first research objective of the proposal aims at investigating the membrane protein interactions in the plant Golgi. The second objective of the proposal consists of identifying specific interacting partners by affinity purification mass spectrometry for partially characterized Golgi membrane protein complexes, such as the cellulose synthase complex. In addition, the training objective of the project is the acquisition of detailed knowledge on quantitative mass spectrometry tools, such as the SWATH-MS technique and related data analysis tools. I also obtained extensive didactic experience by Master student supervision as well as teaching opportunities within the ETH Bachelor student block courses and the international DIA/SWATH course. I gained ample experience in scientific communication, by involvement in organization of scientific meetings, session and outreach activities.
In order to study the protein interaction landscape of Arabidopsis, I apply protein separation techniques in conjunction with state of the art mass spectrometry technologies for the measurement of protein elution profiles and inference of protein complexes. The recent SWATH-MS methodology developed by the host laboratory offers comparable specificity and selectivity as targeted workflows, delivers deeper proteome coverage and high-throughput acquisition. The data generated with this approach was analyzed by computational tools developed in the host laboratory that enable inference of protein complexes.

I carried our research on the establishment of a comprehensive tools in order to establish the objectives of the proposal, namely I focused on the generation of a pan-Arabidopsis spectral library that contains spectrum query parameters in order to analyze the mass spectrometry data obtained from SWATH acquisition. The generation of such large scale library required gathering sizable data from different organs and tissues, as well as different growth conditions. In summary, I collected data from 496 mass spectrometry measurements and compiled a repository that covers 13.919 unique proteins which accounts for 50.6% of the total Arabidopsis proteome. This resource will contribute significantly to the simplified and reproducible analysis of Arabidopsis proteome samples across studies and laboratories. The results from this task are currently being prepared for publication and the repository data will be made available as an open resource. This repository is of crucial relevance to the further study on the protein interaction landscape, which I conducted by using a technique that allows high-throughput assignment of protein interactions. The methodological design is based on separation of protein complexes in conjunction with SWATH MS. The separation of intact complexes was achieved by Size Exclusion Chromatography (SEC).

Additionally, I obtained plant lines for the study of specific Golgi membrane protein complexes, namely the cellulose synthase and the xylan synthase complexes. The work on identifying these biosynthetic machineries was carried within a supervision of a Master student and led to the discovery of novel interacting partners involved in the assembly of the cellulose synthase machinery which provides details in the knowledge on plant cell wall synthesis. Additionally I performed affinity purification of another Golgi-localized complex: the xylan synthesis complex and the obtained data support the idea of distinct primary and secondary cell wall xylan synthesis complexes.
Primarily this project delivers a resource in from of a large scale peptide spectral library that allows for worldwide application of the SWATH mass spectrometry and broadly the DIA technology for reproducible and quantitative measurements of the Arabidopsis proteome. The database resulting from this work presents a major advance in making Arabidopsis accessible to highly reproducible, quantitatively accurate proteomic measurements. This is particularly important because for this species the availability of antibodies for protein quantifications is very sparse. This is an essential step to accelerate the field of plant proteomics, which usually lags behind the mammalian or yeast proteomics.

Secondarily, the protein interaction work on the cellulose synthase complex has also led to progress beyond the state of the art. The results of the affinity purification of the complex have pointed towards novel protein interactors involved in the cellulose synthesis complex that are currently investigated in detail by collaborators.

Tertiary, and most significantly, based on the project’s progress we believe that the methodology of size exclusion chromatography with SWATH acquisition applied on Arabidopsis leads to a significant understanding of plant biosynthetic machineries and eventually of their activities. This will deepen our current understanding of the plant molecular biology, especially on systems level, in order to develop crops that are resistant to environmental challenges, as well as varieties of plants for production of biofuels. This is a challenge that has enormous socio-economic impact.
Assembly + trafficking of CSC to the plasma membrane