Biosynthesis and transport of flavonol sophorosides in Arabidopsis thaliana anthers Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Herrn Stephan Grunewald geb. am 10.05.1989 in Berlin Gutachter: 1. PD. Dr. Thomas Vogt 2. Prof. Dr. Ingo Heilmann 3. Prof. Dr. Enrico Martinoia Verteidigungsdatum: 18.01.2021 II Table of contents Table of contents List of abbreviations ....................................................................................... V List of tables ................................................................................................. VII List of figures............................................................................................... VIII 1 Introduction ............................................................................................. 1 1.1 The role of transport in plant secondary metabolism ............................................................ 1 1.2 Transporters of specialized metabolites ................................................................................. 2 1.2.1 ABC-transporters ............................................................................................................. 2 1.2.2 MATE-transporters .......................................................................................................... 3 1.2.3 The nitrate/peptide family (NPF-transporters) ............................................................... 4 1.3 Flavonoids: highly diverse compounds ................................................................................... 5 1.4 Biosynthesis of flavonol-3-O-sophorosides in A. thaliana ...................................................... 6 1.5 Flavonoid glycosylation ........................................................................................................... 8 1.5.1 Biological relevance of flavonoid glycosylation ............................................................... 8 1.5.2 Plant glycosyltransferases ............................................................................................... 9 1.5.3 Flavonol sophorosides ..................................................................................................... 9 1.6 Tapetum and pollen – A special relationship ........................................................................ 10 1.6.1 Angiosperm pollen development .................................................................................. 10 1.6.2 A special cell layer: the tapetum ................................................................................... 11 1.6.3 Composition of the pollen wall ..................................................................................... 13 1.7 Flavonoid transport in plants ................................................................................................ 14 1.8 Aim of this investigation ........................................................................................................ 17 2 Materials and methods ........................................................................... 18 2.1 Materials ................................................................................................................................ 18 2.1.1 Chemicals and biochemical reagents ............................................................................ 18 2.1.2 Media ............................................................................................................................. 18 2.1.3 Vectors ........................................................................................................................... 18 2.1.4 Bacterial strains ............................................................................................................. 19 2.1.5 Preparation of electrocompetent cells ......................................................................... 20 2.1.6 Plants and growth conditions ........................................................................................ 20 2.1.7 Isolation of T-DNA insertion lines .................................................................................. 20 2.2 Molecular methods ............................................................................................................... 21 I Table of contents 2.2.1 Isolation of genomic DNA .............................................................................................. 21 2.2.2 Isolation of plasmid DNA ............................................................................................... 21 2.2.3 Polymerase chain reaction (PCR) .................................................................................. 21 2.2.4 Agarose gel electrophoresis .......................................................................................... 22 2.2.5 RNA extraction and cDNA synthesis .............................................................................. 22 2.2.6 Quantitative real-time polymerase chain reaction (qRT-PCR) ...................................... 23 2.2.7 Classical cloning for recombinant protein expression .................................................. 24 2.2.8 Golden Gate Cloning ...................................................................................................... 25 2.2.9 Restriction analysis ........................................................................................................ 28 2.2.11 Site-directed mutagenesis ............................................................................................. 29 2.2.12 Sequencing and in silico analysis ................................................................................... 29 2.2.13 Transformation of electrocompetent cells ................................................................... 30 2.2.14 Generation of transgenic Arabidopsis lines .................................................................. 30 2.3 Microscopic methods ............................................................................................................ 31 2.3.1 Subcellular localization .................................................................................................. 31 2.3.2 Transient expression in N. benthamiana leaves ........................................................... 31 2.3.3 Isolation of protoplasts ................................................................................................. 32 2.3.4 Transformation of protoplasts ...................................................................................... 33 2.3.5 Promotor-GFP localization ............................................................................................ 33 2.3.6 Immunolabelling of anther cross-sections .................................................................... 34 2.3.7 Transmission electron microscopy (TEM) ..................................................................... 35 2.4 Protein biochemical methods ............................................................................................... 35 2.4.1 Protein concentration determination ........................................................................... 35 2.4.2 SDS-PAGE ....................................................................................................................... 35 2.4.3 Immunoblot analysis ..................................................................................................... 36 2.4.4 Heterologous expression and partially purification of glycosyltransferases ................ 37 2.4.5 Standard flavonol glycosyltransferase assays ............................................................... 38 2.4.6 Preparation of 14C-labelled flavonol glucosides ............................................................ 38 2.4.7 Transporter uptake assays with 14C-labelled flavonol glucosides ................................. 39 2.5 Analytical methods ................................................................................................................ 40 2.5.1 Harvesting of pollen material from Arabidopsis ........................................................... 40 2.5.2 Preparation of pollen and anther extracts .................................................................... 40 2.5.3 High performance thin-layer chromatography ............................................................. 40 2.5.4 High performance liquid chromatography .................................................................... 41 2.5.5 Gas chromatography-mass spectrometry ..................................................................... 41 II Table of contents 2.6 Biological assays .................................................................................................................... 41 2.6.1 Pollen viability assay ...................................................................................................... 41 2.6.2 In vitro pollen germination assay .................................................................................. 42 3 Results .................................................................................................... 43 3.1 The flavonoid glycosyltransferases UGT78D2 and UGT79B6 ................................................ 43 3.3 Transport and biosynthesis of flavonol sophorosides........................................................... 45 3.3.1 UGT79B6 is highly expressed in tapetal cells ...............................................................