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Biophysikalische Untersuchung Von Phloem-Lokalisierten Carriern Und Kaliumkanälen Und Deren Interaktion Im Modellsystem Der Xenopus Oozyte

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Biophysikalische Untersuchung Von Phloem-Lokalisierten Carriern Und Kaliumkanälen Und Deren Interaktion Im Modellsystem Der Xenopus Oozyte Biophysikalische Untersuchung von Phloem-lokalisierten Carriern und Kaliumkanälen und deren Interaktion im Modellsystem der Xenopus Oozyte Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Dietmar Geiger aus Coburg Würzburg 2004 Eingereicht am: 13.10.2004 Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Ulrich Scheer 1. Gutachter: Prof. Dr. R. Hedrich 2. Gutachter: Prof. Dr. A. J. E. van Bel 3. Gutachter: Prof. Dr. E. Bamberg Tag des Promotionskolloquiums:…………………………. Doktorurkunde ausgehändigt am:……………………………… Inhaltsverzeichnis Inhaltsverzeichnis 1. Einleitung...............................................................................................1 1.1 Das Phloem .......................................................................................1 1.2 Funktionen des Phloems......................................................................4 1.3 Phloembeladung.................................................................................5 1.3.1 Symplastische Phloembeladung......................................................7 1.3.2 Apoplastische Phloembeladung.......................................................9 1.3.3 Sekundär aktive Transporter des Phloems .....................................10 1.3.4 Apoplastische Phloementladung....................................................15 1.3.5 Phloembeladung durch Polyoltransporter .......................................16 1.4 Phloem-lokalisierte Kaliumkanäle .......................................................18 1.4.1 Kaliumkanäle der KAT1-Unterfamilie .............................................22 1.4.2 Die AKT2/3-Unterfamilie..............................................................24 1.5 H+-ATPasen energetisieren den Transport über die Phloemmembran ......27 1.5.1 Transportmechanismus und Regulation von H+-ATPasen..................29 1.5.2 Physiologische Bedeutung der H+-ATPasen beim Phloemtransport.....31 1.6 Zielsetzung......................................................................................33 2. Ergebnisse ...........................................................................................35 Kapitel I: The Pore of Plant K+-Channels is Involved in Voltage and pH Sensing: Domain-Swapping between Different K+ Channel α-Subunits ..........35 Kapitel II: Outer Pore Residues Control the H+ and K+ Sensitivity of the Arabidopsis Potassium Channel AKT3 .......................................................46 Kapitel III: Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis.......................................................................57 Kapitel IV: The K+ Channel KZM1 Mediates Potassium Uptake into the Phloem and Guard Cells of the C4 Grass Zea mays ................................................69 Kapitel V: Poplar Potassium Transporters Capable of Controlling K+ Homeostasis and K+-Dependent Xylogenesis .............................................79 Kapitel VI: Differential Expression of Sucrose Transporter and Polyol Transporter Genes during Maturation of Common Plantain Companion Cells ..93 Kapitel VII: The new Arabidopsis transporter AtPLT5 mediates H+-symport of numerous substrates including myo-inositol, glycerol and ribose................ 108 I Inhaltsverzeichnis Kapitel VIII: Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under Control of Sucrose Gradient and pmf........... 138 3. Ergebnisse unveröffentlichter Arbeiten................................................... 160 Kapitel IX. Elektrophysiologische Charakterisierung von KAT2 ................... 160 1. Funktionelle Charakterisierung in Xenopus Oozyten........................... 160 2. Spannungsabhängigkeit und Selektivität von KAT2............................ 161 3. Regulation von KAT2 durch den extra- und intrazellulären pH-Wert..... 164 4. Diskussion.......................................................................................... 167 4.1 Charakterisierung von KZM1 aus der KAT1-Unterfamilie der pflanzlichen Shaker-Kaliumkanäle ........................................................................... 169 4.2 Biophysikalische Charakterisierung von KAT2..................................... 172 4.3 Einfluss von AKT2/3 auf die Phloemphysiologie .................................. 174 4.3.1 Struktur-Funktionsanalyse der Porenregion von AKT3 durch den Porenaustausch zwischen AKT3 und KST1............................................ 174 4.3.2 Identifikation der molekularen Grundlage der pH- und Kalium- sensitivität von AKT3......................................................................... 