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DISS ETH NO. 16953 Proteome analysis of tobacco BY-2 cell culture plastids and Capsicum annuum chromoplasts: Protein profiling, quantification and novel strategies for the detection of proteins from non- model organisms A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY for the degree of DOCTOR OF NATURAL SCIENCES presented by Muhammad Asim Siddique M.Sc (Hons) Agronomy University of Agriculture Fasialabad, Pakistan Born 31st December 1973 Pakistan accepted on the recommendation of Prof. Wilhelm GRUISSEM, examiner Prof. Nikolaus AMRHEIN, co-examiner Dr. Sacha BAGINSKY, co-examiner Zurich 2007 To Prof. Walter Siegenthaler ii Table of contents ABSTRACT ........................................................................................................................................... V ZUSAMMENFASSUNG..................................................................................................................... VI ABBREVIATIONS............................................................................................................................. VII 1. GENERAL INTRODUCTION .................................................................................................... 1 1.1 PLASTIDS................................................................................................................................ 1 1.1.1 Plastids from tobacco Bright Yellow (BY-2) Cell Culture ................................................ 5 1.2 PROTEOMICS STRATEGIES: DEFINITION AND CONCEPTS ......................................................... 7 1.2.1 Approaches in proteomics ................................................................................................ 8 1.2.2 Plastid proteomics .......................................................................................................... 10 1.3 AIM OF THE RESEARCH ......................................................................................................... 12 2. MATERIAL AND METHODS.................................................................................................. 13 2.1 MATERIALS .................................................................................................................................. 13 2.1.1 Chemicals and materials...................................................................................................... 13 2.1.2 Plant material ...................................................................................................................... 13 2.2 METHODS ..................................................................................................................................... 14 2.2.1 Cultivation of BY-2 cells ...................................................................................................... 14 2.2.2 Preparation and isolation of plastids................................................................................... 14 2.2.2.1 BY-2 plastids................................................................................................................................ 14 2.2.2.2 C. annuum chromoplasts .............................................................................................................. 15 2.2.3 Assessment of plastid purity ................................................................................................. 15 2.2.3.1 BY-2 plastids................................................................................................................................ 15 2.2.3.2 C. annuum chromoplasts .............................................................................................................. 17 2.2.4 Characterization of different plastid types........................................................................... 17 2.2.4.1 BY-2 plastids................................................................................................................................ 17 2.2.4.2 C. annuum chromoplasts .............................................................................................................. 19 2.2.5 Fractionation and isolation of plastid proteins.................................................................... 19 2.2.5.1 BY-2 plastids................................................................................................................................ 19 2.2.5.2 C. annuum chromoplasts .............................................................................................................. 25 2.2.6 Protein identification by Mass Spectrometry (MS) .............................................................. 25 2.2.6.1 ESI-Ion Trap Mass Spectrometry ................................................................................................. 25 2.2.6.2 MALDI-TOF/TOF Mass Spectrometry........................................................................................ 28 2.2.7 Identification of proteins from MS/MS data......................................................................... 31 2.2.7.1 SEQUEST search engine.............................................................................................................. 31 2.2.7.2 MASCOT search engine .............................................................................................................. 32 2.2.7.3 Manual data interpretation............................................................................................................ 33 2.2.7.4 Quality scoring for MS-spectra .................................................................................................... 34 2.2.7.5 De Novo peptide sequencing........................................................................................................ 34 2.2.7.6 MS-BLAST .................................................................................................................................. 35 2.2.8 Bioinformatics analysis........................................................................................................ 36 2.2.8.1 Protein localizations ..................................................................................................................... 36 2.2.8.2 Metabolic pathway modeling ....................................................................................................... 37 3. RESULTS .................................................................................................................................... 38 3.1 BY-2 PLASTIDS ............................................................................................................................. 38 3.1.1 BY-2 plastid isolation and purity ......................................................................................... 38 3.1.2 Characterization of the BY-2 plastids .................................................................................. 39 3.1.3 BY-2 plastid –Complete proteome analysis ......................................................................... 41 3.1.3.1 Shotgun proteome analysis........................................................................................................... 41 3.1.3.2 2D-PAGE ..................................................................................................................................... 54 3.1.4 Functional proteome analysis .............................................................................................. 62 3.1.4.1 Blue Native (BN)-PAGE.............................................................................................................. 62 3.1.4.2 NP-40 insoluble fraction .............................................................................................................. 67 3.1.5 Overview of the BY-plastid proteome................................................................................... 72 3.2 CAPSICUM ANNUUM CHROMOPLAST .............................................................................................. 73 3.2.1 Isolation and purification C. annuum chromoplasts............................................................ 73 3.2.2 Protein fractionation............................................................................................................ 74 3.2.3 Detection of proteins in different fractions .......................................................................... 76 3.2.4 Functional classification of identified proteins.................................................................... 82 3.2.5 PepNovo and MS- BLAST search ........................................................................................ 85 iii Table of contents 3.3 COMPARISONS OF THE DIFFERENT PLASTID TYPES ........................................................................ 90 4. DISCUSSION .............................................................................................................................. 95 4.1 CHARACTERIZATION OF PLASTIDS ................................................................................................ 96 4.1.1 BY-2 plastid.......................................................................................................................... 96 4.1.2 C. annuum chromoplast ....................................................................................................... 96 4.2 PROTEOME COVERAGE: DIFFERENT APPROACHES AND ENCOURAGING RESULTS ........................... 97 4.2.1 BY-2 plastid.......................................................................................................................... 97 4.2.2 C. annuum chromoplast ..................................................................................................... 102 4.3 PLASTID COMPARISONS .............................................................................................................. 107 4.4 CURRENT STATUS OF PLASTID PROTEOMICS................................................................................ 109 REFERENCES ................................................................................................................................... 110 CURRICULUM VITAE ....................................................................................................................... A ACKNOWLEDGEMENTS .................................................................................................................. B SUPPLEMENTARTY TABLE 1 ......................................................................................................... C SUPPLEMENTARY TABLE 2............................................................................................................. J SUPPLEMENTARY TABLE 3............................................................................................................ P iv Abstract Abstract Plastids, essential cell organelles, are present in all living cells of plants, except pollen. They are responsible for many of the essential biosynthetic and metabolic activities required for the basic architecture and functions of plant cells. Depending on the tissue type, they are differentiated into different forms. Proplastids are progenitors of all plastid types and act as precursors for differentiated plastid types like chloroplasts, etioplasts and amyloplasts. Chromoplasts generally occur in mature tissue and derive from pre-existing mature plastids. During fruit ripening, chloroplasts differentiate into nonphotosynthetic chromoplasts and during this process chlorophyll is degraded and large amounts of carotenoids accumulate. The aim of this work is to advance the understanding of the proteome and biochemical functions of different plastid types. For this work, I used plastid from cultured BY-2 cells as a model system for undifferentiated heterotrophic plastids to answer questions related to plastid development, differentiation and function. For this purposes, I used different approaches like complete proteome analysis (shotgun and 2DPAGE) and functional proteome analysis (BN-PAGE and NP-40 insoluble fraction) to increase the protein detection rate from this plastid type. In total, 197 plastidic proteins were detected with the combination of these approaches. For the analysis of the C. annuum chromoplast proteome, two approaches (shotgun and MS-BLAST) were applied to increase the proteome coverage from this plastid. These approaches have identified 149 and 26 plastid proteins, respectively. In addition to the identification of proteins, I also compared the proteomes of different plastid types, i.e., BY-2 plastids (own research), C. annuum chromoplasts (own research), etioplasts (von Zychlinski et al., 2005) and already published chloroplast proteomes (Kleffmann et al., 2006). This comparative proteome analysis provided information about prevalent metabolic activities in different plastid types. v Zusammenfassung Zusammenfassung Plastiden sind charakteristische pflanzliche Zellorganellen, die für essentielle Funktionen der Pflanzenzellen verantwortlich sind. Je nach Gewebe- und ZellTyp findet man Plastiden in unterschiedlichen Differenzierungszuständen. Man unterscheidet Proplastiden aus meristematischen Geweben, Chloroplasten aus photosynthetisch aktiven Blattzellen, Chromoplasten aus Blütenblättern und Früchten und Amyloplasten aus Speichergeweben. Ziel der vorliegenden Arbeit ist es, ein besseres Verständnis der metabolischen Funktionen unterschiedlicher Plastidentypen zu erhalten und die molekularen Grundlagen der Plastiden-Differenzierung zu verstehen. Zu diesem Zweck haben wir das Proteinmuster von undifferenzierten Plastiden einer BY-2 Zellkultur und vollständig ausdifferenzierten Chromoplasten analysiert. Zur Proteom-Analyse der BY-2 Plastiden wurden wir unterschiedliche Methoden eingesetzt, um einen möglichst grossen Teil des Proteoms analysieren zu können und zusätzlich quantitative Information über die identifizierten Proteine zu erhalten. Insgesamt konnten wir 197 Proteine aus BY-2 Plastiden identifizieren. Zur Analyse des Chromoplasten-Proteoms wählten wir die Proteom-Analyse Methode mit der höchsten Sensitivität, die sogenannte „Shotgun“-Methode. Da das Genom von Capsicum annuum L. nicht sequenziert ist, wurde wir für die Datenanalyse eine alternative Suchstrategie verwendet, die eine Datenbank-unabhängige Identifikation von Proteinen auf Grundlage von Massenspektren ermöglicht. Insgesamt konnten wir 151 Proteine aus vollständig differenzierten Chromoplasten identifizieren. Ein detaillierter Vergleich der Proteome der beiden Plastidentypen miteinander und mit den bereits veröffentlichten Proteomen von Etioplasten und Chloroplasten hat deutliche Unterschiede in der Proteinausstattung der verschiedenen Plastidentypen gezeigt. Dies kann als Ausdruck der spezialisierten Funktion der Plastiden in unterschiedlichen Gewebetypen verstanden werden. Trotz dieser Unterschiede wurde eine grosse Zahl von Proteinen identifiziert, die in allen Plastidentypen vorkommen. Diese Proteine haben grundlegende Funktionen (z. B. im Kohlenhydratstoffwechsel), die für alle Plastidentypen wichtig sind unabhängig von deren Differenzierungszustand. vi Abbreviations Abbreviations 2, 4 D 2, 4-Dichlorophenoxyacetic acid ACN Acetonitrile ABA Abscisic acid Brij 35 Polyoxyethylene (35) lauryl ether BY-2 Bright Yellow -2 BN-PAGE Blue native polyacrylamide gel electrophoresis BLAST Basic Local Alignment Search Tool Ccs Capsanthin-capsorubin synthase CHAPS 3-(3-Cholamidopropyl) dimethylammonio)-1-propane sulfonate Crtr-b β-Cycle hydroxylase ECL Enhanced chemiluminescent EM Electron Miscrope DDT Dithiothreitol DXS 1-Deoxy-D-xylulose 5-phosphate synthase DXR 1-Deoxy-D-xylulose 5-phosphate EDTA Ethylenediaminetetraacetic acid GGPS Geranylgeranyl diphosphate synthase ICAT isotope-coded affinity tagging IspD 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase iTRAQ isobaric tagging for relative and absolute quantitation LC-ESI-MS/MS Liquid Chromatography- Electrospray Ionization-Tandem Mass spectrometry Lcy-b Lycopene-β-cyclase HEPES 4-(2-Hydroxyethyl) piperazine-1 -ethanesulfonic acid MALDI-TOF/TOF Matrix-assisted laser desorption ionization tandem timeof-flight MS Mass Spectrometry MS/MS Tandem Mass Spectrometry MS-BLAST Mass spectrometry-driven BLAST NEP Nucleus-Encoded plastid RNA Polymerase vii Abbreviations Nxs Neoxanthin synthase PEP Plastid-Encoded Polymerase PERCOLL Polyvinylpyrrolidone coated silica colloidal PMSF Phenylmethylsulfonylfluoride PVP Polyvinylpyrrolidone SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis TBP Tributylphosphate Tris Tris (hydroxymethyl) aminomethane Zds ξ-Carotene desaturase viii General Introduction 1. General Introduction 1.1 Plastids Plastids are an important and essential group of plant cell organelles. They are found only in plant and algal cells (Kirk and Tilney-Bassett, 1978). Plastids distinguish plant cells from those of other eukaryotes. The origin and evolution of plastids has been an important subject in biological sciences. Plastids represent the endosymbiotic remnants of a free-living cyanobacterial progenitor, which lost the vast majority of its ancestral cyanobacterial genes after primary plastid endosymbiosis (Goksoyr, 1967; Taylor, 1974; Martin et al., 2002; reviewed in Lopez-Juez and Pyke, 2005). In order to function, plastids depend on the cell nucleus for most of their proteins. Plastids have their own genetic system (DNA) but their gene expression and differentiation are largely controlled by the cell nucleus (Tanaka et al., 1996). The plastid genome is much smaller than the genome found in the nucleus. In most plastids, the genome size is around 120-150 kb (Sugiura, 1992). However, the proteome of plastids is estimated to consist of 2000-4000 proteins and despite significant efforts, less than half of the predicted number of plastid proteins has been confirmed (Martin and Hermann, 1998; Leister, 2003, 2005; Kleffmann et al., 2004, Millar et al., 2006). Plastids are highly polyploid and proplastids contain ca. 20 copies of the genome (~400 per meristematic cell), while chloroplasts contain around 100 copies (10000 copies per cell) [Sugiura, 1992]. A comprehensive list of fully sequenced chloroplast genomes is currently available at http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html. Plastid DNA is organized in a particular structure called “nucleoid” or “plastid nucleus”. These particles are dense, heterogeneous and consist of various DNA-binding proteins, plastid DNA and uncharacterized RNA (Briat et al., 1982). Plastid DNA exists in discrete regions in the form of nucleoids associated with the inner envelope. A single small nucleoid is present in the center of proplastids in meristematic cells, whereas nucleoids are replicated 1 General Introduction while attached to the envelope membranes. In mature leaf cells, only a small number of nucleoids are present within the stroma in close association with the thylakoid membranes (Sato et al., 2003). Depending on cell and tissue type, plastids develop and differentiate from progenitor plastids (proplastids) into different plastid types (chloroplast, etioplast, chromoplast or amyloplast) that are responsible for important biosynthetic and metabolic activities. Among these are photosynthetic carbon fixation and the synthesis of fatty acids, pigments, starch, amino acids and vitamins etc. Plastids are classified into different categories according to their structure (morphology), pigment composition (color), and other developmental aspects (Thomson and Whately, 1980). The most fundamental distinction between plastid types is based on their primary energy metabolism, i.e. heterotrophy versus autotrophy. Although autotrophy is restricted to photosynthetically active chloroplasts, several distinct heterotrophic plastid types can be found in different plant tissues e.g., elaioplasts, chromoplasts, amyloplasts and etioplasts. These plastids are end products of a developmental program that is determined by the cell and tissue type (elaioplasts, amyloplasts and chromoplasts) as well as by environmental factors (etioplasts) [Neuhaus and Emes, 2000]. Proplastids, which are found in meristematic cells of roots and shoots, are the progenitors of all other plastids. All plastids derive from this small, colorless and undifferentiated proplastid but little is known about this plastid type in isolated form (Kirk and Tilney-Bassett, 1978; Pyke and Waters, 2004; reviewed in Lopez-Juez and Pyke, 2005). In the shoot meristem, there are 720 proplastids and in the root cap there are 20-40 proplastids per cell present (Pyke, 1999). They are generally 1.0-1.5 µm long and perhaps about 0.7 µm thick with relatively few internal structures. The main function of this plastid type is to act as the precursor of differentiated plastid types such as the chloroplast, etioplast and amyloplast.Therefore proplastid development in living plants is a crucial stage for plastid differentiation. There is little known about the precise biochemical function of proplastids. It seems likely that they might play some important role in the carbohydrate metabolism of the 2 General Introduction meristematic cells (Kirk and Tilney-Bassett, 1978; reviewed in Lopez-Juez and Pyke, 2005). Amyloplasts are mature plastids and most of their internal volume is filled with starch. They are a specialized type of leucoplasts. They are found in roots and storage tissues such as cotyledons, endosperm and tubers (Tetlow et al., 2004). Amyloplasts function as storage plastids and they presumably synthesize starch as a reserve when carbohydrates are available in excess and break down their starch into free sugars or sugar derivatives when the plant is in need of carbohydrates (Neuhaus and Emes, 2000; Pyke and Waters, 2004). Etioplasts are those plastid types that are formed in the leaf cells of plants growing in the dark. They can rapidly differentiate into photosynthetically active chloroplasts upon illumination (Leech, 1986). Etioplasts are filled with a matrix or stroma consisting of a uniform background of proteins and many ribosomes. The most prominent feature of the etioplast is the presence of large quasi-crystalline bodies, build up of interconnected membranous tubules in regular bodies, which are referred to as prolamellar bodies. It also contains high level of protochlorophyllide and during chloroplast development these crystalline bodies are disassembled and develop into thylakoids (lammellae) [Kirk and Tilney-Bassett, 1978; Pyke and Waters, 2004]. It is likely that a wide variety of metabolic and regulatory pathways are active in etioplasts, and most likely carbohydrate metabolism is the key function of etioplasts (Pyke and Waters, 2004; von Zychlinski et al., 2005). Chloroplasts are highly structured plastid types that carry out photosynthesis and amino acid biosynthesis in the green parts of plants. The size of a chloroplast is between 5-10 µm in diameter and 3-4 µm in thickness. Their number per cell varies from 10 to over 100 depending on species (reviewed in Lopez-Juez and Pyke, 2005). Chloroplasts are highly structured organells and contain three distinct membranous systems; the outer and the inner envelope membranes that surround the organelle and separate the stroma from the cytosol, and the thylakoid membrane network, which contains the photosynthetically active protein complexes (reviewed in Soll and Schleiff, 3 General Introduction 2004). Chloroplasts are also a center of plant metabolism and many biosynthetic activities like starch synthesis, reduction of nitrite, biosynthesis of fatty acids are localized to chloroplasts (Neuhaus and Emes, 2000). Chromoplasts are special among heterotrophic plastids because they develop from photosynthetic (autotrophic) chloroplasts during fruit ripening or petal development (reviewed in Camara et al., 1995). The primary function of chromoplast is for carotenoid synthesis and storage that gives fruit and flower their characteristic colors. They are red, orange or yellow depending on the pigments they accumulate. Chromoplasts are usually found in flower petals, in many fruits and certain roots (Kirk and Tilney-Bassett, 1978). Tomato chromoplasts are the site of the biosynthesis of protein, lipid, carotenoid, starch and sugar (Hansen and Chiu, 2005). During fruit ripening, chloroplasts differentiate into chromoplasts, photosynthetically competent thylakoid membranes are greatly diminished, grana are lost and carotenoids accumulate to high levels. They are also called carotenoid-containing plastids (reviewed in Camara et al., 1995). The main carotenoids in chromoplasts are β-carotene and xanthophylls. In non-photosynthetic chromoplasts the distribution of carotenoids is subject to considerable variation from one species to another (Josse et al., 2000). The carotenoid lycopene, for example, is responsible for the deep red color of the plastids and is believed to have anticancer and anti-cholesterol properties because of its antioxidant properties. In chloroplasts, carotenoids play vital roles in photosynthesis and are indispensable, whereas in chromoplasts they can be considered as secondary metabolites. Carotenoids in plants are also precursors for the synthesis of the hormone abscisic acid (ABA) [Chernys and Zeevaart, 2000]. Several of the enzymes involved in carotenoid synthesis have been cloned and characterized, but only a few other proteins involved in chromoplast metabolism were reported. A superoxide dismutase accumulates during tomato chromoplast development (Livne and Gepstein, 1988) as well as cysteine synthase, which has been cloned from tomato and pepper chromoplasts (Romer et al., 1992). Fully differentiated chromoplasts still have DNA, and some of the plastid DNA-encoded genes are actively transcribed at rates comparable with those in chloroplasts (Kuntz et al., 1989). However, 4 General Introduction translation activities decreases considerably, suggesting that the expression of plastid genes is predominantly regulated at the level of translation during chloroplast to chromoplast conversion. Plant cell functions have been investigated in various cell culture systems. Arabidopsis cell cultures have been generated but so far, synchronization of these cell cultures has been difficult. In addition, their cells are very small and detailed knowledge of their intracellular organization and content is limited. In contrast, the BY-2 cell culture is a well-defined and commonly used model system for studies of cell cycle regulation and the structure of the cytoskeleton (Geelen and Inze, 2001). 