Using an Untargeted Transportomic Approach forExploration of Arabidopsis thaliana ABCGtransporters

Research output: Book/ReportPh.D. thesisResearch

Abstract

Transport proteins are essential for plant fitness and survival because they facilitate the distribution of metabolites important for growth, development, and defence. Within the plant, correct distribution of phytohormones ensures proper physiological responses, while the distribution of defence compounds such as glucosinolates is crucial for deterring herbivores and pathogens. In addition to internal translocation of substrates, transport proteins are also essential for plants to interact with their immediate environment. Exported metabolites are involved in responses to biotic and abiotic stresses, as well as the recruitment of a beneficial microbiome to the plant surface both above and below ground.However, despite the critical functions of transport proteins, there is a significant gap in the identification and characterisation of export proteins.

The research presented in this dissertation explores the Arabidopsis thaliana ATPBINDING CASETTE transporter subfamily G (ABCG) as candidate exporters of the glucosinolate defence compounds.

In the hunt for a glucosinolate exporter among the full-size ABCG transporters, we developed a reverse genetic screen based on diamondback moth bioassays and cuticle extractions. For biochemical analysis, we built a library of full-size ABCG transporters and explored the utilisation and efficacy of various expression hosts to be used for screening purposes.

To increase efficiency and breadth of studies of protein exporters, we developed an untargeted transportomic approach to screen for transport activity using Xenopus laevis oocytes. With a plant extract optimised for injection into the oocyte lumen, we screened for export activity of half-size ABCG transporters from Arabidopsis thaliana by analysing multiple endogenous metabolites simultaneously. With this method, we identified ABCG9 as a novel glucosinolate and hormone transporter and present the initial efforts to characterise its biochemical and physiological role.

With these results, we provide a method applicable for analysis of export activity of theoretically any given transporter. Ultimately, assigning substrates to protein exporters will help to identify new targets for transport engineering that can be used to improve crops and sustainable crop production.
Original languageEnglish
PublisherDepartment of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Number of pages230
Publication statusPublished - 2024

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