175 4.3.3 Bedeutung von AKT2/3 für die Phloemphysiologie von Arabidopsis . 177 4.4 Charakterisierung von PTK2, dem AKT2/3 orthologen Kanal aus dem Kambium der Pappel ............................................................................ 180 4.5 Be- und Entladung von Saccharose durch ZmSUT1............................. 182 4.5.1 Transportkinetiken von ZmSUT1 in Abhängigkeit von Saccharose, pH- Wert und der Spannung..................................................................... 182 4.5.2 Reversibilität des Transports von ZmSUT1................................... 184 4.6 Biophysikalische Analyse des Phloem-lokalisierten Polyoltransporters PmPLT1 .............................................................................................. 187 4.7 AtPLT5, ein unspezifischer Polyoltransporter aus Arabidopsis ............... 191 5. Zusammenfassung .............................................................................. 195 6. Summary ........................................................................................... 199 7. Referenzen......................................................................................... 203 8. Anhang............................................................................................ 232 Veröffentlichungsverzeichnis ................................................................. 232 Lebenslauf .......................................................................................... 234 Eidesstattliche Erklärung....................................................................... 235 Danksagung........................................................................................ 236 II 1. Einleitung 1. Einleitung Erste Spuren pflanzlichen Lebens an Land konnten auf ein Alter von 475 Millionen Jahren datiert werden (Wellman et al., 2003). Beim Übergang von der aquatischen Lebensweise zum Leben an Land mussten sich die Pflanzen einer Reihe neuer Anforderungen stellen. Es begann eine Coevolution von Organen, welche die Anpassung an das Landleben ermöglichten. Wurzeln und Rhizome stellten den Bedarf an Wasser und Nährstoffen aus dem Boden sicher, während sich oberirdische Organe zu Blättern ausbildeten, um Licht für die photosynthetische Reduktion von Kohlendioxid einzufangen. Mit zunehmender Komplexität und Größe der Landpflanzen reichte ein reiner Zell zu Zell Transport durch einfache Diffusion nicht mehr aus, um Photoassimilate effizient umzuverteilen und um die gesamte Pflanze mit Wasser und Mineralien ausreichend zu versorgen. Abb. 1.1: GFP-markierte Phloemgefäße in Arabidopsis GFP-Fluoreszenz in den Phloemgefäßen eines Arabidopsis Rosettenblattes von AtSUC2-Promotor::GFP Pflanzen (von Prof. Sauer erhalten). 1.1 Das Phloem Gefäßpflanzen entwickelten Ferntransportbahnen, um einen effektiven Austausch dieser Absorptions- und Assimilationsprodukte über große Distanzen zwischen Wurzel und Spross sicherzustellen (Abb. 1.1). Zwei Hauptleitsysteme sind dabei in Angiospermen zu unterscheiden, das Xylem und das Phloem, die zusammen die Leitbündel bilden. Angetrieben durch die Wasserpotentialdifferenz im Boden-Pflanze-Luft Kontinuum (Transpirationssog) ermöglichen Xylemgefäße einen Massenstrom von Wasser mit gelösten Mineralien in den Spross. Diese Gefäße weisen starke Verdickungen ihrer Sekundärwände auf und verlieren im ausgereiften, leitenden Zustand ihr Protoplasma. Sie sind also tot, wenn sie ihre 1 1. Einleitung Ausdifferenzierung erreichen. Parenchymatische Zellen umgeben das Xylem und sorgen für die Be- und Entladung der Gefäße. Im Gegensatz zum Xylem ist das zweite Transportsystem höherer Pflanzen, das Phloem, eine funktionelle Einheit zweier lebender Zelltypen, die ontogenetisch durch inäquale Teilung aus einer Mutterzelle entstehen. Zusammen bilden sie einen strukturellen und funktionellen Komplex mit hochspezialisierten, symplastischen Verbindungen (Poren-Plasmodesmen Einheiten, PPU) für den selektiven Austausch von Mikro- und Makromolekülen. Während die longitudinal gestreckten Siebelemente viele ihrer cytoplasmatischen Organellen sowie die Zentralvakuole und den Kern verloren haben, sind die dazugehörigen Geleitzellen überaus aktiv (Sjölund, 1997). Sie besitzen einen Kern und ein sehr dichtes Zytoplasma mit zahlreichen Mitochondrien zur Versorgung der Siebelemente mit Transkripten, Proteinen und Energie (ATP) über verzweigte Poren-Plasmodesmen Einheiten (Knoblauch und van Bel, 1998). Die Einheit aus Siebelementen und assoziierten Geleitzellen nennt man Siebelement- Geleitzell-Komplex (SE/CC Komplex, aus dem Englischen für sieve element/companion cell complex) (Abb. 1.2; van Bel, 2003 und Referenzen darin). Abb. 1.2: Aufbau eines Siebröhren/Geleitzellen Komplexes in Vicia faba, modifiziert nach van Bel, 2003. SE (Siebröhren-Elemente) und CC (Geleitzellen) sind durch zahlreiche Poren/Plasmodesmen-Einheiten (PPUs) verbunden.
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