1.1.1 Plastids from tobacco Bright Yellow (BY-2) Cell Culture Tobacco BY-2 cells are cell line of plant cells, which was established from a callus induced on a seedling of Nicotiana tabacum cv. BY-2 (cultivar Bright Yellow-2 of the tobacco plant) [Nnagata et al., 1992]. Tobacco BY-2 cells were first presentated at the 14th Miles International Symposium at Baltimore in May 1982 (Nagata, 1984). BY-2 cells are non-green, rapidly growing heterotrophic cells. These cells can multiply up to 100 times within a week under conventional cell culture conditions (Nagata et al., 1992). BY-2 cells contain 400-750 mitochondria and 40-100 proplastid like plastids. At the time of transfer, each BY-2 cell contains about 46 spherical plastids, each of which contains 6.6 plastid nucleoids and each plastid nucleoids contains 3.3 copies of plastid DNA on average (Sakai, 2001). Due to these properties, these cells can be easily grown on large scale in the laboratory. Plastids from BY-2 cell culture have many proterties in common with undifferentiated proplastids: • Plastids are undifferentiated with few internal structures and the nucleoids (DNA) are similar to those of proplastids (Sakai, 2001; Philips et al., 2002). • BY-2 plastids reveal a DNA synthesis pattern that is similar to that of proliferating plant cells. Also, the isolated nucleoids from BY-2 plastids have several favorable characteristics such as morphological integrity of the DNAprotein complex, low nucleolytic activity, low RNA content and considerable as 5 General Introduction well as stable transcriptional activities under suitable in vitro conditions (reviewed in Sakai et al., 2004). • RNA polymerase from BY-2 plastids predominatnly transcribes plastidencoded genes from NEP-promoter elements (Kapoor and Sugiura, 1999), which is characteristic for undifferentiated plastids. NEP transcribes housekeeping genes in non-green plastids, while PEP transcribes both housekeeping and photosynthetic genes in chloroplasts (Hajdukiecz et al., 1997; Sakai et al., 2004). Tagetitoxin-insensitive RNA polymerase (NEP) is abundant in proplastid-nuclei, while tagetitoxin-sensitive RNA polymerase (PEP) is abundant in chloroplast nuclei and is responsible for active transcription in chloroplasts (Sakai et al., 2004). • BY-2 plastids have retained the ability to develop and differentiate in a hormone-dependent manner (Miyazawa et al., 1999). Conventionally, BY-2 cells are grown in a liquid culture medium containing auxin (2, 4-D)]. When BY-2 cells are transferred into an auxin-depleted medium and then supplied with cytokinin [benzladenine (BA)], amyloplast formation is synchronously induced with in two days (Sakai et al., 1992), which results in accumulation of starch and increase in cell length and size. In this system, amyloplast formation is primarily triggered by the depletion of auxin and is further facilitated by the addition of cytokinin (Sakai et al., 1992). These properties taken together suggest that BY-2 plastids represent an interesting undifferentiated plastid type with several features that characterize true proplastids. At present in plastid proteomics most of proteome studies were done on fully functional chloroplasts (Ferro et al., 2002, 2003; Peltier et al.,2002; Schubert et al., 2002; Froehlich et al., 2003; Kleffmann et al., 2004) and only few heterotrophic plastid proteomes [rice etioplasts (von Zychlinski et al. 2005); wheat amyloplasts (Andon et al., 2002, Balmer et al., 2006)] were reported. Therefore, I decided to analyze the proteome of BY-2 plastids and C. annuum chromoplasts in order to obtain initial insight into the protein complement and metabolic capacities of different plastid types. 6 General Introduction 1.2 Proteomics strategies: Definition and concepts Proteomics defines an approach for the systematic analysis of all proteins expressed in a cell. In the last few years, the proteomics technology progressed quite fast due to two parallel developments. First, the amount of genomic information has increased. This has paved the way for the large scale analysis of proteins, for which amino acid sequences have been deposited into databases (e.g. the Arabidopsis Genome Initiative, 2000). Second, improvement in mass spectrometry technology, especially in the development of soft ionization techniques for peptide analysis, allowed the high-throughput identification of proteins from small amounts of samples (reviewed in Pandey and Mann, 2000). Scientists aim at the most complete analysis of the protein complement of a cell or a tissue type under certain and well-defined conditions. In principle, two basic proteomics approaches can be distinguished. “Protein profiling” attempts the identification of all proteins that are present in a sample and results in a list of proteins. Combined with sophisticated protein or peptide fractionation strategies, protein profiling is a relatively simple approach for high throughput analyses of the proteome of an organelle or a cell type and provides a snapshot of the major protein constituents (Washburn et al., 2001). “Functional proteomics” concentrates on the identification of specific proteins related to specific biological processes. Although the identification focuses on those proteins that are altered upon a stimulus or signal, the original analysis takes place at the level of the complete proteome. This approach often involves 2D-PAGE. Proteins that differ in abundance and present or absent from one of the differently treated samples, are specifically analyzed and identified. In addition to 2-D PAGE, such changes can be revealed by other differential display techniques such as isotope-coded affinity tagging [(ICAT) Gygi et al., 1999, 2000] or isobaric tagging for relative and absolute quantitation [(iTRAQ) Ross et al., 2004). Another example of functional proteomics is the identification of proteins isolated by affinity separation methods such as antibody affinity precipitation, native purification of protein complexes by Blue-native gel electrophoresis (BN-PAGE), or affinity 7 General Introduction ligand binding (reviewed in Baginsky and Gruissem, 2004). All these approaches are to some extent designed to solve a specific biological question and the experimental design is based on a hypothesis. 1.2.1 Approaches in proteomics In the proteomics field there are several techniques used in combination to identify proteins from a sample. These include mass spectrometry (MS), twodimensional gel electrophoresis (2D-PAGE) and bioinformatics. MS is the heart of all proteomics experiments as it provides the key tools for the analysis of proteins. In the last five years, the development of technology and methodology in the field of MS and proteomics are increased rapidly. The matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the two most commonly techniques used in the proteomics field (reviewed Aebersold and Mann, 2003 and Domon and Aebersold, 2006). Both are soft ionization techniques in which ions are created with low internal energies and thus undergo little fragmentation. In MALDI, samples are crystallized with an organic matrix, most commonly used are 3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2, 5- dihydroxybenzoic acid (DHB), on a MALDI plate (usually a metal plate designed for this purpose). A pulsed laser is used to excite the matrix, which causes rapid thermal heating of the molecules and eventually desorption of ions into the gas phase. MALDI is usually coupled to TOF analyzers, which separates ions according to their flight time down a field-free tube. The timeof-flight (TOF) of ions is directly related to their m/z values and thus a mass spectrum can be acquired. ESI is based on spraying an electricity-generated fine spray of ions into the inlet of a mass spectrometer at atmospheric pressure. This technique ionizes molecules directly from solution, so it can easily be combined with liquid separation methods. ESI is mostly coupled to ion traps (three-dimensional and linear ion traps) and hybrid tandem mass spectrometers like quadrupole time-of-flight (Q/TOF) instruments (reviewed in Domon and Aebersold, 2006). 8 General Introduction MALDI-TOF MS is relatively simple and robust to operate; it has good mass accuracy, high resolution and sensitivity. It is widely used in proteomics to identify proteins from simple mixtures by a process called peptide mass fingerprinting (Cottrell, 1994). This technique is frequently used in conjunction with 2D-PAGE. On the other hand, ESI ion trap MS is the most popular setup for the simultaneous analysis of large number of peptides derived from the enzymatic digest of complex protein mixtures (de Hoog and Mann, 2004). ESI is quite popular in shotgun proteomics. The main advantage of ESI is the production of multiply charged ions, which allows the measurement of high molecular weight peptides that are often neglected in MALDI-TOF analysis. Traditionally, 2D-PAGE has been the primary tool for obtaining a global picture of the expression levels of a proteome under various conditions. In the first dimension, proteins are separated in one direction by isoelectric focusing usually in a tube gel, and then in the second direction by molecular mass using electrophoresis in a slab gel containing sodium dodecyl sulfate (SDS) (Görg et al, 2004). 2D-PAGE allows the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass, solubility and relative abundance. Depending on the gel size and pH gradient used, 2DPAGE can resolve more than 5000 protein spots (~2000 protein spots routinely) simultaneously. One of the greatest strengths of 2D-PAGE is its potential to study proteins that have undergone post-translational modifications (PTM), such as phosphorylation, glycosylation or limited proteolysis, and the fact that it provides quantitative information. Bioinformatics plays an essential role in today’s proteomics field. As the amount of data grows exponentially, there is a parallel growth in the demand for tools and methods for data management, visualization, integration, analysis, modeling and prediction. Many tools have been developed for MS to identify proteins from databases. The most common ones are SEQUEST (http://fields.scripps.edu/sequest) and MASCOT (http://www.matrixscience.com). Both tools rely on the comparison between theoretical peptides derived from the database and experimental MS data. SEQUEST produces a list of possible peptide assignments in a protein 9 General Introduction mixture based on a correlation-scoring scheme (Yates et al., 1995). MASCOT incorporates probability-based scoring and all three types of search are supported, peptide mass fingerprinting (PMF), sequence query and MS/MS ions search (Perkins et al., 1999). The limitations of these programs are that a significant portion of MS/MS spectra cannot be assigned due to various reasons like sequencing and annotation errors in the database search and various modifications like post-translation modifications or chemical modifications (reviewed in Palagi et al., 2006). Due to these limitations in the database search, development of methods of de novo sequencing is an active research field. De novo sequencing methods allow peptide sequencing without prior knowledge of the amino acid sequence. Typically, de novo sequencing algorithms match the spacing of peaks by the mass of one or several amino acids and help to infer the probable peptide sequences that are consistent with the matched amino acids. The popular software packages for de novo sequencing are Lutefisk 1900 (http://www.hairyfatguy.com/lutefisk) [Taylor and Johnson 1997], PEAKS (http://www.bioinformaticssolutions.com/products/peaks) [ Ma et al., 2003], PepNovo (http://peptide.ucsd.edu/pepnovo.py) [Frank and Pevzner, 2005], DeNovoX (http://www.thermo.com/com/cda/product/detail/1,1055,19050,00.html) [Chao et al., 2004] and AUDENS (http://www.ti.inf.ethz.ch/pw/software/audens/) [Grossmann et al., 2005]. A general problem encountered during de novo sequencing is that the isobaric masses of the amino acids leucine and isoleucine cannot be differentiated by mass spectrometry. Also not every fragment ion might be present in the spectrum, thus a possible gap of two or three amino acids has to be matched. Within such a gap, the correct order of the amino acids cannot be estimated. Instead, several top candidate sequences are suggested. 1.2.2 Plastid proteomics Despite the interest in plastid biology, our current understanding of the metabolic functions and capacities of different plastid types is limited. 10 General Introduction Proteomics is one promising way towards a better understanding of plastid biology. Various fractionations and mass spectrometry techniques have been applied to catalogue plastid proteins. Several proteomics studies were conducted with chloroplasts in recent years that provided valuable information on their metabolic capacities (Ferro et al., 2002, 2003; Peltier et al.,2002; Schubert et al., 2002; Froehlich et al., 2003; Rolland et al., 2003; Kleffmann et al., 2004; Friso et al., 2004; Majeran et al., 2005; Peltier et al., 2005). It has become clear that chloroplast proteome analyses have reached their saturation level, since highly abundant photosynthetic proteins that dominate the proteome of fully developed photosynthetically active chloroplasts hamper the detection of new proteins. For particular example, Rubisco (Ribulose 1, 5bisphosphate carboxylase/oxygenase) is the most abundant protein on the earth. This protein accounts up to 50% of the soluble proteins in leaves and this complicates the analysis of low abundant proteins in green tissues (Rossignol et al., 2006). A valid strategy to circumvent this constraint and to increase the proteome coverage is the use of different plastid types for highthroughput protein identification. Heterotrophic plastids for example do not contain highly abundant photosynthetic proteins and therefore allow the detection of other metabolic activities and regulatory factors. At the same time, the proteomes of different plastid types contain valuable information about plastid type-specific functions. Most of the studies reported to date were conducted with fully functional chloroplasts and only three proteomics approaches used heterotrophic plastid types. These include rice etioplasts (von Zychlinski et al. 2005); wheat amyloplasts (Andon et al., 2002, Balmer et al., 2006) and BY-2 cell culture plastids (own study). A comparison of the proteome data from the different plastid types confirmed that the proteomes of heterotrophic and autotrophic plastids differ considerably which is especially apparent for their distinct energy metabolism. Heterotrophic plastids import metabolites such as ATP and glucose 6-phosphate for essential biosynthetic activities, for example the synthesis of starch from ADP glucose, fatty acids from acetate and amino acids from inorganic nitrogen (Weber et al., 2005). These pathways place a high demand for energy and reducing equivalents on the heterotrophic plastid. 11 General Introduction Substantial evidence has accumulated that heterotrophic plastids generate reducing equivalents by the oxidative branch of the pentose phosphate pathway that is initiated by glucose 6-phosphate. Glucose 6-phosphate as well as ATP are imported from the cytosol by well the characterized plastidic glucose 6-phosphate/phosphate translocator (GPT) and ATP/ADP transporters, respectively (Weber et al., 2005). 1.3 Aim of the research The aim of my work was to increase our knowledge of “plastid proteomes” through the analysis of different plastid types. For this purpose, I used the BY2 plastid as a model system for undifferentiated heterotrophic plastids and Capsicum annuum as an example for chromoplasts. In addition to the identification of proteins, I also analyzed metabolic functions of BY-2 plastids and C. annuum chromoplasts and compared different plastid types in terms of their proteomes. To accomplish this work, I used the information from BY-2 plastid (own research) and C. annuum chromoplasts (own research), together with information from chloroplasts and etioplasts that were already available in our laboratory (Kleffmann et al., 2004; von Zychlinski et al., 2005) and recently published Arabidopsis chloroplast proteomes (Peltier et al., 2002; Schubert et al., 2002; Ferro et al., 2003; Froehlich et al., 2003). All published plastid proteomes are collectively available at http://www.plprot.ethz.ch/ (Kleffmann et al., 2006). This comparative proteome analysis provided information about prevalent metabolic activities in different plastid types. 12 Material and methods 2. Material and methods 2.1 Materials 2.1.1 Chemicals and materials Acetonitrile 5-Aminolevulinic Acid BSA Cellulase Onozuka RS Complete EDTA-free protease inhibitor cocktail ECL Western Blotting Analysis System HEPES Murashige & Skoog Plant salts incl. Vitamins Miracloth Nonidet P40 Pectolyase Y23 Percoll PVP ReadyStrip IPG strip Spermidine SYPRO® Ruby protein stain TBP Trypsin, sequencing grade Brunschwig, Basel, Switzerland Sigma-Aldrich,Switzerland Sigma-Aldrich, Switzerland Yakult Honsha, Tokyo, Japan Roche, Rotkreuz, Switzerland GE Healthcare, formerly Amersham Biosciences Carl-Roth GmbH, Karlsruhe, Germany Duchefa, Haarlem, the Netherlands Calbiochem, Laeufelingen, Switzerland Fluka, Buchs, Switzerland Yakult Honsha, Tokyo, Japan Sigma-Aldrich, Switzerland Sigma-Aldrich, Switzerland Biorad, Hercules, CA, USA Sigma-Aldrich, Switzerland Molecular Probes, Leiden, The Netherlands Fluka (Buchs, Switzerland) Promega, USA All other reagents were purchased at higest analytical grade from Fluka (Buchs, Switzerland), Sigma (Seelze, Germany) and Aldrich (Steinheim, Germany). 2.1.2 Plant material Tobacco bright yellow cell culture (BY-2) was provided by Prof. Pascal Genschik, Institut de Biologie Moléculaire des Plantes, Strasbourg Cedex – France. Red bell pepper (Capsicum annuum L.) fruits were purchased from the local market. 13 Material and methods 2.2 Methods 2.2.1 Cultivation of BY-2 cells The tobacco BY-2 cell culture was grown in the modified Murashige and Skoog medium (MS-medium with vitamins, M 0222) as described previously by Fan and Sugiura (1995) with little modifications. The BY-2 cell culture was diluted 1:100 into a fresh BY-2 growing medium (MS-Medium with vitamins, M 0222, sucrose 3%, KH2PO4 255mg /Liter, 2, 4-D 0.2mg/Liter pH 5.8) every 7th day. The BY-2 cell culture was kept at 130 rpm on a shaker at 27 oC in the dark. 2.2.2 Preparation and isolation of plastids 2.2.2.1 BY-2 plastids BY-2 plastids were isolated as described previously by Kapoor and Sugiura (1999) with little modifications. BY-2 cells were collected after 90-96 hr (~250 g fresh weight) from a suspension culture. Fresh cells were collected by passing through two layers of miracloth (Calbiochem, Switzerland ), washed three times with 0.4 M mannitol, pH 5.0 and digested in three volumes of an enzyme solution (1% Onozuka RS cellulose, 0.1% pectolyase Y-23 containing 0.4 M mannitol pH, 5.6) at 30 oC for 1.5 hr. Protoplasts were subsequently harvested by centrifugation at 500 g at 4 °C for 5 min .Three washing steps followed, using five volumes of ice cold 0.4 M mannitol, pH 5.0. The pellet was resuspended in plastid isolation buffer (0.4 M mannitol, 20mM Tris-HCL, pH 7.6, 0.5 mM EDTA, 1.2 mM spermidine, 7mM 2-mercapoethanol, 0.6 {w/v} PVP and 0.1% {w/v} BSA) and protoplasts were broken by passing the suspension several times through layers of a 40-µm mesh under high pressure. The broken proplastids were analyzed under the light microscope. The broken protoplasts were centrifuged at 500 g for 10 min at 4 °C to pellet the cell debris and nuclei. The supernatant was then filtered through two layers of 20-µm mesh under low pressure. At the end, Percoll was added to the filtrate to a final concentration of 15% and the whole suspension was centrifuged at 10000 g for 20 min at 4 °C. The pellet was subsequently resuspended in plastid isolation buffer and loaded onto a sucrose density 14 Material and methods gradient (30-50-70 % sucrose in 20mM Tris-HCL, pH 7.6, 0.5 mM EDTA, 1.2 mM spermidine, 7 mM 2-mercapoethanol) for further purification from mitochondria, nuclei and cellular debris. A yellow band of BY-2 plastids was collected at the 50-70% sucrose interface and washed two times with plastid isolation buffer without PVP and BSA. After washing, the BY-2 plastids were loaded onto a linear sucrose gradient from 30-70% sucrose for further purification. Pure proplastids were washed two times with plastid isolation buffer and spun down by centrifugation. The pellet was stored at -80 °C for further studies. 2.2.2.2 C. annuum chromoplasts Red fruits (~ 230 g) of Capsicum annuum L. were bought from the local market. Chromoplasts were isolated as described previously by Hadjeb et al (1988) with little modifications by using the Percoll density gradient centrifugation. The fruit was cut into small pieces of about 1 cm2 and blended in ice cold GR buffer (1 mM NaP2O7, 50 mM Hepes, 330 mM Sorbitol, 2 mM EDTA, 1 mM MgCl2, 2mM MnCl2, 2 mM DTT, pH 6.8) using 5 bursts of 5-10 seconds each. Then the solution was filtered through four layers of miracloth and the clear solution was centrifuged at 800 g for 10 min. The supernatant was discarded and the pellet was washed three times with GR buffer. The pellet was resuspended in 2 ml of GR mix and loaded on top of a Percoll density gradient developed with 2.5 ml of each 15%-20%- 30%-40% and 3 ml of 50% Percoll in 0.33 M Sorbitol, 2.5 mM MgCl2, 50 mM HEPES/KOH pH 7.8 with KOH , 2 mM DTT and centrifuged at 7000 g for 30 min. The intact chromoplasts were collected at the 40%-30% interphase and washed twice in the GR buffer and stored at -80 °C for further use. 2.2.3 Assessment of plastid purity 2.2.3.1 BY-2 plastids 2.2.3.1.1 Enzymatic measurement The purity of the BY-2 plastid preparation was assessed by enzymatic activity assays for characteristic marker proteins of other cell organelles (fumarase and catalase measurements), Fumarase activity was measured spectrophotometrically with L-malate as substrate (50 mM L-malate in 50 mM 15 Material and methods potassium phosphate buffer, pH 7.9) as described by Beeckmans and Kanarek (1982). Catalase activity was measured by following the decrease of H2O2 in the sample (0.01 M H2O2 in 0.1 M potassium phosphate buffer, pH 7.0), measured at 240 nm. The measurement of fumarase and catalase reactions were done as described below hereafter: For the fumarase test, 1 ml of reaction buffer (50 mM potassium phosphate buffer pH 7.4) was added to a quartzcuvette which contained 50 µl samples, and for the start of the reaction 20 µl 0.2 M malate was added. The reaction was measured at 240 nm after 5 min. In the catalase assay, 1 ml reaction buffer (0.01 M H2O2 in 0.1 M potassium phosphate buffer, pH 7.0) was added in a quartzcuvette which contained 50 µl samples. The activity was measured at 240 nm after 5 min. 2.2.3.1.2 Western Blot The purity of the BY-2 plastids was also checked by the detection of marker proteins with antibodies. For this purpose, I used the TOC 75 plastid marker protein antibody (provided by Prof. F. Kessler, Université de Neuchâtel, Switzerland) and α-porin mitochondrial protein antibody (provided by Prof. Kunau, Ruhr-University Bochum, Germany). Western blotting was performed by standard means, which is described as follows 12% SDS-PAGE was run at room temperature at 80 V until the protein marker reached the end. Twenty layers of Whatman paper 3 MM (Huber Co, Switzerland) and a nitrocellulose membrane 82 mm (Schleicher and Schuell, Switzerland) were soaked in the electrotransfer buffer (200 ml Methanol, 2.9 g glycine, 5.8 g Tris-base, 0.4g SDS in water). Electrodes were wetted with electrotransfer buffer and a sandwich like 10x Whatman-Gel-Nitrocellulose membrane-10x Whatman was prepared. The electrotransfer was run at 20 V for 1 h and after the run the membrane was blocked for 30 min in TBST buffer (1M Tris-HCl pH 7.5, 8.8 g NaCl, 0.1 %Tween-20 in water) plus 5% milk powder at room temperature. After 30 min, TBST+milk solution was changed and antibodies (TOC 75 and α-porin) were added. The membrane was incubated overnight at 4 oC. On the next day, the membrane was washed 3 times with TBST for 10 min and the membrane was 16 Material and methods incubated for 1 h at room temperature with TBST+5% milk powder+ 2nd antibody. The membrane was again washed 3 times with TBST for 10 min and the membrane was developed according to ECL western blotting analysis system (Amersham Biosciences). 