Steady, ready, go
– proteomics of etioplast inner membranes reveals a high readiness for light
Lisa Blomqvist
Akademisk avhandling för filosofie doktorsexamen i fysiologisk botanik, som enligt beslut i lärarförslagsnämnden i biologi kommer att offentligt försvaras fredagen den 5:e juni 2009, kl 10.15 i föreläsningssalen, Institutionen för Växt- och Miljövetenskaper, Carl Skottbergs Gata 22B, 413 19 Göteborg
Examinator: Professor Adrian K. Clarke
Fakultetsopponent: Professor Karin Krupinska, Christian-Albrechts Universität, Botanisches Institut, Kiel, Germany
Göteborg, Maj 2009 ISBN: 978-91-85529-29-2
Front cover: Scanning electron micrograph of isolated prolamellar bodies
ISBN 978-91-85529-29-2 http://hdl.handle.net/2077/20072 Copyright © Lisa Blomqvist, 2009
Tryck: Chalmers reproservice, Göteborg, Sweden, 2009
Steady, ready, go
– proteomics of etioplast inner membranes reveals a high readiness for light
Lisa Blomqvist
Department of Plant and Environmental Sciences,
University of Gothenburg, Box 461, SE-405 30 Gothenburg, Sweden
Abstract: Light is essential for development of photosynthetically active chloroplasts. In the absence of light proplastids develop into etioplasts which are readily transformed into chloroplasts upon illumination. The etioplast inner membranes (EPIMs) differ significantly from those of chloroplasts regarding composition and structure. EPIMs consist of two laterally separated membrane systems, namely the three-dimensional lattice of tubular membranes, prolamellar bodies (PLBs), and the flat membranes of prothylakoids (PTs) which radiate from the PLBs. PLBs and PTs offer heterogeneity in lipid, pigment and protein composition. This thesis reports on novel proteomic studies of EPIMs and analyses of the light-dependent key enzyme in the chlorophyll biosynthesis, NADPH:protochlorophyllide oxidoreductase (POR).
POR, which constitutes at least 90% of the protein content of PLBs, is known to be important for the formation of the PLB membrane structure. Light activates the POR- mediated reduction of protochlorophyllide into chlorophyllide. This event is the starting point for the dispersal of PLBs and thus the whole rebuilding of the plastid inner membranes during etioplast to chloroplast transition. POR is firmly attached to the membrane and transmembrane helix predictions show that POR is a plausible integral transmembrane protein.
Proteomic analyses were performed on EPIMs isolated from well-defined sections of dark-grown wheat (Triticum aestivum) leaves. Proteins of EPIMs or subfractionated PLBs and PTs were separated and identified by mass spectrometry analyses.
The proteome of PLBs and PTs reveals a far more complex protein composition than previously suggested. In total, 111 proteins were identified in PLBs and PTs. The proteins represent diverse functions such as pigment biosynthesis, photosynthesis and protein degradation. The majority of the identified proteins are directly or indirectly connected to photosynthesis, thus suggesting that PLBs and PTs are well prepared for construction of the photosynthetic apparatus. The spatial separation of certain proteins between PLBs and PTs suggests that photosystem formation is initiated in the PTs. EPIMs contain numerous proteins involved in protection against excess light. Etioplasts are steady in darkness, ready for light and well prepared to go for a fast onset of photosynthesis.
Keywords: chloroplast, etioplast, NADPH:protochlorophyllide oxidoreductase, prolamellar body, proteomics, prothylakoid, transmembrane, Triticum aestivum, wheat
Gothenburg, May 2009 ISBN: 978-91-85529-29-2
Till Daniel,
Klara och Hilda
Steady, ready, go
– proteomics of etioplast inner membranes reveals a high readiness for light
Lisa Blomqvist
This thesis is based on the following papers, which are referred to by their Roman numerals.
(I) Blomqvist LA, Ryberg M, Sundqvist C (2006) Proteomic analysis of the etioplast inner membranes of wheat (Triticum aestivium) by two-dimensional electrophoresis and mass spectrometry. Physiol Plant 128, 368-381*
(II) Blomqvist LA, Ryberg M, Sundqvist C (2008) Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development. Photosynth Res 96, 37-50*
(III) Blomqvist LA, Ryberg M, Sundqvist C, Aronsson H (2009) The proteomes of prolamellar bodies and prothylakoids are well prepared for a fast onset of photosynthesis. (Submitted)
(IV) Blomqvist LA, Töpel M, Ryberg M, Aronsson H (2009) NADPH:protochlorophyllide oxidoreductase (POR) is a plausible integral membrane protein. (Manuscript)
* Reprinted with permission of the respective copyright holder
TABLE OF CONTENTS Abbreviations
1. INTRODUCTION 1
2. PROTEOMICS 2
2.1. Experimental design 3
2.1.1. Sample preparation 3
2.1.2. Protein separation and mass spectrometry (MS) 3 2.2. Proteomics of chloroplasts 4 2.3. Proteomics of etioplasts 4
3. ETIOPLASTS 6
3.1. The significance of studying etioplasts 6 3.2. Etioplast inner membranes (EPIMs) 7
3.3. Pigments of EPIMs 7
3.4. Proteins of EPIMs 8
4. NADPH:PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) 10
4.1. POR isozymes 10
4.2. Structure of POR 10
4.3. Import and localization of POR 11 4.4. Association of POR to the membrane 12
4.4.1. Experimental behaviour of POR 12
4.4.2. POR - an integral transmembrane (TM) protein? 12 4.4.3. POR - an integral monotopic or peripheral
membrane protein? 13
4.5. Regulation of POR 14
4.5.1. Isozyme regulation 14
4.5.2. Is POR regulated by phosphorylation? 14
4.5.3. POR degradation 15
5. ETIOPLAST TO CHLOROPLAST TRANSITION 16 5.1. Biogenesis of the photosynthetic apparatus 16
5.1.1. Significance of prolamellar bodies (PLBs)
during transition 16
5.1.2. Significance of prothylakoids (PTs)
during transition 18
5.2. EPIMs are ready for a life in light – concluding remarks 18
6. Acknowledgements - Tack! 19
7. REFERENCES 20
8. Populärvetenskaplig sammanfattning på svenska 31
Abbreviations
1-D one-dimensional 2-D two-dimensional 3-D three-dimensional
a.a. amino acid(s)
DGDG digalactosyl diacylglycerol EPIM etioplast inner membrane
ESI electrospray ionization
FTICR Fourier transform-ion cyclotron resonance IMAC immobilized metal affinity chromatography MALDI matrix-assisted laser desorption/ionization MGDG monogalactosyl diacylglycerol
MOAC metal oxide affinity chromatography
MS mass spectrometry
MS/MS tandem mass spectrometry
LC liquid chromatography
Ndh NAD(P)H:plastoquinone oxidoreductase Pchlide protochlorophyllide
PLB prolamellar body
POR NADPH:protochlorophyllide oxidoreductase PT prothylakoid
SDR short-chain dehydrogenase
TM transmembrane ToF time-of-flight Trp tryptophan Q quadrapole
