Congratulations to Lukas Schulz for receiving the prize for the best Master Thesis in the plant field in Germany of the year 2022 awarded by the German Botanical Society (DBG).
Lukas Schulz's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Technische Universität München in the year 2022
Title: "Biochemical and Biophysical Characterization of PIN-FORMED8 Protein from Arabidopsis thaliana"
Schulz established a new electro-physical method to characterize transport proteins, characterized the biochemical and biophysical properties of a PIN-FORMED (PIN) Auxin exporter thoroughly and parts of his work were published in a Nature article (DOI: 10.1038/s41586-022-04883-y).
The phytohormone auxin controls essentially all aspects of plant growth and development. The most abundant auxin is Indole-3-acetic acid (IAA). It is distributed through the plant by long distance transport in the phloem and by polar auxin transport (PAT) from cell to cell. IAA is a weak acid and can therefore enter the cell by passive diffusion at a pH of 5. Once in the cell, at neutral pH, the IAA molecules dissociate and auxin efflux carriers are mandatory for export out of the cell. The PIN-FORMED (PIN) efflux carriers are the key players in PAT. In Arabidopsis thaliana, there are eight different PIN proteins divided into three subgroups, the canonical plasma membrane- localized PINs, the noncanonical ER-localized PINs and the intermediate PIN6. Recently, the structure of the non-canonical PIN8 was described in the publication "Structures and mechanism of the plant PIN-FORMED auxin transporter", revealing that PINs form dimers and operate by an elevator-mechanism. I contributed the biochemical and biophysical characterization of PIN8 by solid supported membrane (SSM)-based electrophysiology, which were a part of this thesis to the publication.
To better understand the SSM-electrophysiology measurements, a theoretical model of the proteoliposomes and a mathematical derivation of the electrogenic transport was proposed. Additionally, to further optimize the assay, the sensor preparation process was critically re-evaluated and optimized. A focus of this thesis was also to distinguish between binding and transport current response of the PIN8 substrates IAA and N-1- naphthylphthalamic acid (NPA) in SSM electrophysiology. The results show that the inhibitor NPA induces a binding current, whereas IAA induces a mix of transport- and a binding current. Additionally, binding, transport and inhibition constants were investigated and a rather low apparent affinity for IAA was determined. In order to understand the transport mechanism of PIN8 better, transport properties of PIN8 mutants were determined. The results were additionally confirmed in a Xenopus laevisoocyte-based efflux assay, showing a high degree of correlation between both assays. With the help of the PIN8 mutants and a substrate screening, the substrate specificity of PIN8 was investigated, leading to insights of the properties required to interact with the PIN8 binding pocket.
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Lukas Schulz conducted this work at the Chair of Plant Systems Biology in the working group of PD Dr. Ulrich Z. Hammes.