2.2.3.2 C. annuum chromoplasts The crude extract was resuspended in 2 ml of a GR mix (1 mM NaP2O7, 50 mM Hepes, 330 mM Sorbitol, 2 mM EDTA, 1 mM MgCl2, 2 mM MnCl2, 2 mM DTT, pH 6.8) and loaded on top of a Percoll density gradient developed with 2.5 ml of each 20%-30%-40%-50%-60% Percoll in 0.33 M Sorbitol, 2.5 mM MgCl2, 50 mM HEPES/KOH pH 7.8 with KOH , 2 mM DTT and centrifuged at 7000 g for 30 min. The six bands (intact tissues) were recovered from the top of the gradient (Supernatant), at the interphases of upper side-20%, 20%30%, 30%-40%, 40%-50% and50%-60%. These bands were designated A, B, C, D, E and F (from top to bottom) and they were washed twice in the GR buffer and stored at -80 °C for further use. 2.2.4 Characterization of different plastid types 2.2.4.1 BY-2 plastids 2.2.4.1.1 Electron Microscopy For the characterization of BY-2 plastids, I also checked them under the microscope. Due to the small size of the plastid, light microscopic observations did not provide satisfactory results. Therefore, I performed these observations under the electron microscope. The pure and isolated BY-2 plastids were handed over to the electron microscopy facility (www.em.biol.ethz.ch), and Dr. Ernst Wehrli performed the observations. The protocol is described hereafter. For electron microscopic observations, BY-2 plastids were collected and pre-fixed in 1% (w/v) glutaraldehyde for 2 hr on ice. The pre-fixed cells were then transferred to 1 % (w/v) OsO4 dissolved in 20mM sodium cacodylate (pH 7.2) and fixed for 12h at 4 °C. Fixed plastids were dehydrated by a series of 30%, 50%, 70%, 90%, 99% and 100% ethanol incubations at room temperature for 20 min each. The cells were then infiltrated using an ethanol: propyleneoxide infiltration series (75%:25%, 50%:50%, 25%:75%, 17 Material and methods and twice in 0%:100%, for 20 min each). Then the plastids were gradually infiltrated with Spurr’s resin and a propyleneoxide infiltration series, placed in new Spurr’s resin and then polymerized for 72 h at 70 °C. The sections were cut with a diamond knife, stained with uranyl acetate and lead acetate and examined with an electron microscope (JEM-1200EX, JEOL, Tokyo, Japan). 2.2.4.1.2 Tetrapyrrole measurements Protochlorophyllide analysis was done at Prof. K. Apel’s lab with the help of Mr. Jean-Charles Isner. A detailed report of this work is described hereafter. BY-2 cells were incubated with 10mM ALA (5-aminolevulinic acid hydrochloride) in 50 ml of freshly transferred cell culture. The cell culture was divided into two parts; the first one was harvested after 24 h and the other was harvested after 96 h. Cells were spun down at 500 g and washed with the cell culture medium (section 2.2.1, Cultivation of BY-2 Cells). Cells were again spun down and the supernatants were removed as much as possible. Cells were resupended in 4 ml buffer containing 80% acetone, 0.1% ammonium hydroxide HN4OH in water. Cells were ground with the help of an Ultra-Turrax T25 for 3 times at 5 sec at full speed and the extract was spun down at maximum speed (10000 g) for 5 min. The supernatants were evaporated under N2 until the ca. 20% was remaining and were injected into the HPLC (High Performance Liquid Chromatography) system. In the control cell culture, I followed the same protocol but without feeding ALA to the BY-2 cell culture. Analysis of pigments (intermediates of the tetrapyrrole biosynthetic pathways) were conducted using the reverse phase HPLC procedure outlined by La Rocca et al (2001) using a 5 µm Modulo-Cart QS Zorbax ODS column (250 x 4.6 mm; Laubscher Labs, Switzerland ) fitted to a Spectra system HPLC with a fluorescence detector. Solvent A consisted of 60:40:0.03 (v/v) acetone: water: acetic acid and solvent B was 100:0.03 acetone: acetic acid. Pigments were separated at a flow rate of 1 ml min−1 by a linear gradient programmed as follows (minutes; % solvent A; % solvent B): (0; 100; 0), (5; 100; 0), (20; 0; 100), and (25; 100; 0). All solvents were of HPLC grade. Porphyrins were detected by their fluorescence using the following excitation and emission (em/ex) wavelengths: 18 Material and methods Intermediates Name Excitation Emission Protoporphyrin IX 404 625 Mg-Protoporphyrin IX 416 594 Mg-Protoporphyrin IX 416 594 430 630 6-aminomethyl ester Protochlorophyllide a 2.2.4.2 C. annuum chromoplasts For the characterization of chromoplasts, the recovered bands (A, B, C, D, E and F) were examined under the light microscope using an Axioplan 2 microscope (Zeiss, Wetzikon, Switzerland) to check their purity and intactness. Band D (interphase 30%-40%) contained the highest amount of plastids as compared to the other bands. 2.2.5 Fractionation and isolation of plastid proteins 2.2.5.1 BY-2 plastids 2.2.5.1.1 Fractionation of BY-2 Plastid proteins With the isolated BY-2 plastids I employed extensive protein fractionation techniques, consisting of a serial protein extraction followed by liquid chromatography (LC) and SDS-PAGE (Laemmli, 1970). Starting from intact BY-2 plastids, the proteins were first separated by their differential solubility, a procedure that is referred to as “serial extraction “. During this procedure proteins were solubilized from membranes with buffers of increasing solubilization capacity providing information of membrane association for every identified protein. Here, the proteins were fractionated, using four different buffer compositions, into soluble proteins (OSMO), peripheral (8M urea) and two integral membrane protein fractions, CHAPS and 5% SDS. During the serial extraction procedure after each step, insoluble material was precipitated by ultracentrifugation (100,000 x g for 45 minutes), washed twice and subsequently used for the next extraction step. The buffer compositions of each step were as follows: First step (OSMO) 40 mM Tris/HCl pH 8.0, 5 mM MgCl2, 1 mM DTT (dithiothreitol) and 2 x protease inhibitor cocktail (Roche Diagnostics GmbH, Germany, 2 x of supplier’s recommended 19 Material and methods concentration); second step (8M Urea) 8 M urea, 20 mM Tris-base, 5mM MgCl2, 20 mM DTT and 2 x protease inhibitor; third step (CHAPS) 7 M urea, 2 M thiourea, 20 mM Tris-base, 40 mM DTT, 2% cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPS 1% Brij (3-[(335 (polyoxyethylene (23) lauryl ether), 2 x protease inhibitor; fourth step(SDS) 40 mM Tris base, 5% SDS, 40 mM DTT and 2 x protease inhibitor. ~ 5 µg of samples from each fraction were loaded on the 10% SDS-PAGE and the gel was stained by silver stain to get an idea of protein bands from each fraction. Soluble proteins and peripheral membrane proteins were further fractionated by chromatography liquid on chromatography MonoQ (Bio Rad, using ion Hercules, exchange USA) or (IEX) affinity chromatography on Cibacron Blue Sepharose 3 GA (Sigma, Buchs, Switzerland) to subtract highly abundant purine nucleotide binding proteins. Proteins bound to MonoQ were eluted in six steps ranging from 50 mM, 100 mM, 200 mM, 500 mM, 1M to 2M KCl in a buffer containing 20 mM HEPES/KOH pH 7.9, 8M urea, 100 mM NaCl, 5 mM MgCl2, 10 mM DTT, 2 x protease inhibitor. Where indicated, highly abundant purine nucleotide binding proteins were subtracted from protein fractions by Blue Sepharose affinity chromatography in step 1 buffer prior to IEX chromatography. Bound proteins were eluted with step 1 buffer including 1.5 M KCl. The total amount of proteins in each fraction was determined by Bradford analyses (Bradford, 1976). Proteins from each fraction were further fractionated according to their molecular mass by 10% SDS-PAGE (Laemmli, 1970). Integral membrane proteins (CHAPS and 5% SDS) were directly subjected to SDS-PAGE. 2.2.5.1.2 2D-PAGE with BY-2 Plastid proteins Traditionally, protein solubilization for 2D-PAGE is carried out in a buffer containing chaotropes (7M urea and 2M thiourea), zwitterionic detergents (2% CHAPS), reducing agent (DTT or TBP), Carrier ampholytes (CA) and protease inhibitors (Görg et al., 2004). In the first dimension, buffers are important to reduce streaking and to increase the number of resolved proteins (Görg et al., 2004). BY-2 plastids were resuspended in approximately 100 µl of solubilization buffer containing 40 mM Tris base, 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% Brij 35, 0.4 % carrier ampholytes, 2 mM TBP, 40 mM DTT, 20 Material and methods complete EDTA-free protease inhibitor mixture resulting in a protein concentration of at least 1 µg /µl. Any insoluble material was spun down by ultracentrifugation for 45 min at 100,000 x g, which resulted in a much higher number of spots and good separation of proteins in the first dimension. For the first dimension, 160 µg of protein were loaded onto 24-cm long strips with an immobilized linear pH gradient from 4–7 (Bio-Rad, USA) by in-gel rehydration. Rehydration was performed overnight in solubilization buffer without Tris-base according to the manufacturer’s instructions. Proteins were focused using the IPGhor (GE Healthcare, formerly Amersham Biosciences) by the following voltage gradients Step 1 2 3 4 5 6 7 8 Voltage 500 1000 1000 2000 2000 4000 8000 8000 Type Step/Hold Gradient Step/Hold Gradient Step/Hold Step/Hold Gradient Step/Hold Time(hours) 1 1 2 0.3 2 4 1 5 After the focusing I performed one additional step to refocus the IPG strip at 4000 V for 1 hour in Step/Hold mode. Focused strips were immediately used for the second dimension. The second dimension was performed in house-made homogeneous 12% polyacrylamide gels. The buffer composition for SDS-PAGE gel was (12% monomer solution 37.5:1, 1.5 M Tris-HCl pH 8.8, 0.1 % SDS, 0.05 % ammonium persulfate, 0.033 % TEMED in MilliQ water) and the displacing solution was (0.375 M Tris-HCl pH 8.8, 50% glycerol, few crystals of bromophenol blue in MilliQ water). Before proceeding to the second dimension, the focused IPG strips were equilibrated in dry strip equilibration tray with SDS-equilibration buffer (20 mM Tris-HCl pH 8.8, 6M urea, 30% glycerol, 2% SDS, few crystals of bromophenol in MilliQ water) with 1 % DDT and 2.5 % iodoacetamide (IDA) for 15 min each at room temperature. For the electrophoresis we used Ettan Dalt II unit (GE Healthcare, formerly Amersham Biosciences) with constant Watt in following steps, 21 Material and methods Step 1: 2.5 W/gel for 30 min Step 2: 17 W/gel for 4.5 h. The composition of the SDS electrophoresis buffer was 25 mM Trisbase, 192 mM glycine, 0.1% SDS in MilliQ water. To overlay the gels, 0.5 % Agarose sealing solution (0.5 % Agarose, 0.002% bromophenol blue in 1X SDS Electrophoresis buffer) was used and it was made sure that there were no air bubbles underneath the strip. After electrophoresis, gels were transferred into the fixing solution (10% methanol, 7% acetic acid in MilliQ water) for overnight shaking at 50-100 rpm on an orbital shaker at room temperature. After the fixing solution, gels were stained by the fluorescent protein detection technique (SYPRO® Ruby, Molecular Probes Europe BV, Leiden, The Netherlands). SYPRO® Ruby offers a broad dynamic range for quantitative purposes and it was chosen due to its simplicity and reproducibility, as well as its nonselective nature and excellent compatibility with MS (Patton, 2000). Thereafter, gels were washed three times, two times with MilliQ water and one time with fixing solution for 30 min at room temperature. After staining, the gels were scanned with the help of a Typhoon 9400 scanner (GE Healthcare, formerly Amersham Biosciences). The gels were scanned at 100-200 microns/ pixel and other criteria are as under; Emission filter PMT Laser Sensitivity Focal plane 610 BP 30 SYPRO Ruby, ROX, EtBr 495-525 Blue (488) Normal Platen For further analysis, gels were stored in the fixing solution at 4 oC in the dark. 2D-PAGE was analyzed with the proteome weaver software v3.0 (Definiens, Munich, Germany). For the BY-2 plastids analysis five replicate gels were performed and they were integrated into an average gel. The average gel was used for further proteome analysis. 2.2.5.1.3 Analysis of protein complexes (Blue Native-PAGE) Blue native polyacrylamide gel electrophoresis (BN-PAGE) was first introduced by Schägger and Jagow (1991). It is a charge shift method, where 22 Material and methods the electrophoretic mobility of the proteins is mainly determined by the negative charges of bound Coomassie dye and aminocaproic acid serves to improve the solubilization of membrane proteins. The protein complexes were first solubilized by a solution containing a mild non-ionic detergent. Due to Coomassie blue G 250 negatively charged complexes are separated on a non denaturing gel. With this technique, uniform acrylamide gels are not suitable to separate large protein complex masses. This technique is suitable to separate water soluble and membrane protein complexes in the range of 100 to 1000 kDa. To initiate the BN-PAGE, the same procedure was followed as described previously by Baginsky et al (2001). One hundred micro-liters of the BY-2 plastid protein fractions were incubated with 25 µl of an incubation buffer (300mM Tris-HCL pH 7.0, 10 mM DDT, 5 mM EDTA, 3.75% Serva Blue G dye in water) for 30 min at room temperature. Higher stringency of complex association was achieved by adding 1 M urea and 10 mM DDT to the 100 µl of BY-2 plastid fraction. Insoluble materials were spun down at 10000 g for 5 min. Then fractions were loaded onto a 5% native polyacrylamide gel (5% acrylamide 30/0.8, 0.5M Tris-HCL pH 7.0) and the electrophoresis was performed in the cold room; a voltage of 100 V was applied until the protein samples reached the bottom of the gel (6-12h). The electrophoresis buffer contained 25 mM Tris and 192 mM glycine and the cathode buffer also contained 0.02% Serva blue G. The interesting bands (5) were cut for further MS analysis. 2.2.5.1.4 Preparation of the NP40-insoluble fraction from BY-2 plastid Protoplasts were prepared as described by Nagata et al (1992) with modifications. Three day old cells (~160 g) were collected from 1.5 liter of culture by passing two layers of miracloth (Calbiochem, Switzerland). 500 ml of enzyme solution (1% Onozuka RS cellulose, 0.1% pectolyase Y-23 containing 0.4 M mannitol pH, 5.6) were added and the cells were incubated at 30 °C for 90 min. In the meantime the cell suspension was agitated mildly every 15-20 min. The enzyme solution was removed by centrifugation at 200 x g for 5 min at 4 °C. Thereafter the protoplasts were washed three times with five volumes of ice-cold 0.4 M mannitol, pH 5.0. The protoplast pellet was 23 Material and methods resuspended in 0.4 liter nucleoid isolation buffer (0.5 M Sucrose, 20 mM TrisHcl (pH 7.6), 0.5 mM EDTA, 7 mM 2-mercaptoethanol, 1.2 mM spermidine, 0.4 mM PMSF) and then the protoplasts were disrupted by passing several times through two layers of a 40 µm mesh under high pressure. The disrupted protoplasts were centrifuged at 220 x g for 12 min at 4°C to remove cell debris and the supernatant was filtered several times through layers of 20 µm mesh under pressure. Percoll was added to the filtrate at 7.5% to the final concentration (V/V) and were sedimented the BY-2 plastids by centrifugation at 10000 x g for 20 min at 4°C. The BY-2 plastids were resuspended into 80 ml nucleoid isolation buffer containing 15% (V/V) Percoll and the suspension was filtered through two layers of 20 µm mesh under light pressure. Afterwards, BY-2 plastids were centrifuged at 15000 x g for 20 min at 4°C. The BY-2 plastids were resuspended in 10 ml of nucleoid isolation buffer and filtered through two layers of 20 µm mesh under light pressure. The filtrate was overlaid onto a discontinuous sucrose density gradient (8 ml of each 30%-50%-70% sucrose in nucleoid isolation buffer) and centrifuged at 7000 x g for 30 min at 4°C with a swinging-bucket rotor. A yellow band at the 70-50% sucrose interface was recovered, diluted with 100 ml of nucleoid isolation buffer and filtered through layers of 20 µm mesh under light pressure. After the filtration the suspension was incubated at 26°C for 2 min. Five ml 20% Nonidet P40 (NP40) were added to the suspension and stirred for 15 min at room temperature. Before centrifugation, the clarified solution was chilled on ice and the suspension was centrifuged at 4400 x g for 15 min at 4°C. The suspension was filtered through two layers of 20 µm mesh under light pressure. At the end, the suspension was centrifuged at 38000 x g for 40 min at 4°C to sediment the NP-40 insoluble fraction. The clear supernatant was removed and the NP-40 insoluble fraction was stored at – 80 °C. Proteins from the NP-40 insoluble fraction were fractionated using three buffer compositions, crude, soluble proteins (OSMO) and membrane protein fractions (CHAPS). The buffer composition of each step is as follows: In the crude fraction, the isolated BY-2 plastid nucleoids were dissolved in 10 X Laemmli buffer (0.625 M Tris, 10% SDS, β-mercaptoethanol 20%, glycine 10%, 0.5% bromphenolblue pH 6.8), OSMO ( 40 mM Tris/HCl pH 8.0, 5 mM MgCl2, 20 mM DTT and 2 x protease inhibitor cocktail), CHAPS (7 M 24 Material and methods urea, 2 M thiourea, 40 mM Tris-base, 20 mM DTT, 2% CHAPS, 1% Brij 35, 2 x protease inhibitor). Each fraction (Crude, OSMO and CHAPS) were loaded onto 10% SDS-gels to increase the protein coverage of the BY-2 plastid nucleoids. 2.2.5.2 C. annuum chromoplasts With the isolated chromoplasts, a multidimensional protein fractionation strategy was employed to increase the dynamic range of proteome research. A serial extraction procedure was used for protein fractions as described for the BY-2 plastid, but with some modifications. The proteins were solubilized using three different buffer compositions, soluble proteins (OSMO), peripheral (8M urea) and 5% SDS. The buffer compositions of each step were as follows; First step (OSMO) 40 mM Tris/HCl pH 8.0, 5 mM MgCl2, 1 mM DTT and 2 x protease inhibitor cocktail), second step (8M urea) 8 M urea, 20 mM Tris-base, 5mM MgCl2, 20 mM DTT and 2 x protease inhibitor, third step(SDS) 40 mM Tris base, 5% SDS, 40 mM DTT and 2 x protease inhibitor. In this proteome study, I also included crude extract beside three fractionated steps to increase the coverage of protein identifications. The crude extract was resuspended in 10X Laemmli buffer (0.625 M Tris, 10% SDS, 20% βmercaptoethanol, 10% Glycine, 0.5% bromphenolblue pH 6.8). Proteins from each fraction were further fractionated according to their molecular mass by 10% SDS-PAGE (Laemmli, 1970). 2.2.6 Protein identification by Mass Spectrometry (MS) 2.2.6.1 ESI-Ion Trap Mass Spectrometry In-gel digestion All protein fractions, excluding those obtained by BY-2 2D-PAGE, were analyzed by LCI-ESI-MS/MS (LCQ-Deca XP, Thermo Finnigan, San Jose). Prior to mass spectrometry, all protein mixtures were directly subjected to SDS-PAGE by loading 30 µl per lane onto 10 cm long homogeneous 10% polyacrylamide gels. After electrophoresis, the gel strips were cut into 10 pieces, except C. annuum chromoplast gels, which were cut into 12 segments. Proteins in each section were immediately subjected to in-gel 25 Material and methods tryptic digest (Shevchenko et al., 1996). The buffer composition and protocol for in-gel digestion is as follows: Buffer composition 1x digestion buffer 25mM ammoniumcarbonate pH 7.8 2x digestion buffer (DB) 50mM ammoniumcarbonate pH 7.8 100% Acetonitrile (ACN) 5% Formic Acid (FA) Trypsin-solution 1x digestion buffer trypsin to protein ratio (1:20 W/V) Protocol for in-gel digestion Tryptic digest Incubation time 2x 30 sec Remarks Wash Buffer 100 % ACN 5 min Incubation 1 min Wash 5 min Incubation 1 min Wash 100 % ACN 2x 30 sec Wash 100 % ACN 30 min at 45 °C Incubation 2x DB Supernatant 2x DB 10mM DDT in 2x DB Wash 100 % ACN 2x 30 sec Wash 100 % ACN Incubation Always remove 100 % ACN 2x 30 sec 15 min General remarks 50mM Iodoacetamide in 2x DB 2x 30 sec Wash 100 % ACN 5 min Incubation 2x DB 1 min Wash 5 min Incubation 1 min Wash 100 % ACN 2x 30 sec Wash 100 % ACN 20 min at RT Incubation With open lid Overnight at 25 °C Incubation with trypsin solution 100 % ACN 2x DB 26 Material and methods Elution of tryptic peptides 2x 5 min Incubation 100%ACN 10 min Incubation 5% FA 5 min Incubation 100%ACN 10 min Incubation 5% FA 5 min Incubation 100%ACN Collect supernatant After elution, all tryptic peptides were lyophilized to dryness and stored at -80 °C until MS-analysis. LC-ESI-MS/MS Before identification by mass spectrometry, all tryptic peptides were separated by reversed phase-liquid chromatography (RP-LC) on a C18 matrix. Peptides were separated either on an MTVC15- C18w150 capillary column (Micro-Tech Scientific, Inc., Sunnyvale) for electrospray ionization (ESI) or on laboratory made silica capillary columns with an inner diameter of 75 µm (length 8 cm, BGB Analytik AG, Böckten, Switzerland) for nanospray ionization (NSI) packed with C18 reversed phase material (Magic C18 resins; 5 µm, 200-Å pore; Michrom BioResources, Auburn, CA). Twenty µl (ESI) or 5 µl (NSI) of peptides were resolved in buffer A (5% acetonitrile, 0.5% formic acid, or 0.5% acetic acid in Millipore water), centrifuged at 16000 x g to pellet small remaining gel pieces or alternatively Zip-Tips were used. The samples were loaded manually or alternatively an auto sampler was used. The peptides were eluted with an increasing concentration of acetonitrile (Buffer B, 0.5% formic acid or 0.5% acetic acid in acetonitrile) at a flow rate of 300 nl/min (NSI). RP-LC was coupled online to an LCQDecaXP ion trap mass spectrometer (Thermo Finnigan, San Jose) equipped with either an ESIsource or a nanospray source. Analysis was performed in the positive ion mode and peptides were ionized with a spray voltage of 3–4 kV for ESI and 2.1–2.8 kV for NSI. The below mentioned tune method was used in every analysis. The method was based on default parameters with an automated tuning file in a data dependent manner. The measuring method comprised four scan events. In the first scan event, peptide masses were measured while in the remaining 27 Material and methods scan 2 to 4, the most intense ions from scan event 1 were selected in a window of +/- 2 Da and further fragmented and the spectra of these peptides were acquired. MS running time: 125 min Number of scan events: 4 Scan event details: 1. Mass range 450.0-2000.0 m/z 2. MS/MS on most intense ion from (1) 3. MS/MS on 2nd most intense ion from (1) 4. MS/MS on 3rd most intense ion from (1) Dynamic exclusion was used to avoid measuring the same abundant peptide repeatedly. This function allows for the exclusion of a certain parent mass from MS/MS analysis. The repeat count is the number of spectra that are produced with the same parent mass in a time window of 1 min. Every further peptide with such a parent mass would be ignored during the time window. Repeat count: 2 Exclusion duration: 1.00 min Gradient for the reverse phase LC-Chromatography Time (0.00-125.0min) 0.00 0.02 15.00 75.00 82.00 88.00 98.00 100.00 105.00 125.00 Buffer A (%) 100 100 90 40 20 0 0 100 100 100 Buffer B (%) 0 0 10 60 80 100 100 0 0 0 Flow rate (µl/min) 25 25 50 50 50 50 50 50 50 50 2.2.6.2 MALDI-TOF/TOF Mass Spectrometry Before the start of the MS-analysis, protein spots were excised, digested and targeted to the MALDI plate. For this purpose, the GelPix robot (Genetix, UK) for protein spot excision was used. This robot is designed for image analysis and to excise protein spots from SYPRO Ruby, Silver and Coomassie stained 28 Material and methods gels. The excised spots were automatically transferred into 96 well plates and the 96-well plates were transferred to the Genesis ProTeam 150 platform (Tecan, Zurich, Switzerland) for the performance of in-gel digestion, extraction and purification of peptides and spotting onto MALDI target plates. Buffer compositions and their functions were as follows: Solution D: (20% H2O, 80% acetonitrile) 100 ml Solution G: (50 mM ammonium bicarbonate in70% H2O, 30% acetonitrile) 100 ml Digestion solution: (5 mM Tris-Hcl pH 8.3, 2 mM CaCl2) 4 X 1 ml Trypsin Solution: (6.7 ng/µl trypsin in digestion buffer) 4 X 1 ml Alkylation solution: [100 mM IAA (Iodoacetamide)] 4 X 1 ml Reducing solution: [10 mM TCEP {tris-(2-carboxyethyl) phosphine}] 4 X 1 ml For the digest procedure, the TecanProTeam standard protocol was applied as mentioned below, the tryptic digest was performed at 37 °C for 3 hr using sequencing grade trypsin. Before peptide extraction (after the tryptic digestion step) the plates were stored at -20°C. Time Buffer Temperature remove excess water from the gel plugs 300 sec 50µl solution G 37 °C 1800 sec 5 µl reducing solution 37 °C 1800 sec 5 µl alkylation solution Room temperature (RT) remove reducing and alkylation solutions 120 sec wash gel plugs with RT solution G remove residual of reducing and alkylation solutions 600 sec evaporate residual 60 °C solutions 180 sec incubation RT 600 sec 80 µl solution D RT remove solution D 600 sec evaporate solution D 180 sec incubation 60 °C RT 29 Material and methods 300 sec trypsin solution 7200 sec digestion solution RT 37 °C ZipTip desalting and spotting onto MALDI target plate Solution A (0.1% Trifluoroacetic acid (TFA); wash solution for ZipTips) 25 ml Solution C (Isopropanol; wash solution for the fixed tips) 50 ml Solution J (1% TFA; acidification of digest solution) 50 ml Solution K (20% TFA solution, 80% ACN; pre-wetting solution for ZipTips) 25ml MALDI matrix solution (3-5 mg α-cyano-4-hydroxycinnamic acid, 65% ACN, 35% 0.1% TFA) 1ml After digestion, samples were cleaned up using ZipTips (Millipore, Switzerland) and spotted onto the MALDI plate. Time Buffer ZipTip washing steps 2 x 5 µl Solution K 2 x 5 µl Solution A 900 sec 12 µl Solution J extraction of peptides (with ZipTip) peptides bound to the ZipTip by pipetting up and down 5 x 10 µl sample mix. peptides were washed on the Ziptip with 2 x 5 µl solution A. spotting on the MALDI target 0.8 µl MALDI matrix solution spotted onto the target peptides were eluted by pipetting the matrix solution 3 x up and down on the target The target ready for the instrument 30 Material and methods The samples were analysed on a 4700 Proteomics Analyser MALDITOF/TOF system (Applied Biosystems, Framingham, MA, USA) equipped with an Nd: YAG laser operating at 200 Hz. All mass spectra were recorded from 750 to 4000 Da in positive ion mode. They were generated by accumulating data from 5000 laser pulses. First, MS spectra were acquired from the standard peptides. Subsequently, MS spectra were recorded for all sample spots on the plate and internally calibrated using signals from autoproteolytic fragments of trypsin. Up to five spectral peaks per spot that met the threshold criterion (S/N > 60) were included in the acquisition list for the MS/MS spectra. Peptide fragmentation was performed at a collision energy of 1 kV and a collision gas pressure of approximately 2 x 10-7 Torr. During MS/MS data acquisition a method with a stop condition was used. In this method, a minimum of 3000 laser shots (60 sub-spectra accumulated from 50 shots each) and a maximum of typically 6000 shots (120 sub-spectra) were allowed for each spectrum. The additional laser shots was halted whenever at least 4 ions with a S/N of at least 50 were present in the accumulated MS/MS spectrum, in the region from m/z 200 to 90% of the precursor mass. 2.2.7 Identification of proteins from MS/MS data 2.2.7.1 SEQUEST search engine The MS/MS data generated by the LC-ESI-MS/MS were analyzed by the SEQUEST search engine (Thermo Finnegan, San Jose, USA) in combination with the NCBI (http://www.ncbi.nlm.nih.gov/) data base. SEQUEST is a computer algorithm that converts the character based representation of amino acid sequences in a protein database to a fragmentation pattern that can be used to match fragment ions in a tandem mass spectrum. It initially identifies amino acid sequences in the database that match the measured mass of the peptide ion and predicts the fragment ions expected for each sequence. A score is calculated for each amino acid sequence by matching the predicted ions to the ions observed in the tandem mass spectrum and this score is called Xcorr. 31 Material and methods For the BY-2 plastids, I used the NCBI non-redundant (nr) protein database including the contaminants (download of December 15, 2003) and for the BN-PAGE the same database was used. For the NP-40 insoluble fraction, the NCBI viridiplantae database (download of January 02, 2005) was used and for C. annuum chromoplast proteome analysis, MS/MS data was searched against the NCBI viridiplantae database including the contaminants (download of November 24, 2005). Dta files were created by the SEQUEST software for every MS/MS scan with a total ion count (TIC) of at least 5 x 104, minimal peak count of 35, and a precursor ion mass in the range of 300–2000 m/z. Data were searched against the database indexed for speed restricting, the search to tryptic peptides with modifications (cysteines were allowed to be either unmodified or carboxyamidomethylated). 2.2.7.2 MASCOT search engine The raw files generated by MALDI-TOF/TOF mass spectrometry were searched using the MASCOT (Matrix Science, London, UK) (www.matrixscience.com) search engine. All searches were performed against a non redundant database from NCBI (http://www.ncbi.nlm.nih.gov/) (download from December 15, 2003) including the contaminants. GPS (Global Proteome Server) Explorer software (Applied Biosystems) was used for submitting data acquired with the MALDI-TOF/TOF mass spectrometer for database searching. The following search settings were applied; Maximum number of missed cleavages: 1 Peptide tolerance: 25 ppm Peptide charge: 1+ MS/MS tolerance: 0.2 Da Carboxyamidomethylation (C) of cysteine was set as fixed modification and oxidation (O) of methionine was selected as variable modification. Normally, just a single criterion for positive identification of proteins (based on MALDI-TOF/TOF data and the GPS/Mascot software) is used. For every search, a significant threshold value for the Mascot score is calculated. Protein scores greater than this value are significant (p < 0.05). In other words, the protein score confidence interval is greater than 95 %. 32 Material and methods The significance threshold value for the Mascot score is variable and depends on the database and on a number of search parameters. These are criteria for a positive identification of proteins: 1. The total ion score confidence interval calculation (C.I. %) should be above 90%.The total ion score is the sum of all scores calculated for the MS/MS spectra. However, for in-gel digests of spots, the protein ion score, which includes the scores for the assigned peptide masses, is more relevant. 2. The MS match consisted of a minimum of two peptides. 3. The matched peptides covered at least 20% of the whole protein sequence. 4. 50 ppm or better mass accuracy. 2.2.7.3 Manual data interpretation To exclude any false positive protein identification I manually interpreted each SEQUEST output by filtering the peptide hits using stringent hierarchical criteria as described under: 1. A cross-correlation score (Xcorr) of at least 2.5. 2. An ion coverage (ratio between detected and expected y- or b-ions) of more than 40%. 3. Only fragment-spectra from doubly charged parent-ions were considered 4. Long peptides with more than 50 theoretical fragment ions (b- and y-ions) were not taken into account. 5. Grouping of at least four peptides with an Xcorr value of at least 2.5 to the same protein was rated as a significant protein identification when at least one of them exhibited a CN value (normalized difference in correlation score, giving the difference between the front-ranking hit and the following possible hits) higher than 0.1. 6. The other remaining MS/MS spectra were visually examined for a correct peak assignment and evaluated considering the following standard: i. A gapless assignment of a series of y or b ions to peaks of high intensity. ii. A y-ion with an N-terminal proline that corresponded to a high peak (in most cases the highest peak in the entire MS/MS spectrum) or having a corresponding unassigned doubly charged fragment ion peak. iii. A neutral loss of 18 mass units (loss of water) from ions carrying the amino acids serine or threonine. 33 Material and methods 7. For peptide identification with CN value lower than 0.1, the other spectra of the lower ranking peptide hits were also examined for significance considering the above criteria. Peptide hits without a significant difference to lower ranking hits or with a sequence identical to peptides from other proteins were not considered. 2.2.7.4 Quality scoring for MS-spectra In MS-based proteomics large amounts of data are collected in a single experiment. A small fraction of the data is then used to produce identification of peptides and proteins. A significant portion of the data is missed for peptide and protein identification. The reason for this is that the machine is acquiring the data all the time and many of the MS/MS scans are of low quality and cannot be identified in the database search directly. Nevertheless, there are often many spectra that produce good fragmentation spectra but can still not be identified by the database search engine. For this reason, an approach was employed to identify the quality of the spectra by using the QualScore tool (Nesvizhskii et al, 2006). This computational tool was used to find those high quality spectra that could not be assigned in the first database search. This approach employed on the C. annuum chromoplasts to increase proteome coverage. MS/MS data were first searched by the SEQUEST software against viridiplantae database and peptide assignments to the spectra were processed using the PeptideProphet (Keller et al, 2002). The results obtained from the database search were loaded into the QualScore tool. In this investigation, 83 mass spectrometry runs were analyzed with the outcome of 302430 total spectra. In the first set of search, 4793 were identified with a confidence score of higher than 0.9. In the QualScore run, 8666 spectra were extracted with the quality score of >1 which had not been identified in the first database search. 2.2.7.5 De Novo peptide sequencing I first analyzed the mass spectrometric data from altogether 83 LCQ analytical runs to identify those spectra that were left unassigned by the standard database search described above despite originating from true peptide fragmentation. Collision induced peptide dissociation (CID) generates spectra 34 Material and methods with a set of well defined characteristics that distinguish them from low-quality noise spectra or non-protein contaminant fragmentation. These high quality spectra spectra (8666) were subsequently submitted to a local version of PepNovo v1.03 PepNovo (Provided by Mr. Ari Frank, University of California, San Diego, USA) to extract an amino acid sequence exclusively from the information contained in the MS/MS spectra using PepNovo default parameters (http://peptide.ucsd.edu/pepnovo.py). The parameters for the PepNovo were used as default mode according to the manufacturer’s recommendations. All these parameters were assembled in a tryp_model.txt file. Only those PepNovo results were accepted that received a mean reliability score of at least 0.5. To make the results more accurate, I removed all keratin and trypsin related spectra in the first run and again loaded the PepNovo out file into the MS-BLAST searching software. 2.2.7.6 MS-BLAST The output of the PepNovo is further processed in a way to make it convenient in conjunction with the MS-BLAST. An additional amino acid letter B is added at the N-terminus of the peptide (B in MS-BLAST can be either a lysine or an arginine). Gaps were filled with X, double X, triple X or quadruple X (X in MS-BLAST can be any amino acid) according to the gap length. If a gap was ambiguous, the sequence was written several times to produce a MS-BLAST query according to MS-BLAST rules. Before loading the PepNovo output on the MS-BLAST, I removed all spectra related to keratin and trypsin proteins. All the complete and partial peptide sequences obtained by PepNovo were submitted via the web interface in the MS-BLAST searching software (http://dove.embl-heidelberg.de/Blast2/msblast.html) and searched against the NCBI non-redundant protein database using MS Blast default parameters. The search parameters for MS-BLAST were edited as proposed by the manufacturers (Shevchenko et al., 2001). The outputs of MS-BLAST were displayed in a web page and these results were filtered manually according to the following criteria; • Only accepted the hits as a true positive which had the MS-BLAST score 62 or higher. • Hits were accepted which belong to higher plants. 35 Material and methods • Hits were accepted if they had a plastid or mitochondrial transit peptide. • Only those single hits were accepted which had predicted transit peptide and MS-BLAST score more than 65. In this analysis, I always found several peptides from one particular protein which increases the reliability of this approach. Most of the identified proteins were already identified during the database search and essentially reproduced the database findings. Additionally, a significant number of trypsin and keratin spectra were identified that escaped the original database detection. 2.2.8 Bioinformatics analysis 2.2.8.1 Protein localizations All the identified proteins were assembled into a database in FASTA format and a BLAST search (Altschul et al., 1997) was performed against itself to eliminate redundant protein entries i.e., identical proteins from different organisms (all entries giving an E-value of zero were detected). All identified BY-2 plastid proteins were deposited in a database called PLprot (www.pb.ethz.ch/proteomics). This database contains all information about the Arabidopsis chloroplast and Rice etioplast proteomes. All the identified proteins from every fraction (BY-2 plastids, NP-40 insoluble fraction, BY-2 plastid 2D-PAGE and C. annuum chromoplast) were first carefully analyzed to substantiate their plastid localization. I carefully analyzed the identified proteins in more detail for their plastid localization. In addition to the plastome-encoded proteins, I accepted all proteins as true plastid proteins if they have 1. A predicted plastid transit peptide using the TargetP programme (http://www.cbs.dtu.dk/services/TargetP/) [Emanuelsson et al., 1999]. This programme is well defined to predict the subcellular protein targeting for plastids, mitochondria, the secretory pathway and any other localization. 2. A reported plastid function in the literature. 36 Material and methods 3. An Arabidopsis thaliana orthologue that has an identical plastid transit peptide. 2.2.8.2 Metabolic pathway modeling We classified the proteins into 16 major metabolic pathways. Putative protein functions were assigned by different means like: 1. With the help of publicly available software tools e.g., i) KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.ad.jp/kegg/kegg2.html) ii) Munich information center for protein sequences (http://mips.gsf.de/) 2. Their functions were manually assigned by literature search. 37 Results 3. Results 3.1 BY-2 plastids 3.1.1 BY-2 plastid isolation and purity BY-2 cell culture plastids have many properties in common with undifferentiated proplastids (Introduction, section 1.1.1 Plastids for Bright Yellow (BY-2) Cell Culture). Therefore, this cell culture was used as a model system for proteomics with undifferentiated plastids. BY-2 plastids were isolated by sucrose density gradient centrifugation. Before proceeding to protein fractionation and mass spectrometry, the purity of the isolated BY-2 plastids was first established using enzymatic assays (fumarase and catalase measurements) in combination with Western blot analysis (antibody detection) [Figure 3.1]. After the first sucrose density gradient centrifugation, both fumarase and catalase activities which are diagnostic for mitochondria and peroxisomes, showed one major peak at the interphase between 30% and 50% sucrose (Band A, Figure 3.1, II), while a plastid marker protein (TOC-75) was enriched at the interphase between 50% and 70% sucrose (Band B, Figure 3.1, II). This suggests that peroxisomes and mitochondria are enriched in Band A, and BY-2 plastids are enriched in Band B (Figure 3.1, II). BY-2 plastids (Band B) were recovered and loaded onto a linear sucrose gradient. The twelve fractions (from top to bottom) from linear sucrose gradient were recovered and an only enzymatic assay (fumarase and catalase measurements) was done to check the BY-2 plastid purity (Figure 3.1, III). In the linear sucrose gradient, the fumarase and catalase activity is significantly diminished compared to the step gradient (Figure 3.1, II). No significant fumarase and catalase activities could be measured in the linear sucrose gradient. After the linear sucrose gradient, BY-2 plastids (Fractions 9-11, Figure 3.1, III) were washed, concentrated and used for the subsequent protein fractionation and proteome analysis. 38 Results I II 0.45 mt Fumarase Catalase 0.4 pt A Rel. activity [∆E/6min] 0.35 A Mitochondria Plastids B B 0.3 0.25 0.2 0.15 0.1 0.05 0 1 Cell debris III 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Fractions A B 0.06 Fumarase Catalase Rel. activity ∆E/6 min 0.05 0.04 0.03 0.02 0.01 0 1 2 3 4 5 6 7 8 9 Fractions 10 11 12 B Figure 3.1: Isolation and purification of BY-2 plastids. (I) shows the sucrose step gradient centrifugation of BY-2 plastids. (II) shows the purity of BY-2 plastids. The gradient was fractionated from top to bottom into 1 ml aliquots, and for each fraction the fumarase and catalase activites were determined. Additionally, fractions 4 to 7 (Band A) and 13 to 16 (Band B) were pooled and probed with antibodies against plastid TOC75 and mitochondria α-porin. TOC-75 is a component of the translocon at the outer chloroplast envelope membranes and α-porin is a mitochondrial membrane protein which forms the channels that allow passive diffusion of small hydrophilic solutes. [Inset, pt: Plastid marker (TOC75); mt: Mitochondria (α-porin)]. (III) The 2nd sucrose linear gradient was fractionated into 1 ml aliquots, and for each fraction fumarase and catalase activity was determined. 3.1.2 Characterization of the BY-2 plastids To characterize BY-2 plastids, they were first examined under the electron microscope (EM). The EM observations were made at the electron microscope facility of ETH Zurich (www.em.biol.ethz.ch) [Figure 3.2]. The EM picture shows that BY-2 plastids are small organelles with few internal 39 Results structures, resembling proplastids in morphology and size (Kirk and TilneyBassett, 1978). Figure 3.2: Characterization of the BY-2 plastids. The EM picture shows BY-2 plastids with few internal structures which resemble proplastids (Kirk and Tilney-Bassett, 1978). No other organelles were detected supporting the purity of the preparation. 1 µm The next question to investigate was whether BY-2 plastids accumulate intermediates of the tetrapyrrole pathway and, if so, which compound or intermediate accumulated. In etioplasts, protochlorophyllide A accumulates, which is the last intermediate of chlorophyll biosynthesis in the dark (La Rocca et al., 2001; Kim and Apel, 2004; von Zychlinski et al., 2005). For BY-2 plastids, the analysis was done under three conditions: control, ALA feeding for 24 h, and ALA feeding for 96 hours (Figure 3.3). Four intermediates of the tetrapyrrole biosynthetic pathway, i.e. protoporphyrin IX, Mg-protoporphyrin IX, Mg-protoporphyrin 6-monomethyl ester and protochlorophyllide A were detected. In the control, only protoporphyrin IX accumulated and the other intermediates did not accumulate at all. After ALA-feeding, BY-2 plastids accumulated large amounts of protoporphyrin IX (ProtoIX) but only small amounts of Mg-protoporphyrin IX, Mg-protoporphyrin 6-monomethyl ester and a very small amount of protochlorophyllide A (Pchlide a). The ALA (5aminolevulinic acid) feeding enhanced the accumulation of intermediates of the tetrapyrrole biosynthetic pathways as compared to the control treatment (Figure 3.3). 40 Results Mg-Protoporphyrin IX Mg-Protoporphyrin IX 6-monomethyl ester Heme Phytochromobilin Protochlorophyllide a arbitrary units Protoporphyrin IX arbitrary units 5-Aminolevulinic acid 9 8 7 6 5 4 3 2 1 900 800 700 600 500 400 300 200 100 arbitrary units Glutamate 800 700 600 500 400 300 200 100 Chlorophyll a control + 5mM ALA, 24 h + 5mM ALA, 96 h ProtoIX Mg-ProtoIX Mg-ProtoIX PChlide a MME Figure 3.3: Characterization of the BY-2 plastids. The BY-2 plastids did not accumulate protochlorophyllide a in the tetrapyrrole biosynthetic pathway, even after feeding with ALA. This makes them distinct from other plastid types. The green star shows four intermediates of the tetrapyrrole biosynthetic pathway that were dectected. These observations allowed us to conclude that BY-2 plastids do not accumulate Protochlorophyllide A and they are distinct from etioplasts. Etioplasts are present in the leaf cells of plants growing in the dark, but accumulate high levels of protochlorophyllide A, and upon illumination, etioplasts convert into photosynthetically active chloroplasts (Leech, 1986). 3.1.3 BY-2 plastid –Complete proteome analysis 3.1.3.1 Shotgun proteome analysis 3.1.3.1.1 BY-2 plastid protein fractionation In order to fractionate BY-2 plastid proteins, a multidimensional protein fractionation strategy was first devised to increase the dynamic range of the proteome analysis. In this procedure, which is called “serial extraction”, proteins were solubilized from membranes with buffers of increasing solubilization capacity, providing information about membrane association for every identified protein (Figure 3.4). 41 Results BY-2 plastids Serial Extraction O U C S Blue Sepharose Ion exchange SDS-PAGE cut into 10 pieces in gel digest Reversed Phase Chromatography on C18 ESI-MS/MS Figure 3.4: Fractionation strategy for the BY-2 plastid proteome. The multidimensional chromatrography separated proteins according to their different physical and chemical properties. Abbreviations; O: OSMO, U: 8M urea, C: CHAPS, S: 5% SDS. Here, the proteins were fractionated into soluble protein (OSMO), peripheral (8M urea) and two integral membrane protein fractions, CHAPS and 5% SDS. Protein fractions were analyzed by SDS-PAGE and subsequent silver staining (Figure 3.5). Soluble and peripheral membrane proteins were further fractionated by liquid chromatography using ion exchange chromatography (IEX) on MonoQ (Bio Rad, Hercules, USA) or affinity chromatography on Cibacron Blue Sepharose 3 GA (Sigma, Buchs, Switzerland) to subtract highly abundant purine nucleotide binding proteins [example for the OSMO fraction in Fig 3.5, B]. The soluble proteins make up 17% of the total BY-2 plastid proteins. Peripheral membrane proteins (8M urea) make up 47% of the total BY-2 plastid proteins. The integral membrane protein fraction contains 36% of all BY-2 plastid proteins (Table 3.1). 42 X- FT B S 173 173 111 111 80 61 kDa kDa IE S S ea O SD ur AP H SM M C 5% O 8 FT B Sb I E o un X 5 d IE 0 X 10 0 IE X 20 IE 0 X 50 0 IE X 20 00 Results 49 80 61 49 36 36 25 25 19 19 A. Serial Extraction B. Ion Exchange Chromatography Figure 3.5: Serial extraction of BY-2 plastid proteins. (A) The Silver stain of the proteins of each fraction from the serial extraction procedure. (B) Proteins in the OSMO and urea fractions were further fractionated by ion exchange chromatography (IEX), Blue Sepharose to subtract highly abundant purine nucleotide-binding proteins. All integral membrane proteins were already solubilized with buffer containing 7M urea, 2M thiourea, 1% Brij 35 and 2% CHAPS. No additional proteins were solubilized with 5% SDS (Figure 3.5, A). In contrast, most of the integral membrane proteins from chloroplasts require 5% SDS for solubilization (Kleffmann et al, 2004). These different properties of integral membrane proteins are most likely a consequence of alterations in the lipid composition of plastid membranes upon development and differentiation (Vothknecht and Westhoff, 2001). In addition, BY-2 plastids lack a distinctive internal membrane system comparable to the thylakoid system in fully differentiated chloroplasts (Miyazawa et al., 1999). Table 3.1: Quantitative distribution of BY-2 plastid proteins among different fractions of the serial extraction procedure. Values were determined by Bradford analyses. (n.d: not detected). Solubilization step Osmo 8M urea CHAPS 5% SDS Protein total 8.1 mg 22.7 mg 17.6 mg n.d Percentage 17 % 47 % 36 % n.d 43 Results 3.1.3.1.2 Protein identification The goal of our research was to identify the complete proteome of BY-2 plastids. All identified proteins from BY-2 plastids are listed in Table 3.2, including all contaminants. To exclude false positive protein identification, data were manually checked (materials and methods, section 2.2.7.3 manual data interpretation) and to differentiate between plastidic and contaminants I followed the criteria as explained in material and methods, section 2.2.8.1 “protein localizations”. All identified proteins listed in the Table 3.2 were manually sorted into their putative functional categories (material and methods, section 2.2.8.2 metabolic pathway modeling), including the information about the “serial extraction” step from which each protein was identified, i.e. osmotic shock (O), urea (U) or CHAPS (C). a Table 3.2: List of the proteins identified in the BY-2 plastid. a . Provided is the identifier (“Identifier”), the database accession number (“acc. no”), the organism (“organism”), the number of identified tryptic peptides (“no. of peptides”), protein localization [“Localization” Y: transit peptide by ChloroP (Emanuelsson et al., 1999), reported Pt: reported chloroplast function or Arabidopsis orthologue with a transit peptide, pt-encoded: plastid encoded, co: putative contaminant,1(kleffmann et al.,2004), 2 (Froehlich et al., 2002), 3 ( Ferro et al., 2003), 4 (Peltier et al., 2002), -: any other location] and the information from which fraction the protein was identified (“Fraction”), i.e., osmotic shock (O), urea (U) CHAPS (C) and SDS (S). Organism Proteins identified from BY2 plastids FracNo. of Localizion peptides ation Identifier Accession No. Protein gi|7450428 T03270 Nicotiana tabacum O gi|5701896 CAA47373 Nicotiana sylvestris O 6 Y glutamate-ammonia ligase gi|7437011 T07937 C 2 Y tryptophan synthase beta chain gi|114165 P23981 Chlamydomonas reinhardtii Nicotiana tabacum O 5 Y EPSP synthase 1 gi|5822270 1QGNA Nicotiana tabacum O 5 reported Pt cystathionine gamma-synthase gi|6578124 AAF17705 Canavalia lineata O 10 Y ornithine carbamoyltransferase gi|6319165 AAF07191 Solanum tuberosum O 2 Y branched-chain amino acid aminotransferase gi|2492952 Q42884 O 5 Y chorismate synthase 1 gi|1477480 AAB67843 Lycopersicon esculentum Arabidopsis thaliana O 28 Y carbamoyl phosphate synthetase large chain gi|399333 P31300 Capsicum annuum O 9 Y cysteine synthase gi|100437 A35016 Solanum tuberosum O 12 Y cystathionine gamma-lyase gi|6014908 Q42948 Nicotiana tabacum O 3 Y dihydropicolinate synthase gi|3941322 AAC82334 Medicago truncatula O 2 Y gamma-glutamylcysteine synthetase gi|2252472 CAB10698 Arabidopsis thaliana O 2 Y argininosuccinate lyase gi|2811029 O04866 Alnus glutinosa O 21 Y acetylornithine aminotransferase gi|7431768 T16982 U/C 7 reported Pt glutamate dehydrogenase U/C 46 reported Pt glutamate synthase O 2 reported Pt glycine hydroxymethyltransferase U/C 3 co probable glutathione S-transferase Amino Acid Biosynthesis gi|7431781 S67499 Nicotiana plumbaginifolia Nicotiana tabacum gi|7433550 T05362 Arabidopsis thaliana gi|114208 P25317 Nicotiana tabacum 3 reported Pt probable histidinol-phosphate transaminase 44 Results gi|419757 S30145 Arabidopsis thaliana O/U 27 Y ketol-acid reductoisomerase gi|266463 P29696 Solanum tuberosum O 5 reported Pt 3-isopropylmalate dehydrogenase gi|1708993 P53780 Arabidopsis thaliana U 2 Y cystathionine beta-lyase gi|127041 P23686 Arabidopsis thaliana U/C 8 co S-adenosylmethionine synthetase 1 gi|1170938 P43281 U/C 8 - S-adenosylmethionine synthetase 2 gi|1066499 AAB41904 Lycopersicon esculentum Medicago sativa U/C 29 Y NADH-dependent glutamate synthase gi|2982255 AAC32115 Picea mariana C 4 reported Pt probable NADH-glutamate synthase gi|6685804 O24578 Zea mays U 2 Y adenylosuccinate synthetase gi|1711381 P52877 Spinacia oleracea O 4 Y phosphoserine aminotransferase gi|1168260 P46248 Arabidopsis thaliana O 7 Y gi|7573355 CAB87661 Arabidopsis thaliana O 2 Y aspartate aminotransferase diaminopimelate decarboxylase-like protein gi|482934 CAA54043 Nicotiana tabacum U 1 RP glutathione reductase gi|232202 P30109 Nicotiana tabacum U/C 4 co glutathione S-transferase gi|7387849 O04974 O/U 7 Y 2-isopropylmalate synthase B gi|5931761 CAB56614 U 9 reported Pt acetolactate synthase small subunit gi|7488146 T05416 Lycopersicon pennellii Nicotiana plumbaginifolia Arabidopsis thaliana O/U 2 Y probable phosphoglycerate dehydrogenase gi|7431783 T06228 Glycine max O 10 reported Pt Fe-dependent glutamate synthase gi|7109469 AAF36733 Arabidopsis thaliana O 11 Y putative enolase gi|5123836 CAB45387 Nicotiana tabacum O 6 Y NAD-malate dehydrogenase gi|3121731 O04916 Solanum tuberosum U 2 P09317 Ustilago maydis O 19 reported Pt reported Pt aconitate hydratase gi|120714 gi|2833379 Q42581 Arabidopsis thaliana U 2 gi|7431231 T06401 O/U/C 8 gi|2499497 Q42961 Lycopersicon esculentum Nicotiana tabacum reported Pt Y ribose-phosphate pyrophosphokinase 1 O 13 Y phosphoglycerate kinase gi|6690395 AAF24124 Arabidopsis thaliana O 2 Y phosphoglucose isomerase gi|1351271 P48496 Spinacia oleracea O 11 Y triosephosphate isomerase gi|7489279 T07790 Solanum tuberosum O 35 Y transaldolase gi|7433617 T09541 Capsicum annuum O 44 Y transketolase gi|7484671 T09153 Spinacia oleracea O 31 A41370 Arabidopsis thaliana O 7 Y Y glucose-6-phosphate isomerase gi|99742 gi|4827253 BAA77603 Nicotiana paniculata O 2 Y gi|4584503 CAB40743 Solanum tuberosum O 4 gi|4874272 AAD31337 Arabidopsis thaliana U 4 Carbohydrate Metabolism glyceraldehyde 3-phosphate dehydrogenase malate dehydrogenase 2-dehydro-3-deoxy-phosphoheptonate aldolase plastidic aldolase Starch Metabolism 15 Y starch branching enzyme II 1,2 Y strong similarity to gb|Y09533 involved in starch metabolism alpha-glucan phosphorylase, L isozyme 1 gi|130173 P04045 Solanum tuberosum O gi|2833389 Q43846 Solanum tuberosum O 4 Y soluble glycogen [starch] synthase (SS III) gi|267196 Q00775 Solanum tuberosum O 2 Y granule-bound glycogen [starch] synthase gi|21579 CAA36612 Solanum tuberosum O 16 Y starch phosphorylase gi|6939228 AAF31730 Arabidopsis thaliana O 5 Y gi|1172754 Q05728 Arabidopsis thaliana O/U 6 Y gi|4874278 AAD31343 Arabidopsis thaliana O/U 7 Y gi|1097877 2114378A gi|1170242 P42045 Lycopersicon esculentum Hordeum vulgare gi|3334149 O22436 Nicotiana tabacum Nucleotide Metabolism putative phosphoribosylformylglycinamidine synthase phosphoribosylformylglycinamidine cycloligase member of the phosphoribosyl pyrophosphate synthetase family Porphyrin and Chlorophyll Metabolism U 2 Y aminolevulinate dehydratase U/C 2 Y ferrochelatase U 2 Y MG-protoporphyrin IX chelatase 45 Results gi|2493809 Q42840 Hordeum vulgare U 2 Y coproporphyrinogen III oxidase gi|6856558 AAF29977 Tagetes erecta U 2 reported Pt gi|3157931 AAC17614 Arabidopsis thaliana O 2 reported Pt gi|7438127 T02431 Nicotiana tabacum O/U/C 15 Y isopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase similar to pyrophosphate-dependent phosphofructokinase beta subunit acetyl-CoA carboxylase gi|3023817 Q43793 Nicotiana tabacum U 5 Y glucose-6-phosphate 1-dehydrogenase gi|2497541 Q40545 Nicotiana tabacum O 10 Y pyruvate kinase isozyme A pyruvate kinase isozyme G Other Metabolism gi|2497542 Q40546 Nicotiana tabacum U 2 Y gi|7431333 T03406 Nicotiana tabacum U/C 2 reported Pt probable isocitrate dehydrogenase gi|2454184 AAB86804 Arabidopsis thaliana U 4 Y pyruvate dehydrogenase E1 beta subunit gi|3721540 BAA33531 Nicotiana tabacum U/C 26 Y sulfite reductase gi|5532608 AAD44809 Nicotiana tabacum O/U 11 Y 6,7-dimethyl-8-ribityllumazine synthase phytoene synthase gi|585010 P37294 Synechocystis sp U 2 reported Pt gi|2340166 AAB67319 Arabidopsis thaliana U 2 - glutathione S-conjugate transporting ATPase gi|585421 P38418 Arabidopsis thaliana U 4 Y lipoxygenase gi|1552717 AAB08578 Nicotiana tabacum U/C 2 - squalene synthase gi|585449 P37222 O 5 reported Pt NADP-dependent malic enzyme gi|2498077 P93554 O 7 reported Pt nucleoside diphosphate kinase I U 4 reported Pt putative beta-hydroxyacyl-ACP dehydratase U/C 17 Pt-encoded ATPase alpha subunit gi|4567203 AAD23619 Lycopersicon esculentum Saccharum officinarum Arabidopsis thaliana gi|7525018 NP_051044 Arabidopsis thaliana gi|1583601 2121278A Capsicum annuum U 17 Y zeta carotene desaturase gi|75220859 Q39833 Glycine max U 4 Y alfa-carboxyltransferase precursor gi|100284 A39267 O 2 reported Pt superoxide dismutase (Fe) gi|3121825 O24364 Nicotiana plumbaginifolia Spinacia oleracea O 8 Y peroxiredoxin BAS1 gi|4996602 BAA78552 Nicotiana tabacum O 2 Y thylakoid-bound ascorbate peroxidase gi|134672 P11796 Nicotiana plumbaginifolia U 10 1,2,4 superoxide dismutase (Mn) gi|729409 P41342 Nicotiana tabacum U/C 18 Y elongation factor TU gi|133248 P19683 Nicotiana sylvestris O 7 Y 31 kDa ribonucleoprotein gi|133424 P06271 Nicotiana tabacum U 2 reported Pt DNA-directed RNA polymerase beta chain gi|6831644 Q42362 Musa acuminata O 2 reported Pt 30S ribosomal protein S8 gi|4105131 AAD02267 Spinacia oleracea U 8 Y ClpC protease gi|4887543 CAB43488 Arabidopsis thaliana U 2 Pt-encoded ATP-dependent Clp protease subunit ClpP gi|399212 P31541 U/C 12 Y ATP-dependent Clp protease ATP-binding subunit CLPA homolog CD4A CLPB protein Redox Gene Expression Proteases gi|2493734 P74361 Lycopersicon esculentum Synechocystis sp U 4 reported Pt gi|116527 P12210 Nicotiana tabacum O 2 reported Pt gi|399213 P31542 O 13 Y U 3 Y ATP-dependent Clp protease proteolytic subunit ATP-dependent Clp protease ATP-binding subunit clpA homolog CD4B putative aminopeptidase U 4 reported Pt putative FTSH protease gi|6456169 AAF09157 Lycopersicon esculentum Arabidopsis thaliana gi|2077957 CAA73318 Arabidopsis thaliana gi|100424 S17917 Solanum tuberosum U/C 6 co ADP,ATP carrier protein gi|7489246 T07405 Solanum tuberosum U/C 3 co oxoglutarate/malate translocator gi|124429 P23525 Spinacia oleracea U/C 4 Y 37 KD inner envelope membrane protein Transporters and Envelope Proteins 46 Results gi|7267551 CAB78032 Arabidopsis thaliana gi|2499535 Q41364 Spinacia oleracea gi|6226239 O24381 Solanum tuberosum gi|7229675 AAF42936 gi|4101473 C 2 reported Pt outer envelope membrane protein OEP75 precursor homologue 2-oxoglutarate/malate translocator U/C 2 Y U/C 13 Y plastidic ATP/ADP-transporter Arabidopsis thaliana U 2 Y glucose 6 phosphate/phosphate translocator AAD01191 Arabidopsis thaliana U 5 1,2,3, K -efflux antiporter-1(KEA1) gi|7331143 AAF60293 O/U 10 Y chaperonin 21 gi|2829901 AAC00609 Lycopersicon esculentum Arabidopsis thaliana U 2 reported Pt putative 10kd chaperonin + Chaperones gi|1710807 P08926 Pisum sativum gi|134103 P21240 Arabidopsis thaliana O 19 Y rubisco subunit binding-protein alpha subunit U/C 12 Y rubisco subunit binding-protein beta subunit gi|7441880 T08899 Spinacia oleracea C 6 Y dnaK-type molecular chaperone HSC70-9 gi|1729864 P54411 Avena sativa U 4 1,2 T-complex protein epsilon subunit gi|461944 Q04960 Cucumis sativus U 3 1 DNAJ protein homolog gi|4583546 CAB40381 Arabidopsis thaliana U 2 Y GrpE protein gi|4204861 AAD11550 Triticum aestivum U 6 1,2 heat shock protein 90 gi|1708313 P51818 Arabidopsis thaliana C 2 1,3 heat shock protein 81-3 gi|6018208 AAF01789 Philodina roseola U/C 2 1,2 82-90 kDa heat shock protein heat shock protein 90A Heat Shock-related proteins gi|1515105 CAA68885 Arabidopsis thaliana U 2 1,2 gi|7450157 S74252 Arabidopsis thaliana O 2 1,2 heat shock protein 91 gi|1076758 S49340 Secale cereale U/C 15 Y heat-shock protein 82K gi|113217 P23343 Daucus carota gi|7489185 T03843 Nicotiana tabacum Structural maintenance and plastid positioning U/C 2 1 actin 1 C 6 Y prohibitin gi|6016683 AAF01510 Arabidopsis thaliana O 3 1 putative clathrin heavy chain gi|462013 P35016 Catharanthus roseus U/C 10 1,2 endoplasmin homolog gi|7450791 T09555 Arabidopsis thaliana U/C 2 Y fibrillarin gi|7488227 T04622 Arabidopsis thaliana U 6 co prohibitin-like protein gi|464848 P33628 Picea abies U/C 6 1 tubulin alpha chain gi|1351202 P28551 Glycine max U/C 8 1 tubulin beta chain gi|6723394 CAB66403 Arabidopsis thaliana Hypothetical or unknown proteins U 3 Y putative protein gi|7529745 CAB86930 Arabidopsis thaliana U 2 co putative protein gi|2811028 O04658 Arabidopsis thaliana U/C 2 - hypothetical 47.9 kDa protein gi|1076722 S49173 Hordeum vulgare O 2 reported Pt hypothetical protein gi|5042434 AAD38273 Arabidopsis thaliana U 2 Y hypothetical protein gi|7485588 T01258 Arabidopsis thaliana U 4 Y hypothetical protein hypothetical protein/putative ABC transporter gi|7486039 T02491 Arabidopsis thaliana U 2 1,2 gi|7489244 T07050 Solanum tuberosum O/U 13 Y hypothetical protein R1 gi|7447845 T04985 Arabidopsis thaliana U 2 reported Pt hypothetical protein T16L1.170 probable transaminase putative protein gi|7362740 CAB83110 Arabidopsis thaliana U 2 1 gi|6358779 AAF07360 Arabidopsis thaliana U 2 Y unknown protein gi|6714365 AAF26055 Arabidopsis thaliana U 2 Y unknown protein gi|231660 P12222 Nicotiana tabacum U 6 pt-encoded YCF1 hypothetical 226 kDa protein gi|3915961 P09976 Nicotiana tabacum U 4 pt-encoded YCF2 hypothetical 267 kDa protein hypothetical protein T10I14.140 gi|7486826 T04912 Arabidopsis thaliana C 2 reported Pt gi|7438086 S76557 Synechocystis sp O 4 reported Pt hypothetical protein gi|6911887 CAB72187 Arabidopsis thaliana O 2 co putative protein gi|16245| CAA35887 Arabidopsis thaliana U 2 Y unnamed protein product 47 Results gi|25408620 B84809 Arabidopsis thaliana U 4 - Hypothetical protein Proteins with a reported function in other cell organelles Putative mitochondrial contaminants gi|2689078 AAB88906 Brassica rapa C 2 co cytochrome c oxidase subunit II gi|1076621 S46306 Nicotiana tabacum C 2 co cytochrome b5 gi|477819 B48529 Solanum tuberosum U/C 2 1 ubiquinol-cytochrome-c reductase putative alanine aminotransferase gi|6730768 AAF27157 Arabidopsis thaliana U 2 Y gi|2565305 AAB82711 Tritordeum sp. U 3 Y glycine decarboxylase P subunit gi|4210330 CAA11552 Arabidopsis thaliana U 13 Y 2-oxoglutarate dehydrogenase gi|3928138 CAA10267 Catharanthus roseus U 14 co mitochondrial elongation factor TU gi|1842188 CAA69726 Betula pendula U 2 Y mitochondrial phosphate translocator gi|6714392 AAF26081 Arabidopsis thaliana U 2 Y gi|114421 P17614 U/C 23 Y gi|3334123 Q96250 Nicotiana plumbaginifolia Arabidopsis thaliana putative mitochondrial LON ATP-dependent protease ATP synthase beta chain U/C 2 co ATP synthase gamma chain gi|2117355 S51590 Solanum tuberosum C 2 Y mitochondrial processing peptidase gi|1172555 P42055 Solanum tuberosum U/C 8 co 34 kDa outer mitochondrial membrane protein gi|100961 S17947 Zea mays C 2 co protein URF13 gi|2668492 BAA23769 Arabidopsis thaliana U/C 3 Y metal-transporting P-type ATPase gi|1061420 AAA81348 Vicia faba U/C 2 co p-type H -ATPase gi|1049255 AAA80347 Zea mays U/C 3 co H -pyrophosphatase gi|122101 P04915 Putative membrane contaminants + + Putative nuclear contaminants gi|4883746 AAD31625 Physarum polycephalum Campodea tillyardii gi|7649159 AAF65769 Euphorbia esula O/U/C 30 co histone H4 C 2 co histone H3 U/C 10 co histone H2A nucleolar protein gi|6056371 AAF02835 Arabidopsis thaliana U 2 co gi|7451352 T06379 Pisum sativum U/C 2 co SAR DNA-binding protein 2 gi|1549222 BAA13463 Nicotiana tabacum U/C 2 co NtSar1 protein gi|266989 Q01474 Arabidopsis thaliana C 6 co GTP-binding protein SAR1B gi|1093432 2104177A Helianthus annuus gi|7443459 T03215 Nicotiana tabacum U 2 - translation elongation factor eEF-2 U 3 - translation initiation factor eIF-4A.9 Putative peroxisomal contaminant U 2 co catalase Eukaryotic translation initiation (Cytosolic) gi|2119932 S52017 Nicotiana tabacum gi|1170503 gi|1170506 P41376 P41379 Arabidopsis thaliana Nicotiana plumbaginifolia gi|1170511 P41382 Nicotiana tabacum gi|2500521 Q40468 Nicotiana tabacum gi|1169476 P43643 gi|461998 P30925 Nicotiana tabacum Sulfolobus solfataricus gi|6056373 AAF02837 Arabidopsis thaliana U 21 - eukaryotic initiation factor 4A-1 U 41 - eukaryotic initiation factor 4A-2 U 2 - eukaryotic initiation factor 4A-10 U 5 - eukaryotic initiation factor 4A-15 U/C 3 - elongation factor 1-alpha O/U 8 - elongation factor 2 O 8 - elongation factor EF-2 Ribosomal proteins (cytosolic) gi|7110459 AAF36931 Chrysodidymus synuroideus gi|445612 1909359A Solanum tuberosum C Solanum tuberosum U Cichorium intybus C 3 gi|445613 gi|3986695 1909359B AAC84136 O/U 2 - ribosomal protein S2 2 co ribosomal protein S19 2 - ribosomal protein L7 - ribosomal protein L12 48 Results gi|7488177 T06623 Arabidopsis thaliana gi|1173256 P46299 Gossypium hirsutum gi|6831665 O65731 Cicer arietinum C 2 - 40S ribosomal protein-like 40S ribosomal protein S4 C 3 co U/C 2 - 40S ribosomal protein S5 5 - 40S ribosomal protein S8 gi|3777552 AAC64931 Griffithsia japonica C gi|1173198 P46298 Pisum sativum C 2 co 40S ribosomal protein S13 Arabidopsis thaliana C 2 Y 40S ribosomal protein S14 C 3 Y 40S ribosomal protein S16 gi|1173200 P42036 gi|133808 P16149 Lupinus polyphyllus gi|4887131 AAD32206 Prunus armeniaca U 2 - 60S ribosomal protein L1 C 2 co 60S ribosomal protein L2 gi|132849 P25998 Nicotiana tabacum gi|132944 gi|464621 P22738 P34091 Arabidopsis thaliana Mesembryanthemum crystallinum gi|548774 P35685 gi|6174959 P49210 U 2 co 60S ribosomal protein L3 C 3 - 60S ribosomal protein L6 Oryza sativa U 2 - 60S ribosomal protein L7A Oryza sativa U/C 8 - 60S ribosomal protein L9 3 - putative 60S ribosomal protein L11-1 2 co 60S ribosomal protein L13 gi|7340874 BAA92964 Oryza sativa U gi|1350664 P49627 Nicotiana tabacum C gi|3122673 O23515 Arabidopsis thaliana U/C 2 co 60S ribosomal protein L15 2 co 60S ribosomal protein L17 gi|1710519 P51413 Nicotiana tabacum C gi|585876 Q07761 Nicotiana tabacum U/C 3 Y 60S ribosomal protein L23A (L25) C 2 - 60S ribosomal protein L27 gi|730547 P41101 Solanum tuberosum Most of the proteins were identified in the osmotic shock and urea fractions (53 and 67, respectively) and a considerable number (41) in both the urea and CHAPS fraction (U/C) [Figure 3.6]. This shows that urea was unable to solubilize completely some proteins, which require high stringency for their solubization. Only 17 proteins were identified in the OSMO and the urea overlap fractions, suggesting that the separation of proteins into soluble and peripheral membrane proteins was an efficient strategy. In conclusion, it provides information about the solubility of the identified proteins and their membrane attachment in vivo. Osmo O/U 53 soluble proteins 17 Urea 67 U/C 41 CHAPS 27 integral membrane proteins Figure 3.6: Number of proteins identified in each fraction. Abbreviations are O/U OSMO and urea, U/C urea and CHAPS. Most of the enzymes active in amino acid and carbohydrate metabolism were soluble proteins present in the osmotic shock fraction. Envelope proteins and putative transporters were identified in the urea and the CHAPS fractions, consistent with their function in the envelope membrane. Proteins from all other functional categories including soluble, 49 Results peripheral and integral membrane proteins are distributed in similar fashion. It is notable that most of the identified putative contaminants were derived from the urea and the CHAPS fractions, suggesting that they may have been attached to the outer envelope membrane of the BY-2 plastids (Table 3.2, putative contaminants). In terms of protein localization, the criteria for plastidic proteins were placed as described in Materials and Methods (Material and Methods, section 2.2.8.1 “Protein localization”). In addition to the plastid-encoded proteins, all proteins were accepted as true plastid proteins, which fall into these criteria. Most of the proteins (149 out of 205) fall into these criteria and these proteins are most likely true plastid proteins (Figure 3.7; Table 3.2, column “Localization”). 140 120 No. of proteins 100 80 60 40 al rib os om eu ka ry ot ic ch on dr ito r ea m cl nu m em br an es ia l n un kn ow pl as tid 0 cy to so lic 20 Figure 3.7: Localization of BY-2 plastid proteins. Proteins were designated as true plastid proteins if they fell into the above mentioned criteria (Material and Methods, section 2.2.8.1 “Protein localization”). Twenty four proteins could not be assigned as true plastid proteins, and therefore we analyzed whether orthologues of these proteins had been identified in other plastid proteome studies (Peltier et al, 2002; Schubert et al, 2002; Ferro et al, 2003; Froehlich et al, 20003; Kleffmann et al, 2004) . The idea behind this approach was to provide further evidence for their 50 Results plastid localization or their specific interaction with the plastid outer membrane system. Seven of these proteins were identified in another proteome study, nine of these proteins were identified in two other analyses, two were identified in three other studies and only seven proteins were not detected in previous chloroplast proteome studies (Table 3.2), and these proteins were assigned to unknown localization. Most of these proteins belong to heat shock proteins and structural maintenance classification (Table 3.2). In the BY-2 plastid shotgun-proteome analysis, thirty-two proteins were identified that were likely not of plastid origin. These proteins are related to ribosomal or eukaryotic translation factors and other possible contaminants (Figure 3.7 and Table 3.2). Ribosomal proteins can associate with the outer envelope membrane of plastids (Andon et al., 2002). For this reason, it was possible to detect them in a plastid membrane preparation. In addition to ribosomal proteins, we also detected 25 proteins that have a reported function in mitochondria and nuclei as well as peroxisomal catalase (section 3.1.1 BY2 plastid isolation and purity; Figure 3.7). The possible explanation could be dual targeting of proteins (reviewed in Peeters and Small, 2001), and there are several proteome studies reported contaminations of organelle preparations with highly abundant proteins from other cell organelles (Peltier et al., 2000; Millar et al., 2001; Millar et al., 2006). During plastid preparation, fumarase and catalase tests were employed for the detection of mitochondria and peroxisomes; these assays also detected minor contaminations in the BY-2 plastid preparation (Fig 3.1). 3.1.3.1.3 Functional categorization of the identified proteins The next approach was a functional categorization of BY-2 plastid proteins (Figure 3.8). Most of the identified proteins have a general function in plastid metabolism, and the majority of the proteins are involved in the biosyntheis of amino acids. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase are abundant enzymes in BY-2 plastids. These enzymes are also active in primary nitrogen fixation, which is the basis for the synthesis of other amino acids (Last and Coruzzi, 2000). Several proteins from the 51 Results arginine, the branched-chain amino acid and the aromatic amino acid biosynthesis pathways were identified that utilize reduced nitrogen (Table 3.2). It is likely that the BY-2 plastid provides the rapidly growing and dividing cell with the amino acids required for protein biosynthesis and other cellular functions. 40 35 30 20 15 10 l l th et ica ur a ct Hy po k oc St ru sh He at er on es Ch ap or te rs es Tr an sp as n sio Pr o re s te x do xp er Ot h Re ee Ge n rb o Ca in o ac 0 id hy dr at e St ar ch Nu cle ot id Te e tra py rro le 5 Am No. of proteins 25 Figure 3.8: Functional catogeries of identified BY-2 plastid proteins. The majority of the proteins are involved to amino acid metabolism. The identification of many proteins with a function in protein folding, such as chaperones and heat shock-related proteins, together with the identification of CLP-protease subunits, supports the view of a high protein turnover rate and the need for the folding of newly synthesized proteins at a high rate. The other possibility could be that heat shock-related proteins are characteristics for cell culture conditions. CLP-protease is thought to perform a housekeeping function in plastids and is present in virtually all plant tissues. Its exact role is still unclear, but chloroplast development was impaired in the absence of CLP-protease (Adam, 2000). In addition to proteins involved in dominant 52 Results amino acid biosynthetic pathways, proteins involved in carbohydrate and starch metabolic activities were also common in BY-2 plastids (Figure 3.8). For every plastid type, its interaction with the cytosol and the import of cytosolic compounds and export is of pivotal importance for their metabolism and functional maintenance. Several transporters and proteins of the plastid envelope membrane were identified from BY-2 plastids. The most abundant of these proteins were the ATP/ADP transporter, the oxoglutarate/malate translocator, and the outer envelope protein (OEP) 75 homologue. While OEP 75 is a major component of the plastid protein import machinery (Schnell, 2000; Hiltbrunner et al., 2001; Jarvis and Soll, 2001), the ATP/ADP transporter and the oxoglutarate/malate translocator are involved in metabolite exchange between the plastid and the cytosol (Neuhaus and Wagner, 2000). The identification of these transporters in the BY-2 plastid membranes reflects the energetic requirements of heterotrophic plastids (von Zychlinski et al., 2005). The ATP/ADP transporter imports cytosolic ATP that is generally required for the synthesis of various metabolites such as amino acids and starch in heterotrophic plastids (Neuhaus and Wagner, 2000). Oxoglutarate is the precursor for ammonia assimilation, and recent reports suggest that the oxoglutarate/malate translocator imports carbon skeletons for net glutamate synthesis (Weber and Flügge, 2002). In addition to the above mentioned transporters, glucose 6-phosphate/phosphate translocator was also detected. The possible role of this translocator is the import of glucose 6-phosphate to initiate the oxidative pentose phosphate pathway that provides reducing equivalents, for example for the synthesis of glutamate from oxoglutarate (Schnarrenberger et al., 1995). Only a few plastid-encoded proteins were identified and it is notable that the genes for all identified plastid encoded proteins possessed a promoter that was recognized by the nuclear encoded plastid RNA polymerase (NEP, Table 3.3). While ycf1, clpP and atpB contain an additional promoter element for the plastid-encoded plastid RNA polymerase (PEP), ycf2 and rpoB are exclusively transcribed from an NEP 53 Results promoter (Allison et al., 1996; Hajdukiewicz et al., 1997; Liere and Maliga, 1999). The NEP RNA polymerase has a predominant role for the transcription of the plastome in undifferentiated plastids when the PEP components are not expressed (Allison et al 1996; Hajdukiewicz et al., 1997). Several peptides of ycf1 and ycf2 were identified in gel fractions of proteins with a molecular weight above 200 kDa as judged by SDS-PAGE. This was consistent with the molecular weight predicted for ycf1 (226 kDa) and ycf2 (267 kDa), suggesting that the complete reading frame was transcribed and translated in BY-2 plastids (Drescher et al., 2000). Table 3.3: Promoter elements responsible for the regulation of plastid transcription a. a The assignment of promoter elements was retrieved from information available in the literature (Allison et al., 1996; Hajdukiewicz et al., 1997; Liere and Maliga, 1999). NEP: nucleus encoded plastid RNA polymerase, PEP: plastid encoded plastid RNA polymerase. ycf1 and ycf2: hypothetical protein 1 and 2. clpP: CLP-protease subunit P, atpP, atpP: α, β subunit of ATP synthase; rpoB: β subunit of the plastid encoded plastid RNA polymerase. NEP PEP ycf1 + + ycf2 + - clpP + + atpA + + atpB + + rpoB + - 3.1.3.2 2D-PAGE The next fractionation approach for the BY-2 plastid proteins was 2D-PAGE, which is widely accepted for the quantitative analysis of proteins (Görg et al., 2004). As a starting point for the MS analysis, an intensity factor of 0.090 was selected for spot picking, and 472 spots were picked from three different replicate gels. These selected spots were excised, digested and analyzed by MALDI-TOF-TOF, which resulted in a total of 140 protein identifications (Figure 3.9; Supplementary Table 1). In Supplementary Table 1, all identified proteins are shown along with their spot ID, spot intensity from the different gels, average spot intensity and spot frequency in different gels. These results showed that protein coverage was less than 50% of the spots picked. This is 54 Results due to the stringent criteria to select the proteins after database search (Materials and Methods, 2.2.7.2 MASCOT search engine). The analysis of protein localization followed the same criteria which were used in section 3.1.3 to accept identified proteins as true plastid proteins. In 2D-PAGE, few proteins, which were detected more than one spot at different positions on 2D-PAGE.The possible explanation for this is •One protein has different isoforms that are found in different positions on the 2D-PAGE, •Protein degradation during sample preparation •Post-translational modification (PTM) One particular example “Transketolase 1” has eight different spots in the 2D-PAGE. All spots represent one particular protein (Tranketolase 1; Figure 3.9, B), and thus can be regarded as posttranslationally modified isoforms. Seventy-two proteins were identified from 2D-PAGE but most of these proteins (54) had already been identified in the shotgun-proteome analysis (Figure 3.10, Table 3.4). Newly identified phosphogluconate dehydrogenase and DNA Phosphogluconate dehydrogenase belongs to proteins gyrase the included, subunit oxidative A. 66- pentose phosphate pathway (OPPP), and it catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate with the release of CO2 and the reduction of NADP (Rosemeyer, 1987). The exact role of DNA gyrases in plastid function is presently unknown; a previous study showed that these proteins are critical for nucleoid division by the use of a specific inhibitor (Itoh et al., 1997). 55 Results Figure 3.9: An average 2D-PAGE from BY-2 plastid. A). BY-2 plastid was solubilized and subjected to an immobilized pH gradient 4-7. Red encircled protein spots were identified by MALDI-TOF-TOF.B) shows an enlargement of a rectangular area in A and red encircled protein spots shows an example of one protein (Transketolase 1) which has different isoforms on different positons of 2D-PAGE 56 Results Shotgun-proteomics 173 2D-PAGE 54 72 Figure 3.10: Overlapping proteins found in both shotgun-proteomics and 2D-PAGE of BY2 plastid. The shotgun-proteome and the data from the 2D-PAGE were compared first with respect to the number of proteins found in both approaches, and second, quantatively with respect to spot-intensity in 2DPAGE and number of peptides in shotgun-proteome (Figure 3.11). Based on these comparisons, proteins involved in amino acid and carbohydrate metabolism are abundant in both approaches, suggesting that BY-2 plastids are heterotrophic plastids that provide energy to rapidly growing cells and amino acids for protein synthesis. No proteins of transporters were detected; a possible explanation for this is that it is difficult to detect membrane proteins with 2D-PAGE due to solubility constraints. There are also no proteins detected in 2D-PAGE related to starch, nucleotide and tetrapyrrole pathway as detected in the shotgun-proteome. This comparison showed a good agreement of inferred protein abundances between the shotgun method (i.e. peptide counts) and the 2D PAGE approach (spot intensity). Contradictony results were only obtained for proteins, having unfavourable hydrophobic properties which diminishes their detectability in the 2D PAGE approach. This suggests that different protein analysis methods must be used in order to obtain full proteome coverage for any cell type or organelle under investigation. 57 Results B A ia dr on ch ito M M id ac no i Am l l ia dr on ch ito id ac no i Am He a Ch t sho ap e c ron k and es Heat Cha shock a pero n nes d Proteases te ex pr es si Re do x G en e bo hy dr a al ctur Stru Other metabolism x do Other Re C ar metab olism G e en s es pr x e n io on ases Prote Structural Peroxisomal ar Nucle l ia dr on ch ito M St ru es c G r tu e en al e n io ss e r xp met abo lism Gene expression Redox ral Struc tu teas Pro Ca rb oh yd ra rch Sta otide Nucle m olis tab me er ote as es Ot h St a rch N Te ucle tra ot py ide rro l Transporter Ot h er Carbohydrate id ac no i Am He Ch at s ap ho er ck on a es nd Hy pot het ica l Re do x cid etical Hypoth Pr te Membrane Peroxisomal M a ino Am He Ch at s ap ho er ck on a es nd rs rte po s n Tra bo hy dr a D Membrane lear Nuc l ia dr on ch it o C C ar Tetrapyrrol Figure 3.11: Functional categorization of the BY-2 plastid proteome. A) shows the classification on the basis of protein numbers found in 2D-PAGE. B) shows the classification on the basis of spot intensity of proteins found in 2D-PAGE. C) shows the classification on the basis of protein numbers found in shotgun proteome. D) shows the classification on the basis of number of peptides found in shot-gun proteome. In two approaches, amino acid and carbohydrate metabolism represent a major contribution. 58 te Results Table 3.4: List of proteins identified by 2D-PAGE. a a only those proteins which have a significant MASCOT score (Material and methods, 2.2.7.2 MASCOT search engine) were taken into account. Plastid localization predication was performed with ChloroP (Emanuelsson et al., 1999). Putative function was manually assigned. Provided is the identifier (“identifier”, the Swiss prot accession number), the organim (“organism”), average spot intensity (“average spot intensity” spot intensity detected by the proteome weaver software). Identifer Protein Q94CE2 Q9LQU9 Q8L6J9 Q8GXH6 Q9FS26 Q40360 O82030 Q9SUU0 Q43127 Q9LEC8 Q9MT29 Q42884 O25716 Q9FYW9 Q43593 Q9SDP4 Q89MR7 Q8DAR4 Q9C550 P09114 P09342 Q949X7 Q9SNY8 O46887 Amino Acid synthesis putative monodehydroascorbate reductase aspartate-semialdehyde dehydrogenase (F10B6.22) putative carbamoyl phosphate synthase large subunit putative 3-isopropylmalate dehydrogenase plastidic cysteine synthase 1 NADH-dependent glutamate synthase histidinol-phosphate aminotransferase glycine hydroxymethyltransferase glutamine synthetase glutamate dehydrogenase B cystathionine gamma-synthase isoform 2 chorismate synthase 1 aspartate carbamoyltransferase adenylosuccinate synthetase acyl-[acyl-carrier protein] desaturase ATP-sulfurylase. 5'-phosphoribosyl-5-aminoimidazole synthetase 3-phosphoshikimate 1-carboxyvinyltransferase 2-isopropylmalate synthase acetolactate synthase II acetolactate synthase I putative diaminopimelate decarboxylase branched-chain amino acid aminotransferase ATP phosphoribosyltransferase Q9SE20 Q9C6Z3 Q8SA22 Q82MT5 Q9FPL3 Q8RX97 Q9LIR4 Q9LFG2 Q40475 Q88AE0 Other metabolism zeta-carotene desaturase pyruvate dehydrogenase E1 beta subunit, putative putative pyruvate kinase putative acyl-CoA thioesterase phosphoribosylaminoimidazolecarboxamide formyltransferase ferritin 1 dihydroxy-acid dehydratase diaminopimelate epimerase biotin carboxylase subunit acyltransferase family protein O78327 Q941R1 P46225 Q84LB6 Q9SXX5 Q944T4 Carbohydrate metabolism transketolase 1 Transaldolase triosephosphate isomerase succinyl CoA ligase beta subunit plastidic aldolase glyceraldehyde 3-phosphate dehydrogenase 1 Organism Av. spot Intensity Arabidopsis thaliana Arabidopsis thaliana Nicotiana tabacum Arabidopsis thaliana Solanum tuberosum Medicago sativa Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Nicotiana plumbaginifolia Solanum tuberosum Lycopersicon esculentum Helicobacter pylori Lycopersicon esculentum Olea europaea Allium cepa Bradyrhizobium japonicum Vibrio vulnificus Arabidopsis thaliana Nicotiana tabacum Nicotiana tabacum Arabidopsis thaliana Solanum tuberosum Thlaspi goesingense 0.265 0.451 0.361 0.135 0.149 0.277 0.270 0.425 0.293 0.224 0.143 0.288 0.176 0.187 0.225 0.265 0.113 0.267 0.365 0.224 0.166 0.176 0.204 0.430 Lycopersicon esculentum Arabidopsis thaliana Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Arabidopsis thaliana Pseudomonas syringae Nicotiana tabacum Pseudomonas syringae 0.346 0.239 0.236 0.163 0.232 0.231 0.252 0.180 0.252 0.215 Capsicum annuum Lycopersicon esculentum Secale cereale Lycopersicon esculentum Nicotiana paniculata Fragaria ananassa 0.602 0.242 0.166 0.145 0.168 0.822 59 Results O82058 Q84NI5 Q9SH69 glucose-6-phosphate isomerase Aconitase 6-phosphogluconate dehydrogenase Arabidopsis thaliana Lycopersicon pennellii Arabidopsis thaliana 0.333 0.102 0.187 Q8RVF8 thioredoxin peroxidase Nicotiana tabacum 0.486 Q7XUX0 P68158 Q851Y8 Q40450 Q9HJB6 OSJNBa0027P08.15 protein elongation factor Tu translational elongation factor Tu chloroplast elongation factor TuA DNA gyrase subunit A Oryza sativa Nicotiana tabacum Oryza sativa Nicotiana sylvestris Thermoplasma acidophilum 0.213 0.206 0.222 0.461 0.196 Q43594 Q8VXC9 Q9ZSD7 tubulin beta chain alpha-tubulin Actin O50036 P21240 P21239 Q9M5A8 Heat shock and chaperones heat shock protein 70 rubisCO subunit binding-protein beta subunit rubisCO subunit binding-protein alpha subunit chaperonin 21 Q9LJL3 O98447 P31542 Proteases zinc metalloprotease ClpC protease ATP-dependent Clp protease ATP-binding subunit clp Q8L5C8 Q41445 P29677 Q9AXQ2 P29197 Q05045 Q05046 P17614 P05495 Q01899 Q03685 Putative mitochondrial contaminants putative mitochondrial NAD-dependent malate dehydrogenase Solanum tuberosum mitochondrial processing peptidase. Solanum tuberosum mitochondrial processing peptidase alpha subunit Solanum tuberosum mitochondrial processing peptidase beta subunit Cucumis melo chaperonin HSP60, mitochondrial Arabidopsis thaliana chaperonin CPN60-1, mitochondrial Cucurbita maxima chaperonin CPN60-2, mitochondrial Cucurbita maxima ATP synthase beta chain, mitochondrial Nicotiana plumbaginifolia ATP synthase alpha chain, mitochondrial Nicotiana plumbaginifolia heat shock 70 kDa protein, mitochondrial Phaseolus vulgaris Luminal-binding protein 5 (BiP 5) Nicotiana tabacum 0.134 0.282 0.204 0.268 0.301 0.140 0.613 0.229 0.545 0.220 0.196 O65852 isocitrate dehydrogenase Nicotiana tabacum 0.205 Q40467 Eukaryotic translation initiation (cytosolic) eukaryotic initiation factor 4A-14 Nicotiana tabacum 0.206 Redox Gene expression Structural maintenance and plastid positioning Oryza sativa Nicotiana tabacum Brassica napus 0.279 0.221 0.159 Spinacia oleracea Arabidopsis thaliana Brassica napus Lycopersicon esculentum 0.518 0.520 0.468 0.269 Arabidopsis thaliana Spinacia oleracea Pelargonium zonate spot virus 0.150 0.169 0.265 60 Results A comprehensive overview of the identified proteins from characteristic heterotrophic metabolic pathways is shown in Figure 3.12. Few of the proteins are shown as examples, which were found in both the shotgun-proteome analysis and 2D-PAGE. The number of identified peptides for each identified protein is given in brackets (1st) together with the staining intensity on a 2D-PAGE map (2nd) [Figure 3.12]. Envelope transporter (13/-) ATP/ADP translocator (2/-) Hexose-/xylose phosphate/phosphate translocator (-) Triose phosphate/phosphate translocator (-) Phosphoenolpyruvate/phosphate translocator (-) Sugar transporter (-) MEX 1 (-) MIP family protein G-6-P Triose phosphate PEP OPPP (5/-) (-) (-/0.187) (-) (-) (35/0.242) (44/0.521) Glyoxylate metabolism (2/0.102) Aconitase 1 (2/0.205) Isocitrate dehydrogenase Glucose 6-phosphate 1-dehydrogenase 6-phosphogluconolactonase 6-phosphogluconate dehydrogenase Ribose 5-phosphate isomerase Ribulose 5-phosphate 3-epimerase Transaldolase Transketolase Calvin Cycle (-) Rubisco small and large (13/-) Phosphoglycerate kinase (19/0.822) Glyceraldehyde-3-phosphate DH (11/0.166) Triose phosphate isomerase (2/0.168) Aldolase (-) Fructose-1,6-bisphosphatase (44/0.521) Transketolase (see OPPP) (-) Sedoheptulose bisphosphatase (-) Ribose 5-phosphate isomerase (see OPPP) (-) Ribulose-5-phosphate-3-epimerase (see OPPP) (-) Phosphoribulokinase precursor Partial glycolysis/PDC (10+2/0.236) Pyruvate kinase (4/0.239) Pyruvate dehydrogenase (E1) (-) Dihydrolipoamide S-acetyltransferase (E2) (-) Dihydrolipoamide dehydrogenase (E3) (3/-) Malate (8/-) Malate DH (5/-) Malic enzyme NADPH E-4-P (+ PEP) Malate/oxoglutarate transporter 2-oxoglutarate Ammonium assimilation (10/-) fdx dependent GOGAT (29/0.277) NADH glutamate synthase (46/0.293) Glutamine synthetase (7/-) Aspartate aminotransferase (-) Ferredoxin-NADP+ reductase Amino acid metabolism 32 different enzymes Shikimate/aromatic amino acid metabolism (7/-) Phospho-2-dehydro-3-deoxyheptonate aldolase (-) 3-dehydroquinate synthase (-) 3-dehydroquinate dehydratase/ shikimate 5-dehydrogenase (-) Homologue to shikimate kinase (2813.m00166) (4/0.267) 3-phosphoshikimate 1-carboxyvinyltransferase (5-enolpyruvylshikimate-3-phosphate synthase 1) (5/0.288) Chorismate synthase (-) Chorismate mutase (2/-) Tryptophan synthase, beta subunit R-5-P; NADPH Hexose phosphate Acetyl-CoA; NADH Purine+pyrimidine biosynthesis 3 different enzymes Starch metabolism 6 different enzymes Fatty acid metabolism 1 different enzyme Figure 3.12: schematic illustration of metabolic pathways in heterotrophic plastids. Abbreviations; E-4-P: erythrose-4-phosphate; G-6-P: glucose 6-phosphate; OPPP: oxidative pentose pathway; PDC: pyruvate dehydrogenase complex; PEP: phosphenole pyruvate; R-5-P: ribulose-5-phosphate; DH: dehydrogenase; GOGAT: glutamate synthase; MEX: maltose exporter; MIP: major instrinic protein; PDC: pyruvate dehydrogenase complex. . 61 Results 3.1.4 Functional proteome analysis 3.1.4.1 Blue Native (BN)-PAGE For the analysis of native protein complexes of BY-2 plastids, BN-PAGE was applied. BN-PAGE separates both acidic and basic water soluble proteins at a fixed pH 7.5 (Schägger et al., 1994). The pH 7.5 used in BN-PAGE represents the physiological range of the pH in intracellular compartments and allows the separation of protein complexes that are susceptible to acidic and/or basic conditions, which results in the dissociation of subunits and/or loss of enzymatic activities. The anionic triphenylmethane dye CBB G-250 is added to the cathode buffer or often directly to the sample shortly before the run. The CBB dye binds to essentially all membrane proteins and many soluble proteins in a manner that preserves protein–protein interactions. CBB confers a negative net charge to the protein complex and largely diminishes any artificial aggregation of detergent-solubilized hydrophobic membrane proteins during the electrophoretic run (Heuberger et al., 2002). Consequently, protein complexes and individual proteins migrate to the anode due to excessive negative charge and are separated by the sieving effect of the polyacrylamide gel according to the molecular mass of protein complexes (Schägger et al., 1994). The isolated BY-2 plastids were applied onto BN-PAGE and five visible bands were selected for the MS-analysis (Figure 3.13 and Table 3.5). Proteins hits were accepted only if they had two peptides identified either in one band or in two bands. For the plastid proteins and contaminants, the same criteria was followed that are mentioned in the BY-2 shotgun-proteome analysis. In the end, a total of 50 proteins were detected in all five bands and most of the hits (42) had already been identified in the BY-2 plastid shotgun-proteome analysis. Eight proteins were uniquely identified in the BN-PAGE bands including ATP-phosphoribosyltransferase, leucine aminopeptidase 2 and leucine aminopeptidase 1. ATP-phosphoribosyltransferase is involved in the histidine biosynthesis pathway (Ohta et al., 2000) and leucine aminopeptidase is most likely involved in the processing and regular turnover of intracellular proteins (Callis and Vierstra, 2000). 62 Results 1 KDa 669 2 I IV 443 II V 200 III Figure 3.13: BN-PAGE analysis of protein complexes.Two sample conditions (material and methods, 2.2.5.1.3, analysis of protein complexes (BN-PAGE) were analyzed. Five bands were selected for MS-analysis (Table 3.5). Table 3.5: List of proteins identified from BN-PAGE a. a only those peptides which have Xcorr >2.5 and dCn >0.1 in the SEQUEST database search were considered as identified. Provided are molecular weight range (‘’Molecular weight range’’ approximate molecular weight range), the identifier (“Identifier”), the NCBI database accession number (“acc. no”), the organism (“Organism”), the number of identified tryptic peptides (“No. of peptides”) and protein name (“Protein”). Molecular weight range 500-700 Band No I Identifier gi|2506277 Acc. No P08927 Organisms No of peptide Pisum sativum 64 Protein rubisco subunit binding-protein beta subunit I gi|1351030 P21239 Brassica napus 31 rubisco subunit binding-protein alpha subunit I gi|7489279 T07790 Solanum tuberosum 2 transaldolase I gi|7447845 T04985 Arabidopsis thaliana 2 probable transaminase I gi|3914996 Q96255 Arabidopsis thaliana 2 phosphoserine aminotransferase I gi|3687228 AAC62126 Arabidopsis thaliana 2 malate oxidoreductase heat-shock protein, 82K, I gi|1076758 S49340 Secale cereale 2 I gi|14916972 Q96291 Arabidopsis thaliana 2 2-cys peroxiredoxin BAS1 I gi|21264504 Q05728 Arabidopsis thaliana 2 phosphoribosylformylglycinamidine cyclo-ligase I gi|2492952 Q42884 2 chorismate synthase 1 I gi|5532608 AAD44809 Lycopersicon esculentum Nicotiana tabacum 1 6,7-dimethyl-8-ribityllumazine synthase I gi|2497542 Q40546 Nicotiana tabacum 1 pyruvate kinase isozyme G I gi|126736 P22178 Flaveria trinervia 1 NADP-dependent malic enzyme I gi|4827253 BAA77603 Nicotiana paniculata 1 plastidic aldolase I gi|5123836 CAB45387 Nicotiana tabacum 1 NAD-malate dehydrogenase Contaminants I I 400-500 gi|7433550 gi|1093432 Q9SUU0 2104177A Arabidopsis thaliana Helianthus annuus 3 2 glycine hydroxymethyltransferase catalase. II gi|27735252 P21240 Arabidopsis thaliana 17 rubisco subunit binding-protein beta subunit II gi|1351030 P21239 Brassica napus 4 rubisco subunit binding-protein alpha subunit II gi|2117700 S58083 Solanum tuberosum 7 transketolase II gi|7431231 T06401 5 malate dehydrogenase II gi|4827253 BAA77603 Lycopersicon esculentum Nicotiana paniculata 5 plastidic aldolase II gi|419757 S30145 Arabidopsis thaliana 4 ketol-acid reductoisomerase heat shock protein 70 II gi|1928991 AAC03416 Citrullus lanatus 4 II gi|7484979 T05572 Arabidopsis thaliana 3 glucose-6-phosphate isomerase II gi|4926868 AAD32948 Arabidopsis thaliana 3 putative phosphoserine aminotransferase II gi|7331143 AAF60293 3 chaperonin 21 II gi|2129963 S51946 Lycopersicon esculentum Nicotiana tabacum 2 pyruvate kinase II gi|2204087 CAA74175 Arabidopsis thaliana 2 enoyl-ACP reductase 63 Results II gi|7447845 T04985 Arabidopsis thaliana 2 II II probable transaminase gi|2252472 CAB10698 Arabidopsis thaliana 2 argininosuccinate lyase gi|1362055 S57786 Medicago sativa 2 phosphogluconate dehydrogenase II gi|9081770 BAA12349 Arabidopsis thaliana 2 monodehydroascorbate reductase II gi|2499497 Q42961 Nicotiana tabacum 1 phosphoglycerate kinase II gi|99696 S18600 Arabidopsis thaliana 1 glutamate--ammonia ligase II gi|6578124 AAF17705 Canavalia lineata 1 ornithine carbamoyltransferase II gi|7489279 T07790 Solanum tuberosum 1 transaldolase II gi|2688839 AAB88880 Thlaspi goesingense 1 ATP phosphoribosyltransferase II gi|4150965 CAA09478 Asparagus officinalis 1 glutamate dehydrogenase II gi|1174745 P46225 Secale cereale 1 triosephosphate isomerase II gi|2811029 O04866 Alnus glutinosa 1 acetylornithine aminotransferase II gi|134672 P11796 Nicotiana plumbaginifolia 1 superoxide dismutase [Mn] Contaminants 200-300 500-700 400-500 III gi|6690395 AAF24124 Arabidopsis thaliana 6 phosphoglucose isomerase III gi|7489279 T07790 Solanum tuberosum 4 transaldolase III gi|1708311 Q08080 Spinacia oleracea 4 stromal 70 kDa heat shock-related protein, III gi|7433617 T09541 Capsicum annuum 2 transketolase III gi|20532373 P46248 Arabidopsis thaliana 2 aspartate aminotransferase III gi|7109469 AAF367 Arabidopsis thaliana 2 putative enolase III gi|7706790 AAB35826 2 enolase; 2-phospho-D-glycerate hydrolase III gi|1170040 P42770 Echinochloa phyllopogon Arabidopsis thaliana 2 glutathione reductase III gi|2499497 Q42961 Nicotiana tabacum 1 phosphoglycerate kinase III gi|1174745 P46225 Secale cereale 1 triosephosphate isomerase III gi|419757 S30145 Arabidopsis thaliana 1 ketol-acid reductoisomerase 2 hypothetical protein 2 superoxide dismutase[Mn] Contaminants Arabidopsis thaliana III gi|7267526 Q9LDL8 III gi|100284 A39267 IV gi|2506277 P08927| Pisum sativum 64 IV gi|134101 P08824 Ricinus communis 69 rubisco subunit binding protein alpha subunit IV gi|5532608 AAD44809 Nicotiana tabacum 17 6,7-dimethyl-8-ribityllumazine synthase IV gi|15229559 NP_189041 Arabidopsis thaliana 2 heat shock protein 60 (HSP60) IV gi|7431768 T16982 1 glutamate dehydrogenase IV gi|6578124 AAF17705 Nicotiana plumbaginifolia Canavalia lineata 1 ornithine carbamoyltransferase V gi|27735252 P21240 Arabidopsis thaliana 26 rubisco subunit binding protein beta subunit V gi|1351030 P21239 Brassica napus 19 rubisco subunit binding protein alpha subunit V gi|20197607 AAD15433 Arabidopsis thaliana 9 putative aspartate aminotransferase V gi|7431231 T06401 8 malate dehydrogenase Nicotiana plumbaginifolia rubisco subunit binding protein beta subunit V gi|5701896 CAA47373 Lycopersicon esculentum Nicotiana sylvestris 8 glutamate--ammonia ligase V gi|7447845 T04985 Arabidopsis thaliana 7 hypothetical protein T16L1.170 V gi|555970 AAA50249 5 glutamine synthetase V gi|3914996 Q96255 Lycopersicon esculentum Arabidopsis thaliana 4 phosphoserine aminotransferase V gi|4827253 BAA77603 Nicotiana paniculata 4 plastidic aldolase V gi|2492530 Q42876 4 Leucine aminopeptidase 2 (LAP 2) V gi|2688839 AAB88880 Lycopersicon esculentum Thlaspi goesingense 3 ATP phosphoribosyltransferase V gi|2811029 O04866 Alnus glutinosa 2 acetylornithine aminotransferase V gi|231536 P30184 Arabidopsis thaliana 2 Leucine aminopeptidase 1 (LAP 1) 64 Results V gi|2497540 P55964 Ricinus communis 2 pyruvate kinase isozyme G V gi|6690395 AAF24124 Arabidopsis thaliana 1 phosphoglucose isomerase V gi|134672 P11796 1 superoxide dismutase[Mn] V gi|7433550 T05362 Nicotiana plumbaginifolia Arabidopsis thaliana 1 glycine hydroxymethyltransferase V gi|5532608 AAD44809 Nicotiana tabacum 1 6,7-dimethyl-8-ribityllumazine synthase V gi|1097877 2114378A 1 aminolevulinate dehydratase V gi|3219164 BAA28783 Lycopersicon esculentum Arabidopsis thaliana 1 glutamine amidotransferase/cyclase In order to assess the complex association of proteins detected from the different bands, I analyzed the known complex associations for some identified proteins by literature searches. Detected in Band I (MW range ~500-700 kDa), the CPN60 family consists of two groups, type I chaperonins that are present in eubacteria (GroEL), choloroplasts (Rubisco binding protein) and mitochondria (Hsp 60), and type II chaperonins that are present in eukaryotic cytosol and in archaea (Baneyx et al., 1995). The chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, α and β. Additionally, chloroplasts contain two structurally distinct co-chaperonins; one of these, chcpn10, is probably similar to the mitochondrial and bacterial co-chaperonins and is composed of 10 kDa subunits. The second, ch-cpn20 (which is also called CPN21), is composed of two cpn10-like domains that are held together by a short linker (Hirohashi et al., 1999; Peltier et al., 2005). In this study, CPN60 α and β are very abundant proteins and are dominant at 500-600 kDa and also found at 400-500 kDa (Table 3.5). The CPN21 is also identified at ~ 400-500 kDa (Figure 3.13, Lane 1) and in oligomeric state it is present in multiple form and complexed with CPN60 (Baneyx et al., 1995; Peltier et al., 2005). Peltier et al (2005) did not identify CPN21 in the CPN 60 complex but it was rather identified at 150-170 kDa complex (Peltier et al., 2005).The detection of cpn60 in band I is therefore consistent with data available in the literature. 6, 7-dimethyl-8-ribityllumazine (DMRL) synthase is involved in vitamin B2 synthesis (riboflavin) and its oligomeric state is a hexamer in spinach isoforms (Fornasari et al., 2004). In a recent plastid proteome study, this enzyme was also found at 738 kDa (Peltier et al., 2005), which is in line with our observations (Table 3.5) 65 Results 2-Cys peroxiredoxin BSA1 is probably involved in an antioxidant enzyme particularly in the developing shoot and photosynthetic leaf. Its oligomeric state is a homodimer (Baier and Dietz, 1997). Peltier and collegues found protein complexes with this enzyme at 360, 219, 119, 110 and 50 kDa and they proposed its oligomeric states monomer, dimer or decamer (BernierVillamor et al., 2004; Peltier et al., 2005). I found this enzyme at 500-700 kDa, which shows some variance in the measured native molecular weight in our approach compared to the data available in the literature. This might be a result of different complex associations in different plastid types (Peltier et al used chloroplasts) or result from the different native gel conditions that were used. While Peltier and colleagues used colourless native conditions, I added a blue dye in order to introduce a charge in the complex. This way, native complex associations in vitro may vary and give conflicting results, as is the case for 2-cys peroxiredoxin. Other examples that showed a higher moleculr mass in this study compared to other studies include phosphoribosylformylglycinamidine cyclo-ligase (AIR synthase), that is involved in purine biosynthesis. Its oligomeric state is unknown but Peltier and colleagues found this enzyme in different molecular mass positions such as 142, 114, 93 and 39 kDa (Peltier et al., 2005) but not at a molecular mass above 500 kDa. Furthernore, transaldolase, which is involved in carbohydrate metabolism was identified with a native mass of 91 kDa (Peltier et al., 2005) and was detected as a high molecular mass complex in this study. Prominent proteins detected in Band II (~400-500 kDa), comprise transketolase and keto-acid reductoisomerase. Transketolase is involved in the Calvin cycle, and its oligomeric state is dimeric. The native molecular weight is 149 kDa (Gerhardt et al., 2003). Keto–acid reductoisomerase is involved in branched chain amino acid metabolism, and it is a homodimer in its oligomeric state. The native molecular weight is 149 kDa (Halgand et al., 1999). As mentioned above, the contradicting molecular masses could result from different complex associations. This could suggest that these two enzymes form stable complexes in BY-2 plastids with currently unidentified partners, potentially with one or several of the other proteins identified from Band II. Targeted experiments to verify complex association, for example TAP 66 Results Tagging strategies or other native purification methods are required for their further ineraction. Alternatively, two selected proteins could be produced in yeast two-hybrid assays (reviewed in Serebriiskii and Kotova, 2004). Glutathione reductase identified in Band 3 (~200-300 kDa), maintains high levels of reduced glutathione in the chloroplast. The possible native complex is 186 kDa (Peltier et al., 2005) which fits with our observations. Superoxide dismutase [Mn] and superoxide dismutase [Fe] both are homodimers (Bannister et al., 1987). Three types of superoxide dismutase (SOD) have been characterized based on the nature of the metal co-factor present at the catalytic site, i.e. copper/zinc (CuZnSOD), iron (FeSOD), or manganese (MnSOD) SODs. CuZnSOD is generally found in the cytosol and chloroplasts, MnSOD in mitochondria, whereas FeSOD is present within the chloroplasts of some plants (Bannister et al., 1987). In this approach MnSOD was detected at two positions 400-500 kDa and 200-300 kDa. Our data therefore suggest that SOD may form different dynamic complexes with currently unknown interaction partners. 3.1.4.2 NP-40 insoluble fraction A group of Japanese scientists developed an in vitro transcription system using DNA-protein complexes isolated from nucleoids (BY-2 plastids) for comparative analyses of plastid transcription in various plastid types (Nemoto et al., 1988, 1990; Sakai, 2001; Sakai et al., 2004). The group also developed a method to isolate nucleoids from BY-2 plastids without any contaminations such as mitochondria and cell nuclei. In their research, they compared the molecular architecture of the isolated nucleoids from the BY-2 plastids to that of chloroplasts (Sakai, 2001). Nucleoids are dense, heterogeneous proteinnucleic acid particles, in which plastome replication and transcription occur (Briat et al., 1982). I isolated the NP-40 insoluble protein fraction that should be enriched in nucleoids and thus in transcriptionally active proteins (Sakai et al., 2004). NP40 insoluble proteins were recovered, washed with a buffer without detergents to wash away copurifying soluble proteins (Osmo) and analyzed by shotgun mass spectrometry. The MS-analysis NP-40 insoluble fractions were further 67 Results fractionated into three fractions; Crude (Cr), osmo (O) and CHAPS (C) [Figure 3.14]. Cr e o PS ud sm HA O C 173 kDa 111 80 Figure 3.14: Serial extraction of NP40-insoluble fraction 61 from BY-2 plastid. SDS-PAGE of three fractions from NP40insoluble fraction. 49 36 25 19 One-hundered and one proteins were identified in total from the NP4Oinsoluble fraction (Table 3.6). 59 proteins of which had already been detected in the previous shotgun-proteome analysis. Most of the identified proteins are active in plastid metabolism and some are of ribosomal origin. Nine ribosomal proteins are of plastidic origin. Ribosomal proteins are thought to associate with nucleoids, an assumption that is based on the identification of a 28 kDa ribosomal-like protein as a part of nucleoid preparations from pea chloroplasts (Oleskina et al., 1999). Our results show that the nucleoid isolation did not enrich transcriptionally active proteins as expected, but nonetheless resulted in the identification of previously unidentified proteins further adding to the coverage of the BY-2 plastid proteome. Table 3.6: List of proteins identified from NP40-insoluble fraction from BY-2 Plastid a . only peptides with a significant SEQUEST score (i.e., Xcorr >2.5, dCn >0.1) were considered in this approach. Protein hits were accepted only if two peptides were detected. Localization prediction was performed with ChloroP (Emanuelsson et al., 1999). Putative protein functions were manually assigned as mentioned in previous BY-2 plastid shotgun-proteome analysis. Proteins with a reported function in other cell organelles were considered as putative contaminants. Provided are the identifier (“Identifier”), the database accession number (“acc. no”), the organism (“organism”), the number of identified tryptic peptides (“no. of peptides”), protein localization [“Localization” Y: transit peptide by ChloroP (Emanuelsson et al., 1999), reported Pt: reported chloroplast function or Arabidopsis orthologue with a transit peptide, pt-encoded: plastid encoded, co: putative contaminant, -: any other location]. The information of each fraction step from which proteins were identified (“Fraction”), i.e., osmotic shock (O), CHAPS (C) or crude extract (Cr). 1 those proteins which also found in Phinney and Thelen (2005). a 68 Results Identifier Accession No. Organism Fraction No of peptides Localization Protein gi|12323852 AAG51893 Arabidopsis thaliana O/C Metabolism 5 Y gi|124367 P09342 Nicotiana tabacum O/C 4 gi|37983566 AAR06290 Solanum tuberosum O/C 4 Y 5'-aminoimidazole ribonucleotide synthetase gi|1066499 AAB41904 Medicago sativa O/C 24 Y NADH-dependent glutamate synthase gi|21535791 CAC85727 Nicotiana tabacum C/Cr 22 Y gi|15231092 NP_191420 Arabidopsis thaliana C 2 Y putative carbamoyl phosphate synthase large subunit ketol-acid reductoisomerase gi|7431333 T03406 Nicotiana tabacum C 6 reported Pt probable isocitrate dehydrogenase gi|14547928 Q9SZX3 Arabidopsis thaliana C 2 Y argininosuccinate synthase gi|40457328 AAR86719 Nicotiana attenuata C 3 reported Pt glutamine synthetase gi|56562177 CAH60891 Lycopersicon esculentum Cr 10 Y carbonic anhydrase gi|15223294 NP_171617 Arabidopsis thaliana O 2 Y gi|18403597 NP_566720 Arabidopsis thaliana C/Cr 3 Y pyruvate dehydrogenase E1 component alpha subunit pyruvate kinase gi|12643998 P10871 Spinacia oleracea Cr 19 Y gi|3914583 Q43832 Spinacia oleracea Cr 14 Y ribulose bisphosphate carboxylase/oxygenase activase ribulose bisphosphate carboxylase small chain 2 gi|57012986 Q9AWA5 Solanum tuberosum C 6 Y starch-related R1 protein gi|24745908 BAC23041 Solanum tuberosum C 4 Y glucose 6-phosphate dehydrogenase gi|32481059 AAP83926 Lycopersicon esculentum O/C 5 Y transaldolase gi|3122858 O04130 Arabidopsis thaliana C 3 Y D-3-phosphoglycerate dehydrogenase, putative gi|3023817 Q43793 Nicotiana tabacum C 2 Y glucose-6-phosphate 1-dehydrogenase gi|1168411 P16096 Spinacia oleracea fructose-bisphosphate aldolase gi|51963380 XP_506284 Oryza sativa gi|5123836 CAB45387 gi|2529376 reported Pt dihydrolipoamide S-acetyltransferase acetolactate synthase I Cr 5 Y O/C 8 Y predicted P0710F09.129 gene product Nicotiana tabacum C 7 Y NAD-malate dehydrogenase AAB81104 Spinacia oleracea Cr 4 Y sedoheptulose-1,7-bisphosphatase gi|15232625 NP_190258 Arabidopsis thaliana C 2 Y TOC75; protein translocase gi|53793514 BAD54675 Oryza sativa gi|100424 S17917 Solanum tuberosum gi|231660 P12222 Nicotiana tabacum gi|21537164 AAM61505 Arabidopsis thaliana gi|15221815 NP_175845 gi|30687628 NP_850297 gi|3721540 BAA33531 Nicotiana tabacum O/C 15 Y sulfite reductase gi|22296371 BAC10140 Oryza sativa O/C 8 Y putative 29 kDa ribonucleoprotein A gi|21700195 BAC02896 Nicotiana tabacum O/C 6 reported Pt tobacco nucleolin gi|38017095 AAR07943 Nicotiana benthamiana C 2 Y DNA gyrase B subunit gi|15224298 NP_181287 Arabidopsis thaliana O/C 4 reported Pt RNA binding / nucleic acid binding C 3 - putative iron inhibited ABC transporter 2 O/C 33 co ADP,ATP carrier protein precursor C 2 pt-encoded hypothetical 226 kDa protein ycf1 O/C 2 - unknown Arabidopsis thaliana C 3 - unknown protein Arabidopsis thaliana C/Cr 5 any other location unknown protein Nucleoids related proteins 1 1 1 gi|133247 P28644 Spinacia oleracea Cr 21 Y 28 kDa ribonucleoprotein gi|1015370 AAA79045 Spinacia oleracea Cr 2 Y 24 kDa RNA binding protein 1 69 1 1 Results Heat shock and chaperone gi|46093890 AAS79798 Nicotiana tabacum O/C 104 Y heat shock protein 90 gi|50947075 XP_483065 Oryza sativa O/C 4 Y heat shock protein, putative gi|20835 CAA38536 Pisum sativum O/C 13 reported Pt HSP70 gi|15228802 NP_191819 Arabidopsis thaliana C 3 reported Pt gi|1351030 P21239 Brassica napus O/C/Cr 11 Y heat shock protein binding / unfolded protein binding rubisco subunit binding-protein alpha subunit gi|18378982 NP_563657 Arabidopsis thaliana C/Cr 25 Y nuclear encoded CLP protease 1 gi|53801462 AAU93933 Helicosporidium sp. C 4 reported Pt plastid clpB gi|18423214 NP_568746 Arabidopsis thaliana C 16 Y CLPC; ATP binding / ATPase gi|15237059 NP_193769 Arabidopsis thaliana C/Cr 19 Y ATP binding / GTP binding / translation elongation factor Proteases Structural maintenance and plastid positioning gi|11967906 BAB19779 Nicotiana tabacum O/CCr 18 any other location alpha tubulin gi|1351202 P28551 Glycine max O/C 47 any other location tubulin beta chain gi|15289940 BAB63635 Oryza sativa O/C/Cr 16 any other location putative actin gi|1946329 AAC49690 Nicotiana tabacum C/Cr 3 Y prohibitin gi|7269413 CAB81373 Arabidopsis thaliana O/C 6 Y fibrillarin-like gi|218312 BAA01975 Nicotiana sylvestris O 6 Y chloroplast elongation factor TuB gi|119143 P13905 Arabidopsis thaliana O/C/Cr 25 Y elongation factor 1-alpha gi|134034 P19954 Spinacia oleracea O/C 4 Y plastid-specific 30S ribosomal protein 1 gi|20867 CAA77595 Pisum sativum Cr 3 Y plastid ribosomal protein CL15 gi|2792007 CAA75149 Spinacia oleracea Cr 9 Y chloroplast ribosomal protein L4 gi|5725342 CAA63650 Spinacia oleracea Cr 9 Y ribosomal protein S5 gi|48428519 P82278 Spinacia oleracea C/Cr 8 Y 30S ribosomal protein S9 gi|50400727 P82192 Spinacia oleracea Cr 6 Y 50S ribosomal protein L5 gi|57471704 AAW50983 Triticum aestivum O/C 2 reported Pt ribosomal protein L11 gi|11497599 NP_055005 Spinacia oleracea Cr 12 pt-encoded ribosomal protein L12 gi|11497513 NP_054921 Spinacia oleracea Cr 3 pt-encoded ribosomal protein S2 gi|11497530 NP_054938 Spinacia oleracea Cr 3 pt-encoded ribosomal protein S4 gi|12023 CAA33932 Oryza sativa O/C 3 pt-encoded ribosomal protein L14 Gene expression Redox and electron transport gi|47027073 AAT08751 Hyacinthus orientalis C/Cr 5 reported Pt 2-cys peroxiredoxin-like protein gi|131386 P12359 Spinacia oleracea Cr 2 Y oxygen-evolving enhancer protein 1 gi|22276 CAA43663 Zea mays C gi|47026912 AAT08677 Arabidopsis thaliana gi|27734400 P59169 Arabidopsis thaliana gi|15226944 NP_180441 Arabidopsis thaliana gi|15231304 NP_190184 Arabidopsis thaliana O/C/Cr Other functions 3 Y ferritin 1 Putative nuclear contminants O/C 7 co histone H2A O 4 co histone H3.3 O/C/Cr 25 co histone H4 29 - DNA binding 1 70 Results Putative mitochondrial contaminants gi|26391487 P83483 Arabidopsis thaliana gi|1582354 2118337 Nicotiana tabacum O/C 7 co ATP synthase beta chain, mitochondrial C 6 Y A Ac-CoA carboxylase:SUBUNIT=biotin carboxylase 1 mitochondrial elongation factor Tu gi|1149571 CAA61511 Arabidopsis thaliana C 6 co gi|10798640 CAC12820 Nicotiana tabacum C 4 co gi|15227257 NP_180863 Arabidopsis thaliana O/C 2 reported Pt mitochondrial 2-oxoglutarate/malate carrier protein hydrogen-transporting ATP synthase gi|516166 CAA56599 Solanum tuberosum C 2 co 34 kDA porin gi|10177333 BAB10682 Arabidopsis thaliana C 4 Y 2-oxoglutarate dehydrogenase gi|12644189 P29197 Arabidopsis thaliana O/C 6 co chaperonin CPN60, mitochondrial gi|3334404 O23654 Arabidopsis thaliana C 2 any other location vacuolar ATP synthase catalytic subunit A gi|13623892 BAB41076 Nicotiana tabacum O/C 9 co gi|449171351 BAD12168 Nicotiana tabacum MAR-binding protein Putative plasma membrane C 2 any other location 14-3-3 a-1 protein gi|15232776 NP_187595 Arabidopsis thaliana Cr 3 any other location CDC48 (cell division cycle48) gi|15231130 NP_191434 Arabidopsis thaliana C 6 - gi|1170503 P41376 Arabidopsis thaliana Eukaryotic translation initiation O/C 9 - citrate (SI)-synthase/ transferase eukaryotic initiation factor 4A-1 gi|1170507 P41380 Nicotiana plumbaginifolia C 2 - gi|1170511 P41382 Nicotiana tabacum O/C 19 - eukaryotic initiation factor 4A-10 gi|2500518 Q40465 Nicotiana tabacum C 2 - eukaryotic initiation factor 4A-11 gi|2500521 Q40468 Nicotiana tabacum C 14 - eukaryotic initiation factor 4A-15 gi|50931205 XP_475130 Oryza sativa O/C 2 co putative 40S ribosomal protein S4 gi|15128437 BAB62621 Oryza sativa O 2 co putative 40S ribosomal protein S5 gi|15233226 NP_191088 Arabidopsis thaliana O/C 26 co RNA binding / structural constituent of ribosome gi|40287492 AAR83860 Capsicum annuum O/C 2 co putative ribosomal protein gi|40556671 AAR87752 Capsicum annuum O/C 4 co small subunit ribosomal protein S16 gi|15214300 Q9ZSR8 Brassica napus C 2 co 40S ribosomal protein SA (p40) gi|12229886 O04204 Arabidopsis thaliana O 3 co 60S acidic ribosomal protein P0-A gi|17367847 Q9XF97 Prunus armeniaca C 2 co 60S ribosomal protein L4 (L1) gi|40287508 AAR83868 Capsicum annuum O 8 co 60S ribosomal protein L12 gi|40287526 AAR83877 Capsicum annuum O/C 3 co 60S ribosomal protein L19 gi|2500376 Q42351 Arabidopsis thaliana C 3 co 60S ribosomal protein L34 gi|2058273 BAA19798 Oryza sativa O 2 co YK426 eukaryotic initiation factor 4A-3 Ribosomal related proteins I next compared the identified proteins from the NP-40 insolube fraction with those found in a previous study of pea nucleoids (Phinney and Thelen, 2005). In their study, Phinney and Thelen identified 35 proteins that are related to nucleoids and this classification was based on literature searches and functional predictions. Nine proteins overlap between the study reported here and the study reported by Phinney and Thelen [Table 3.6 “1”]. The interesting candidates from this classification are sulfite reductase, DNA gyrase subunit A and DNA binding proteins. It has been reported that these proteins are associated with plastid-nucleoids (Sato et al., 2003). 71 1 Results 3.1.5 Overview of the BY-plastid proteome For the BY-2 plastid proteome-analysis, quantative (shotgun and 2D-PAGE) and functional (BN-PAGE and NP-40 insoluble fraction) approaches were applied. All identified proteins from these approaches were assembled into a single FASTA file and searched against the proteins identified in the shotgunproteome analysis. The rationale of this analysis was to assess the BY-2 plastid proteome coverage and the efficiency of the different proteomics approaches employed (Table 3.7). In total, 428 proteins were detected from the BY-2 plastid, 323 plastidic proteins, 197 of which are unique (Table 3.7). Although most of the proteins had already been identified in the shotgunproteome analysis, there were a few hits only in the alternative approaches. Most of these new hits include proteins of amino acid metabolism but also ribosomal proteins from the NP-40 insoluble fraction. Table 3.7: No. of proteins identified in BY-2 plastid proteome using different approaches. BY-Plastid Total No.of approaches proteins Plastidic Contaminants Unique Unique contaminants plastid hits Shotgun 205 149 56 56 149 2D-PAGE 72 59 13 2 16 BN-PAGE 50 46 4 1 7 NP-40 insoluble 101 69 32 17 25 428 323 105 76 197 fraction Total 72 Results 3.2 Capsicum annuum chromoplast 3.2.1 Isolation and purification C. annuum chromoplasts Several studies have already been conducted on chromoplasts of various plant species. Our source of chromoplasts was C. annuum. Chromoplasts from different plant species behave differently during their isolation, e.g., daffodil chromoplasts can be recovered from sucrose gradients, whereas tomato chromoplasts are fragile and break during sedimentation in sucrose or Percoll gradient ((Hadjeb et al., 1988). Capsicum annuum chromoplasts are more stable during Percoll density gradient centrifugation (Hadjeb et al., 1988). C. annuum chromoplasts were isolated by Percoll density gradient centrifugation using a step gradient developed with 20%-30%-40%-50% and 60% Percoll in GR buffer (Material and Methods, section 2.2.3.2). Visual examination revealed that chromoplasts migrate to positions of different Percoll densities in the gradient (Figure 3.15, I). In order to assess the relative amount of chromoplasts compared to other cell organelles in each fraction, I isolated material from the bands A, B, C, D, E and F (from top to bottom) and identified the major protein constituents in each band by shotgun mass spectrometry as described (Materials and Methods, section 2.2.6.1) [Figure 3.15, II). Localization of the identified proteins was predicted using TargetP (http://www.cbs.dtu.dk/services/TargetP/), protein annotations and literature search. I grouped the identified proteins into five major categories they are plastid (Pt), mitochondria (Mit), nuclear (Nu), unknown (Un), secretoy pathway (SP). This analysis demonstrated that Band D (interphase 30%-40%) contains most of the plastid proteins and in general less contaminating proteins from other cell organelles as compared to other isolated bands (Figure 3.15, I; Supplementary Table 2). This can be attributed to the differential centrifugation steps prior to density gradient centrifugation that efficiently removed the mass of the other cell organelles from the plastid preparation. The light microscopy observation also revealed that Band D contains a homogeneous preparation of mostly intact chromoplasts with only minor contaminations from other cell organelles (Figure 3.15, III). 73 Results I II 60 A 50 B C 30% D 40% E No. of proteins 20% 40 Pt Mit 30 Nu Un 20 SP 50% F 10 60% 0 A B Percoll density gradient III C D E F Protein distribution in different fractions of percoll density gradient Band D Figure 3.15: Isolation and purification of C. annuum chromoplasts. (I) Isolated chromoplasts were subjected to Percoll density centrifiguation (60%-50%-40%-30%-20%). (II) Each interphase (6) was subjected to SDS-PAGE, tryptically digested and loaded to the LCESI-MS/MS. The best ratio between plastid proteins and those other cell organelles was detected in Band D. (III) Fluorescence microscopy of organelles isolated from band D revealed chromoplasts with a size is ranging from 3-4 µm. Abbreviations Pt: Plastid; Mit: Mitochondria; Nu: Nuclear; Un: Unknown; SP: Secretory pathway. 3.2.2 Protein fractionation Having established the presence of relatively pure chromoplasts in the Band D (30%-40% Percoll interphase), I modified the Percoll density gradient with minor modifications in order to yield higher purity chromoplasts and to upscale isolation procedure (Material and Methods, section 2.2.2.2). It was reported that intact chromoplasts could be obtained, unless extreme dilutions of the Percoll gradient centrifugation are achieved (reviewed in Camara et al., 1995). Thus, I removed the 60% concentration at the bottom and added 15% concentration at the top of the Percoll density gradient to increase chromoplast intactness and purity. The band at the 30%-40% Percol 74 Results interphase was recovered, washed, concentrated and used for subsequent B) 10% 20% Isolation of chromoplasts 30% Serial extraction (Four steps) 40% 50% SDS-PAGE cut into 12 sections in gel digestion 173 111 80 61 49 36 25 19 C) Solubilization step Reversed phase chromatography on C18 Cr C. annuum KDa A) ud e Os mo 8M u 5% rea SD S proteome studies (Figure 3.16, B). OSMO 8M urea 5% SDS Protein total 1.56 mg 0.50 mg n.d Percentage 75.73 24.27 n.d ESI-MS/MS Figure: 3.16: Isolation and fractionation of C. annuum chromoplast proteins. A). Fractionation strategy for C. annuum chromoplast proteins. B). Organelle fractions in the Percoll density gradient and a silver stain gel from each fraction of serial extraction. C). Quantative distribution of chromoplast proteins among different fractions as determined by Bradford analysis. (n.d: not detected). Chromoplast protein extract was fractionated into soluble proteins (OSMO), peripheral (8M urea) and integral membrane proteins (5% SDS) (Figure 3.16, B). The relative amount of soluble (OSMO) and membrane proteins was determined by Bradford analysis (Figure 3.16, C). Soluble proteins make up 76 %, and the membrane proteins make up 24 % of the total chromoplast proteins. These values indicate that isolated chromoplasts contained mostly soluble proteins and were largely intact after purification. In contrast to this, the 5% SDS fraction contains fewer membrane proteins. A possible explanation for this is that thylakoids are disassembled and photosynthetic proteins are degraded during chloroplast to chromoplast conversion (reviewed in Lopez-Juez and Pyke, 2005). As compared to the chloroplast, which has a developed inner membrane system, the picture is therefore different for the chromoplast. In a pervious chloroplast proteome study, 137 proteins were identified in the envelope fraction and 58 were found to be unique to the plastid (Kleffmann et al., 2004). This represents the major 75 Results protein mass in chloroplasts and requires SDS for their solubilization (Kleffmann et al., 2004). 3.2.3 Detection of proteins in different fractions After MS-analysis, 182 proteins identified from all fractions including contaminants (Table 3.8). Protein localization was predicted using the same criteria as explained in material and methods (section 2.2.8.1). In total, 149 proteins were considered as true plastid proteins because these proteins fall into a criterion that is set of plastid proteins (Material and Mehtods, section 2.2.8.1 “Protein localization”). Thirty-three proteins were most likely not of chromoplast origin and were considered putative contaminants. A few of these proteins are storage and nutrient related proteins (Table 3.8). All identified proteins were manually sorted by their putative functions and considering the fraction from which these proteins were identified i.e., osmotic shock (O), 8M urea (U), 5% SDS (S) and crude (C), the list comprises those proteins that were detected in an unfractionated chromoplast preparation (Figure 3.17, Table 3.8, column “fractions”). Most of the proteins were identified from the Osmo and Crude fractions (87 and 39 respectively) and a small number of proteins (10) overlapped in both the Osmo-Urea and Osmo-SDS fractions. This also suggests that the separation of proteins into soluble and peripheral membrane proteins is an efficient strategy (Figure 3.16, B). Abundant proteins were detected in distinct fractions, which is a wellknown phenomenon in proteomics approaches where fuzziness in their fractionation is frequently observed. Most of the identified proteins in the OSMO fractions are active in carbohydrate and amino acid metabolism, respectively (Table 3.8). The urea and SDS fractions contain proteases and some potential transporters suggesting their function at the plastid outer membrane system (Table 3.8). The abundant proteins in the SDS fraction are capsanthin/capsorubin synthase, lethal leaf spot 1-like protein and transketolase 1 all of which were identified with many peptides in this fraction (49 peptides from CCS, 9 peptides from lethal leaf spot protein 1, and 6 peptides from transketolase 1, respectively). 76 Results 8M urea 12 OSMO 10 Crude 87 39 8 6 10 10 SDS Figure 3.17: Number of proteins identified in each fraction; 10 proteins found in OSMO and 8M, 10 proteins found in OSMO and SDS, 6 proteins found in 8M urea and SDS, 8 proteins found in each fraction [Mix] (OSMO, 8M urea and SDS). 39 proteins are unique to the crude fraction. a Table 3.8: List of the proteins identified from C. annuum chromoplasts. a only peptides which have significant score (i.e., Xcorr >2.5, dCn >0.1) in SEQUEST database search were taken in this approach. Protein hits were accepted only if two peptides were detected from this protein. Localization prediction was performed with ChloroP (Emanuelsson et al., 1999). Putative protein functions were manually assigned as mentioned in pervious approahes (BY-2 plastid proteome). Proteins with a reported function in other cell organelles were considered as putative contaminants. Provided is the identifier (“Identifier”), the NCBI database accession number (“acc. no”), the organism (“organism”), the number of identified tryptic peptides (“no. of peptides”), protein localization [“Localization” Y: transit peptide by ChloroP (Emanuelsson et al., 1999), RP: reported chloroplast function or Arabidopsis orthologue with a transit peptide, PE: plastid encoded, co: putative contaminant,-: any other location]. The information from each fraction step the protein was identified (“Fraction”), i.e., osmotic shock (O), urea (U), SDS (S) or crude extract (C).b homologues to transketolase precursor, c homologues to fructose-bisphosphate aldolase, d homologues to Ferritin, e formylglycinamide ribonucleotide amidotransferase, f belongs to shortchain dehydrogenases family. Identifier Acc. No Organism Fraction No. of peptides Localiztion Carbohydrate metabolism O 11 RP Protein gi|30407706 AAP30039 Lycopersicon pennellii gi|82400156 ABB72817 Solanum tuberosum O/U/S/C 104 aconitase gi|1388021 AAB71613 Solanum tuberosum O gi|54287592 AAV31336 Oryza sativa gi|3915873 P13708 Glycine max gi|12585317 Q9M4G5 Solanum tuberosum O/C 5 Y gi|18412307 NP_565203 Arabidopsis thaliana O/C 6 Y ST1(mercaptopyruvate sulfurtransferase 1) gi|30686602 NP_194193 Arabidopsis thaliana O/C 4 Y Y PGI1(chloroplastic phosphoglucose isomerase) oxoglutarate:malate antiporter Y phosphoglycerate kinase precursor-like 2 RP UDP-glucose pyrophosphorylase O 9 Y putative UDP-sulfoquinovose synthase O 2 RP sucrose synthase (Sucrose-UDP glucosyltransferase) phosphoglucomutase gi|30684152 NP_568283 Arabidopsis thaliana gi|52851186 CAH58641 Plantago major C 2 O/U/C 17 gi|15219980 NP_178093 Arabidopsis thaliana RP malate dehydrogenase C 2 Y malic enzyme/ oxidoreductase gi|585449 P37222 gi|13345411 AAK19325 Lycopersicon esculentum Dunaliella salina O/C 8 RP NADP-dependent malic enzyme U/C 4 RP fructose-bisphosphate aldolase isoenzyme 2 fructose-bisphosphate aldolase, chloroplast gi|78099750 Q40677 Oryza sativa O 4 RP gi|18420348 NP_568049 Arabidopsis thaliana U/S/C 13 Y fructose-bisphosphate aldolase gi|1781348 CAA71408 Solanum tuberosum U/S/C 12 RP homologous to plastidic aldolases 77 Results gi|10279993 CAC09959 Nicotiana tabacum O/U/S 6 Y unnamed protein product unknown gi|77745483 ABB02640 Solanum tuberosum O/U/S/C 20 RP gi|3559814 CAA75777 Capsicum annuum O/U/C 208 Y transketolase 1 gi|51340062 AAU00727 O 16 Y glucose-6-phosphate isomerase gi|10441272 AAG16981 O/S/C 67 Y transaldolase gi|120661 P09043 Lycopersicon esculentum Lycopersicon esculentum Nicotiana tabacum O/C 20 Y gi|20455491 P25857 Arabidopsis thaliana O/U/S/C 12 Y gi|1402885 CAA66816 Arabidopsis thaliana C 2 Y gi|1351282 P48497 Stellaria longipes O/C 6 RP glyceraldehyde-3-phosphate dehydrogenase A glyceraldehyde-3-phosphate dehydrogenase B glyceraldehyde-3-phosphate dehydrogenase triosephosphate isomerase, cytosolic gi|1351271 P48496 Spinacia oleracea O/C 11 Y triosephosphate isomerase, chloroplast gi|30693852 NP_851113 Arabidopsis thaliana O 2 - phosphogluconate dehydrogenase gi|38604456 AAR24912 O 5 Y fructokinase 3 gi|15237303 NP_200104 Lycopersicon esculentum Arabidopsis thaliana O/C 3 Y pyruvate kinase Starch metabolism gi|13124867 AAK11735 Solanum tuberosum O/C 15 Y starch associated protein R1 gi|1169912 P30924 Solanum tuberosum O/C 18 Y 1,4-alpha-glucan branching enzyme gi|130173 P04045 Solanum tuberosum O/C 39 Y gi|1730557 P53535 Solanum tuberosum O 18 Y alpha-1,4 glucan phosphorylase, L-1 isozyme alpha-1,4 glucan phosphorylase, L-2 isozyme Amino acid metabolism gi|37983671 AAR06294 Nicotiana tabacum O 2 Y adenylosuccinate synthase gi|17944 CAA46086 Capsicum annuum O/C 25 Y o-acetylserine (thiol)-lyase gi|25990362 AAN76499 Phaseolus vulgaris O 39 Y aspartate aminotransferase gi|81074221 ABB55364 Solanum tuberosum S/C 4 Y aspartate aminotransferase-like gi|2970447 AAC05981 Glycine max S/C 5 Y aspartokinase-homoserine dehydrogenase gi|15220397 NP_176896 Arabidopsis thaliana O/C 16 Y lactoylglutathione lyase gi|266346 Q01292 Spinacia oleracea O/C 5 Y ketol-acid reductoisomerase gi|1084412 S22527 Nicotiana sp O 2 RP glutamate-ammonia ligase gi|12644435 Q43155 Spinacia oleracea O/C 64 Y gi|15227306 NP_181655 Arabidopsis thaliana O/C 6 Y gi|37953301 AAR05449 Capsicum annuum O/C 3 RP Ferredoxin-dependent glutamate synthase(Fd-GOGAT) Ferredoxin-dependent glutamate synthase 2(GLU2) alanine aminotransferase gi|18391069 NP_563853 Arabidopsis thaliana O 2 Y ATP phosphoribosyl transferase 2 gi|76556492 CAJ32461 Nicotiana tabacum O 6 Y putative chloroplast cysteine synthase 1 gi|2492953 Q42885 Lycopersicon esculentum O 5 Y gi|21535791 CAC85727 Nicotiana tabacum O/C 8 Y gi|15233135 NP_191710 Arabidopsis thaliana O 2 - gi|30688090 NP_197598 Arabidopsis thaliana O 2 Y gi|18028086 AAL55967 Raphanus sativus O 2 - gi|22296426 BAC10194 Oryza sativa S 2 Y chorismate synthase 2 (5enolpyruvylshikimate-3-phosphate phospholyase 2) putative carbamoyl phosphate synthase large subunit 1-aminocyclopropane-1-carboxylate synthase 5-methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase phospholipid hydroperoxide glutathione peroxidase putative phospho-2-dehydro-3deoxyheptonate aldolase 1 gi|16797908 AAL29212 Capsicum annuum gi|12003283 AAG43518 Perilla frutescens gi|14422255 CAC41366 Brassica napus gi|1174466 P46253 Solanum tuberosum Lipid metabolism O/S/C 24 RP putative acyl-CoA synthetase O/C 9 Y malonyl-CoA:ACP transacylase O 12 RP enoyl-[acyl-carrier protein] reductase O/U/C 7 Y acyl-[acyl-carrier-protein] desaturase(stearoyl-ACP desaturase) 78 Results Photosynthesis and carbon fixation gi|27804768 AAO22558 Oryza sativa gi|18414603 NP_567487 Arabidopsis thaliana gi|12082782 AAG48610 O 7 Y sedoheptulose-1,7-bisphosphatase O/C 2 Y C 5 Y FAD binding / disulfide oxidoreductase/ oxidoreductase photosystem II 22 kDa protein precursor U/S/C 5 RP RbcL C 3 Y nonphotochemical quenching O/C 31 Y O/U/S/C 169 RP O/S 10 Y ribulose bisphosphate carboxylase small chain ribulose 1,5-bisphosphate carboxylase large subunit ribulose-phosphate 3-epimerase gi|66876461 AAY58013 Solanum sogarandinum Cuscuta sandwichiana gi|42571761 NP_973971 Arabidopsis thaliana gi|59800169 P69249 Nicotiana tabacum gi|7240236 AAA85290 Glaucidium palmatum gi|2499728 Q43843 Solanum tuberosum gi|21912927 CAC84143 Nicotiana tabacum O 6 Y thioredoxin peroxidase gi|11908048 AAG41453 Arabidopsis thaliana C 3 Y putative 2-cys peroxiredoxin protein gi|11558242 CAC17803 Phaseolus vulgaris O/U/C 5 Y peroxiredoxin gi|81301599 YP_398895 C 3 - cytochrome b/f complex subunit IV gi|33413303 CAE22480 Nicotiana tomentosiformis Lycopersicon esculentum O/C/S 33 Y superoxide dismutase [Fe] gi|1097877 2114378A gi|2493810 Q42946 Lycopersicon esculentum Nicotiana tabacum gi|15218820 NP_171847 Arabidopsis thaliana gi|19875 CAA46787 Nicotiana tabacum gi|15222443 NP_177132 Arabidopsis thaliana gi|14135052 CAC38942 Nicotiana tabacum O/C gi|11878280 AAG40879 Nicotiana tabacum gi|55792566 AAV65378 Prototheca wickerhamii gi|81076111 ABB55387 Nicotiana tabacum gi|75320739 Q5IZC8 Arabidopsis thaliana U/C 2 Y chloroplastic protein TOC75-4 gi|21587 CAA47430 Solanum tuberosum S 2 Y triose phosphate translocator gi|8347246 AAF74567 Solanum tuberosum S 6 RP hexose transporter gi|15224474 NP_178586 Arabidopsis thaliana O 2 Y transporter gi|119194 P17745 Protein synthesis and gene expression O/U/C 11 Y Arabidopsis thaliana gi|3122071 Q41803 Zea mays Redox and electron transport Porphyrin and chlorophyll metabolism O/C 9 Y aminolevulinate dehydratase O 2 Y coproporphyrinogen III oxidase O 6 Y coproporphyrinogen oxidase O/U/C 7 Y O/C 5 Y glutamate-1-semialdehyde 2,1aminomutase porphobilinogen synthase 11 Y unnamed protein product O 2 Y O 2 Y phosphoribosylaminoimidazolecarboxamide formyltransferase Plastid phosphoribosylaminoimidazolecarboxamide formyltransferase Nucleotide Metabolism Transporters and Envelope proteins S S/C 2 4 Y ADP,ATP carrier protein precursor-like elongation factor Tu - elongation factor 1-alpha gi|68566314 Q43364 Nicotiana sylvestris C 2 Y elongation factor TuB gi|68566313 Q40450 Nicotiana sylvestris C 2 Y elongation factor TuA gi|15231362 NP_190203 Arabidopsis thaliana gi|15450585 AAK96564 Arabidopsis thaliana gi|2497541 Q40545 Nicotiana tabacum O/C 7 Y pyruvate kinase isozyme A gi|27807824 BAC55280 Nicotiana tabacum O/C 2 Y nucleoside diphosphate kinase gi|27462474 AAO15447 O/C 13 Y GcpE protein gi|15239983 NP_199191 Lycopersicon esculentum Arabidopsis thaliana O 2 Y APS4 Other Metabolism O 2 C 3 - oxidoreductase - AT5g50700/MFB16_9 79 Results gi|11387206 Q9THX6 gi|15240796 NP_196364 Lycopersicon esculentum Arabidopsis thaliana gi|1279231 CAA65784 Capsicum annuum gi|18399513 NP_565491 Arabidopsis thaliana gi|38231570 AAR14689 gi|40644130 gi|18418270 O/C 3 Y Y putative L-ascorbate peroxidase(thylakoid lumenal 29 kDa protein) peptidmethionine sulfoxide reductase3 O/C 2 O/U/S/C 210 Y plastoglobules associated protein C 2 Y THF1 O/C 3 Y PII-like protein CAC83765 Lycopersicon esculentum Nicotiana tabacum O/C 10 Y allene oxide cyclase NP_567934 Arabidopsis thaliana O/C 4 Y AGD2 (aberrant growth and death2) gi|55773756 BAD72439 Oryza sativa O/C 2 - gi|2281235 AAB64055 Acanthopanax trifoliatus Brassica napus O/U 2 Y pentatricopeptide repeat-containing proteinlike maturase (matK) gi|14388188 AAK60339 gi|16973465 AAL32300 gi|68510418 AAY98500 U/C 4 Y biotin carboxylase U/S/C 48 Y lethal leaf spot 1-like protein C 2 Y senescence-inducible chloroplast staygreen protein 1 Sn-1 gi|860903 CAA55812 Lycopersicon esculentum Lycopersicon esculentum Capsicum annuum O/C 21 RP gi|42561732 NP_563750 Arabidopsis thaliana C 2 Y ARA;GTP binding gi|78191402 ABB29922 Solanum tuberosum C 2 Y unknown gi|1052778 CAA51786 Pisum sativum C 2 Y ferritin gi|729478 P41344 Oryza sativa U/C 10 Y ferredoxin--NADP reductase gi|50918869 XP_469831 Oryza sativa S/C 6 Y putative glucosyltransferase gi|30697397 NP_200868 Arabidopsis thaliana O/C 2 Y chloroplast biogenesis 4 gi|46488698 AAS99589 Elaeis guineensis O 3 RP gi|121145 P80042 Capsicum annuum O/S/C 24 RP 1-deoxy-D-xylulose-5-phosphate reductoisomerase geranylgeranyl pyrophosphate synthetase gi|19071768 AAL80006 C 10 RP beta carotene hydroxylase C 5 Y zeta-carotene desaturase O/U/S/C 293 Y capsanthin/capsorubin synthase 4-diphosphocytidyl-2C-methyl-D-erythritol synthase Probable 1-deoxy-D-xylulose-5-phosphate synthase (DXP synthase) Carotenoid biosynthesis gi|1176437 AAB35386 Sandersonia aurantiaca Capsicum annuum gi|7488963 S71511 Capsicum annuum gi|18395376 NP_565286 Arabidopsis thaliana O 2 RP gi|30315812 O78328 Capsicum annuum U 2 Y gi|3808101 CAA09935 Capsicum annuum U/S/C 6 RP protease gi|14787784 CAC44257 Nicotiana tabacum C 3 RP FtsZ-like protein gi|15485610 CAC67407 C 3 Y Clp protease 2 proteolytic subunit gi|30694608 NP_850962 Lycopersicon esculentum Arabidopsis thaliana O/C 5 Y ATPREP2; metalloendopeptidase gi|18378982 NP_563657 Arabidopsis thaliana O/C 6 Y CLPP5 (nuclear encoded protease 1) gi|18391391 NP_563907 Arabidopsis thaliana C 3 Y CLPR2 gi|2492515 Q39444 Capsicum annuum gi|399213 P31542 gi|18417676 NP_568314 Lycopersicon esculentum Arabidopsis thaliana gi|38679319 AAR26481 gi|42563757 NP_187466 Lycopersicon esculentum Arabidopsis thaliana gi|15218932 NP_176194 Arabidopsis thaliana O/S 4 Y unknown protein gi|15241645 NP_199315 Arabidopsis thaliana O 2 Y unknown protein gi|50935265 XP_477160 Oryza sativa O 2 - unknown protein gi|53793250 BAD54474 Oryza sativa O/C 2 Y unknown protein Proteases U/S/C 17 RP Cell division protein ftsH homolog U/C 13 Y O/C 7 Y ATP-dependent Clp protease ATP-binding subunit clpA homolog CD4B ATP binding / ATPase/ nucleosidetriphosphatase/ nucleotide binding Structural related proteins C 3 Y C 2 RP harpin binding protein 1 structural molecule Unknown and Hypothetical proteins 80 Results gi|15292887 AAK92814 Arabidopsis thaliana U 2 Y unknown protein gi|30699237 NP_565149 Arabidopsis thaliana C 2 Y unknown protein gi|15229871 NP_189995 Arabidopsis thaliana C 4 Y unknown protein gi|12323666 AAG51799 Arabidopsis thaliana O 2 Y hypothetical protein gi|55297107 BAD68751 Oryza sativa O/C 2 Y hypothetical protein Heat Shock and Chaperones gi|77556324 ABA99120 Oryza sativa gi|68989120 BAE06227 gi|2654208 AAB91471 Lycopersicon esculentum Spinacia oleracea O/S 4 - putative heat-shock protein O/U/C 19 Y heat shock protein O/S/C 67 Y heat shock 70 protein gi|1351030 P21239 Brassica napus O/U/S/C 31 Y P21241 Brassica napus O/U/C 57 Y rubisco subunit binding-protein alpha subunit rubisco subunit binding-protein beta subunit gi|134104 gi|7331143 AAF60293 Lycopersicon esculentum Oryza sativa O/C 3 Y chaperonin 21 precursor gi|77554415 ABA97211 C 2 Y chaperone protein DnaK ATP hydrolysis/synthesis gi|28261702 NP_783217 Atropa belladonna U/C 40 PE ATP synthase CF1 alpha chain gi|81238327 NP_054506 Nicotiana tabacum U/C 47 PE ATP synthase CF1 beta chain gi|114610 P00834 Nicotiana tabacum C 4 PE ATP synthase epsilon chain gi|20068317 CAC88030 Atropa belladonna C 3 PE ATPase subunit I gi|114407 P05495 gi|28261730 NP_783245 Nicotiana plumbaginifolia Atropa belladonna gi|2811029 O04866| gi|1345679 P49316 gi|79313237 gi|1644306 Contaminants U 3 - ATP synthase alpha chain, mitochondrial S/C 5 co cytochrome f Alnus glutinosa O 3 co O 3 - NP_001030698 Nicotiana plumbaginifolia Arabidopsis thaliana Acetylornithine aminotransferase, mitochondrial catalase isozyme 2 O/C 3 - catalytic CAB03628 Nicotiana tabacum O/U/C 6 - viral envelope protein gi|22327994 NP_200914 Arabidopsis thaliana gi|548493 P35339 Zea mays gi|51572270 AAU06816 Quercus robur O/C 5 - histone deacetylase U 2 - exopolygalacturonase precursor O/S/C 14 - dehydrin 3 resistance protein, putative gi|77556304 ABA99100 Oryza sativa U/C 4 - gi|37780232 AAP45718 Cichorium endivia O/C 3 - RGC2-like protein gi|6606266 AAF19148 Aegilops ventricosa O/C 2 co Vrga1 gi|15430867 AAK98602 Actinidia deliciosa O/C 2 - D12 gi|38345710 CAD41832 Oryza sativa O/C 2 - OSJNBb0085C12.12 gi|78708062 ABB47037 Oryza sativa O/C 4 - gi|15232660 NP_188188 Arabidopsis thaliana C 2 - retrotransposon protein, putative, unclassified unknown protein gi|15241422 NP_199225 Arabidopsis thaliana C 8 - CRA1 (CRUCIFERINA); nutrient reservoir gi|15219582 NP_171884 Arabidopsis thaliana C 9 - CRU2 (CRUCIFERIN 2); nutrient reservoir gi|15235321 NP_194581 Arabidopsis thaliana C 13 - CRU3 (CRUCIFERIN 3); nutrient reservoir gi|30690736 NP_195388 Arabidopsis thaliana C 2 - nutrient reservoir gi|16604374 AAL24193 Arabidopsis thaliana C 3 - AT3g22640/MWI23_1 gi|112741 P15459 Arabidopsis thaliana S 7 - 2S seed storage protein 3 precursor gi|112743 P15460 Arabidopsis thaliana C 5 - 2S seed storage protein 4 precursor gi|10178154 BAB11599 Arabidopsis thaliana C 5 - oleosin, isoform 21K gi|15232227 NP_189403 Arabidopsis thaliana C 3 - unknown protein gi|15239409 NP_200875 Arabidopsis thaliana C 2 - structural constituent of ribosome gi|15237562 NP_201198 Arabidopsis thaliana C 3 - gi|26454606 P93207 Lycopersicon esculentum C 2 - ATP binding / protein kinase/ protein serine/threonine kinase/ protein-tyrosine kinase 14-3-3 protein 10 81 Results gi|50899322 XP_450449 Oryza sativa O 2 co putative cytochrome P450 gi|1071660 CAA63710 Capsicum annuum O 3 - annexin gi|78099751 P17784 Oryza sativa O 3 - gi|27475610 CAD23248 Medicago truncatula O/S 2 RP fructose-bisphosphate aldolase, cytoplasmic squalene monooxygenase 2 gi|30696286 NP_176151 Arabidopsis thaliana O/C 6 RP ATP binding 3.2.4 Functional classification of identified proteins The identified plastid proteins (149) were functionally classified and most of the identified proteins have a general function in the plastid metabolism (Figure 3.18). The majority of the proteins are involved in carbohydrate metabolism (Figure 3.18, Table 3.8). I have identified many tryptic peptides from transketolase, transaldolase, chloroplast phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase A and triosephosphate isomerase with many peptides signifying their high abundance. In amino acid metabolism, I obtained several hits from glutamate synthase, cysteine synthase and amino acid synthesis. The carotenoid biosynthetic pathway is an important pathway in plastids. I identified the carotenoid biosynthetic pathway as the predominant individual pathway and achieved coverage greater than 40% (Figure 3.19, Table 3.8). Capsanthin/capsorubin synthase (CCS) is the most abundant chromoplast enzyme with 293 detected peptides (Table 3.8). In this study, a large number of tryptic peptides (210) from plastoglobule-associated proteins were detected, which belong to the plant fibrillins family, in different protein fractions (Table 3.8). It is suggested that plastoglobules represent a functional metabolic link between the inner envelope and the thylakoid membranes (reviewed in Rossignol et al., 2006). Besides the metabolic and energetic biochemical pathways, I also detected proteins that are related to photosynthesis and carbon fixation. In this group, the ribulose 1, 5bisphosphate carboxylase large subunit is an abundant protein and I detected 169 peptides in different protein fractions. In general, eight proteins with a potential function in protein degradation were detected, four of which belong to the Clp protease system which could be responsible for the degradation of chloroplast proteins that are not required in chromoplasts (Table 3.8) [Sakamoto, 2006]. 82 Results 35 30 20 15 10 er ot h de tra ns po ge rte ne r ex pre ss ion ca rot en oi d pr o tea se s str uc t ur al he atSh oc AT k Ps yn th e sis un kn ow n le x nu cle ot i red o rap yrr o tet id ixa tio n lip nf PS /ca r bo sta rch am ino hy dr a te 0 ac id 5 ca rbo No. of proteins 25 Figure 3.18: Distribution of the identified proteins from C. annuum chromoplasts according to their various functional aspects. Most of the proteins belong to the carbohydrate and amino acid metabolism. . 83 Results GA3P DXS DOXP DXR MEP ispD ispE A B ispF C Ipi IPP Pyruvate DMADP +3 IPP Ggps GGPP Psy Phytoene Psd Phytofluene Psd Zeta-carotene Zds Neurosporene Zds Lcy-e Lycopene δ-Carotene Lcy-b Lcy-b γ-Carotene Lcy-b α-Carotene β-Carotene CrtR-b CrtR-e CrtR-b Lutein Zeaxanthin Capsanthin CCS Zep1 Capsorubin CCS VNCED VP14 ABA AO ABA-aldehydexanthoxin Vde1 Zep1 Antheraxanthin Vde1 Zep1 Violaxanthin NXS Neoxanthin Xanthoxin Figure 3.19: The carotenoid biosynthesis pathway in plants. The enzymes found in the SEQUEST data base search are shown in red and bold. Blue and bold enzymes are found in MS-BLAST approach. Substrates are shown in black and bold. Abbreviations: GA3P: Glyceraldehyde-3-phosphate, DXS: 1-deoxy-D-xylulose 5-phosphate synthase, DOXP: 1-deoxy-D-xylulose 5-phosphate, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase, MEP: 2-C-methyl-D-erythritol 4-phosphate, ispD: 4-diphosphocytidyl-2Cmethyl-D-erythritol synthase, A: 4-diphosphocytidyl-2C-methyl-D-erythritol, ispE: 4diphosphocytidyl-2C-methyl-D-erythritol kinase, B:4-diphosphocytidyl-2c-metgyl-D-erythritol 2-phosphate, ispF:2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, C:2C-methyl-Derythritol 2,4-cyclodiphosphate,IPP: isopentenyl diphosphate, Ipi: isopentenyl phosphate isomerase, DMADP: dimethylallyl diphosphate, Ggps: Geranylgeranyl diphosphate synthase ,GGPP: Geranylgeranyl diphosphate , Psy: phytoene synthase, Pds: phytoene desaturase, 84 Results Zds: ξ-carotene desaturase, Lcy-e: lycopene-ε-cyclase, Lcy-b: lycopene-β-cyclase, Crtr-b: βcycle hydroxylase, Crtr-e: ε-cycle hydroxylase, Zep1: zeaxanthin epoxidase 1, Ccs :capsanthin-capsorubin synthase, Vde: violaxanthin deepoxidase, Nxs: neoxanthin synthase, VNCED(VP14):9-cis-epoxycarotenoid dioxygenase, AO: aldehyde oxidase, ABA: abscisic acid. 3.2.5 PepNovo and MS- BLAST search It is well known that in mass spectrometry a large amount of data is generated and only a small portion of this data is used to identify peptides and proteins. However, no database search engine (Sequest, Mascot, Peptide mass mapping) has proved to be 100% reliable in identifying proteins, especially from unsequenced organisms. There are several reasons for this: 1. There are deficiencies in the scoring application in the database search tools. 2. Not all proteins from different organisms are present in the database or with with an alternative splicing not annotated in the database. 3. There are sequence variations of proteins between different organisms. 4. Various modifications, such as post-translational modification or chemical modification, occurred. 5. Presence of contaminants and coeluting peptides. 6. Bad quality spectra with noise or unusual fragmentation. 7. Incorrect or imprecise precursor mass. 8. Missed or exotic cleavage sites. 9. transpeptidation 10. Sequencing errors propagated in the database (especially when obtained by automatic translation of genomic data). 11. Any other nonforeseeable event where a peptide spectrum does not exactly correspond to any candidate peptide from the database. In order to assess how many peptides were not detected in the standard database search, I devised an alternative data analysis strategy to identify peptides in a database-independent fashion. This strategy comprises a database-independent MS/MS spectrum quality scoring to identify peptidederived spectra that were not identified in the standard database search 85 Results (Nesvizhskii et al., 2006). Such high quality spectra were subsequently analyzed using de novo sequencing (Frank and Pevzner, 2005). The de novo sequencing results are then filtered on the basis of a reliability threshold before they are submitted to MS-BLAST searches to identify peptides on the basis of homology (Shevchenko et al. 2001) [Figure 3.20). From the 83 mass spectrometry runs of C. annuum chromoplasts, 302430 total spectra were produced. In the Sequest database search, 4793 spectra were already identified with a confidence of higher than 0.9 (Keller et al., 2002). With the help of QUALSCORE, I identified 8666 high-quality peptide spectra that had previously not been identified in the database search (SEQUEST). - unassigned spectra + II II 4793 spectra identified Score I MS/MS data analysis 83 Database (vp) Quality /scoring QUALSCORE High quality Spectra (8666) III Hypothetical Protein (Q9C9I7) De novo sequencing PepNovo Sequence tags K A N L L G E S Y A G E A P S Homology searches MS-BLAST Protein identification Figure 3.20: (I) The outline shows the protein identification with the help of different bioinformatic tools. (II) Distribution of spectra in different categories. 1: all spectra generated by LC-ESI-MS/MS; 2: high quality spectra already identified with Sequest database; 3: bad quality spectra not identified; 4: high quality spectra but not identified in previous database searches. These spectra were used for further analysis. (III) One example The results provided 187 unique peptides from 94 different proteins. To obtain even greater precision, we erased all those proteins (61) which were identified in previous SEQUEST database searches and this contributed 134 peptides in this approach (Supplementary table 3). Forty-four peptides from 86 Results 26 different proteins were not identified in the first database search and contributed 17% to the total C. annumm chromoplast proteome (Table 3.9). Additionally, seven new hits (nine peptides) were also not identified in the first database search and these hits belong to putative contaminants (Supplementary Table 3, new putative contaminants). Some of newly identified proteins are specific to chromoplasts e.g., chromoplast-specific lycopene beta-cyclase, neoxanthin synthase, chromoplast-specific carotenoid-associated protein and these enzymes are specific to the carotenoid biosynthesis pathways (Figure 3.19). A few proteins are of hypothetical or unknown origin and the majority of them belong to the Arabidopsis thaliana genome (Table 3.9). 87 Results Table 3.9: List of proteins identified from the MS-BLAST. a a Only those MS-BLAST output were considered which have MS-BLAST scor >62 for multiple hits, >65 for single hits and the proteins did not identify in pervious SEQUEST database search. Provided is the identifier (‘’Identifer’’, the Swiss Prot accession number), the organism (‘’Organism’’), protein name (‘’Protein’’), protein localization [‘’Localization’’ P: transit peptide by ChloroP (Emanuelsson et al., 1999), co: putative contaminant,-: any other location], the number of identified tryptic peptides (‘’no. of peptides’’), significant score (‘’MS-BLAST score’’), PepNovo sequence output (‘’Query’’) and MS-BLAST result (‘’Subject’’). Identifier O22774 Q9LKR1 organism Arabidopsis thaliana Pisum sativum Protein name Localization No of peptides MS BLAST score putative chloroplast outer envelope 86like - 4 105 SHVVQQSLGQAVGDLR SHIVQQSIGQAVGDLR 3 69 68 72 104 BXXPLPYMLSSMLQSR BXXXSSDEPLAAQVLYR BXXPLPYMLSSMLQSR BSHVVQQSLGQAVGDLR RSPPLPYLLSWLLQSR RLGHSAEDSIAAQVLYR RSPPLPFLLSSLLQSR RSHIVQQAIGQAVGDLR BXXPLPYMLSSMLQSR BXXXSSDEPLAAQVLYR BXXPLPYMLSSMLQSR TVLQELLVQQH VDASGYASDMLEYDQPR BXXHEEMESSLVCEDG BNPTPAPTEALSLL RAPPLPYLLSWLLQSR RLGFTTEESIAAQVLYR RVPPLPYLLSSLLQSR TVLQELIVQQH VDASGFASDFIEYDKPR KVEHEEFESSIVCDDG KNPTPAPTEALTLL chloroplast protein import component Toc159 - Query Subject Q6RJN8 Q8LB69 Q9M424 Physcomitrella patens Arabidopsis thaliana Solanum tuberosum chloroplast Toc125 thylakoid formation 1 neoxanthin synthase P P 1 1 2 Q96398 Cucumis sativus chromoplast-specific carotenoidassociated protein P 2 69 68 80 77 95 84 86 Q9FV32 Lycopersicon esculentum chromoplast-specific lycopene betacyclase P 3 71 84 BXXXVELLTQLESK BXXHEEMESSLVCEDG RAEIVELITQLESK KVEHEEFESSIVCDDG VDASGYASDMLEYDQPR BXXXWHGFLSSR BXXXELSLTTGNAGGR BNPTPAPTEALSLL AVVEEEPPQE BXXXVELLTQLESK VDASGFASDFIEYDRPR KPHYWHGFLSSR KGGHELSKTTGNAGGR KNPNPAPTEALTLL AVVEEEPPKE RAEIVELITQLESK ADSFYGTNR ADSFYGTNR Q9SUJ7 O99019 Arabidopsis thaliana Solanum demissum copper/zinc superoxide dismutase light-induced protein P P 1 3 96 67 83 77 65 71 O24024 Lycopersicon esculentum plastid-lipid-Associated Protein P 5 68 88 Results 71 62 86 65 BXXXVELLTQLESK DSFYGTNR BNPTPAPTEALSLL BXXXVELLTQLESK RAEIVELITQLESK DSFYGTNR KNPTPAPTEALTLL RAEIVELITQLEAK VDWPGLGYAMRP MSAAALATPALARP QDPAEAVSAGD VDWPGLGYAMRPK BXXQLNSALDDLSTQL BSPAEGAYSEGLLNA BXXPGVTATDVNVDEVR BYLGDSVEDQTEQLVK VDWPGLGYSDRP MAAAALAAAALARP QDPAQAVSAGD VDWPGLGYSARPK KIAQLNSAIDDVSSQL RSPAEGAYSEGLLNA KAQPGLTATNVNVDEVR KFVSESVEDQTEQVLK Q6ES31 Q6ZFQ4 Q6H694 Q9CA53 Oryza sativa Oryza sativa Oryza sativa Arabidopsis thaliana hypothetical protein hypothetical protein hypothetical protein P0708h12.37 hypothetical protein P P P P 1 1 1 2 Q9C9I7 Arabidopsis thaliana hypothetical protein P 2 Q94JQ4 Arabidopsis thaliana P 1 Q8RX74 Q9LPI8 Q9C7S3 Q8W480 Q8L7R2 Arabidopsis thaliana Arabidopsis thalaniana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana P P P P 1 1 1 1 1 110 64 65 70 98 PWEGQGQQGQQGFR PTTEVLDSWEL LNSALDDLSTQL BXXQLNSALDDLSTQLR ANLGPNLDMLGCAVDGLGD PWEGQGQQGQQGFR PTTEVINFWEL LNSAIDDVSSQL KIGQLNSAIDNVSSRLR ANLGPGFDFLGCAVDGLGD Q9FLK4 Arabidopsis thaliana 1 69 QEVSLHVVAVAQEVHVPTK QDISIELVEVAKEVHLSTK Q8RU74 Q94F93 Q6K439 Lycopersicon esculentum Brassica napus Oryza sativa P P P 1 1 1 87 87 86 BAPSQAPTVVEVDLPT BYPAMPTVMDLNQLR BNPTPAPTEALSLL KASSQAPTVVEVDLGT RYEAFPTVMDINQIR RNPTPAPTEALTLL Q94CN9 Oryza sativa AT3g20390/MQC12_15 (Translational inhibitor protein, putative) AT4g28520/F20O9_210 F6N18.14/At1g32760 hypothetical protein F13A11.2 hypothetical protein 3-hydroxy-3-methylglutaryl-coenzyme A reductase dimethylaniline monooxygenase (Noxide-forming)-like protein dehydroquinate synthase putative 3-keto-acyl-ACP dehydratase putative chloroplast drought-induced stress protein,34 kD putative lipoamide dehydrogenase. 68 70 70 81 78 96 94 74 P 1 70 BXXLLEGDVVGGT KTAIIEGDVVGGT 89 Results 3.3 Comparisons of the different plastid types In the present plastid proteome study, two unsequenced plant species were studied. For both plastid types, shotgun proteomics was the preferred approach, but it has some limitation towards the detection of abundant proteins and provides some limited quantitative information (Baginsky et al, 2005). A comparative analysis of shotgun proteomics data from different plastid types is therefore suitable to reveal prevalent metabolic activities and plastid type specific functions. In order to identify proteins, which are common for different plastid types, I BLAST-searched different plastid proteomes against each other. In a first round, the BY-2 plastid proteome (173) was compared to 149 proteins identified from the C. annuum chromoplast. This comparison revealed plastid specific differences between the two proteomes (Figure 3.21). The BLAST search showed 83 homologous proteins in both plastid types. In addition, the protein profiles suggested the predominant functions of these two plastid types. The majority of proteins identified in BY-2 plastids are active in amino acid metabolism, whereas most of the identified proteins from chromoplasts are active in carbohydrate metabolism. Heat shock and chaperones made a big contribution in BY-2 plastids as compared to chromoplasts, and this might be attributed to the characteristics of the cell culture conditions. There are few functional categories that were not covered in the BY-2 plastid proteome studies and are specific to chromoplast, such as lipid metabolism, ATP synthesis, carotenoid biosynthesis and photosynthesis and carbon fixation. These results suggest that different plastid types have specific functions related to their differentiation and development process. Results 40 BY-2 plastid 35 C. a chromoplast 173 173 83 149 25 20 15 10 bo h Ca r in o ac id 0 yd ra te St ar ch Nu cl eo tid Te e tra py rr ol e O th er G en Re e do ex x pr es si on Pr ot He ea at se Tr sh s an oc sp k & or Ch te rs ap er on es St ru ct H ur yp al ot he tic al AT Li pi P d sy nt he si Ca s ro t e P/ no S id & Ca rb on 5 Am No of proteins 30 Figure 3.21: Proteome comparison between BY-2 plastid and C. annuum chromoplast. 83 proteins are present in both plastid types. Proteins were manually assigned to their putative function. To minimize technical differences, contaminants were not considered in this BLAST set. In the second BLAST search set, I compared the BY-2 plastid proteome (173) to 1337 experimentally identified Arabidopsis chloroplast proteins from the complete chloroplast (Kleffmann et al., 2004), the chloroplast envelope membrane (Ferro et al., 2003; Froehlich et al., 2003), the thylakoid membrane and lumen (Peltier et al., 2002; Schubert et al., 2002) and 240 proteins from Rice etioplasts (von Zychlinski et al., 2005). Based on BLAST searches, 118 BY-2 plastid proteins were homologues to complete chloroplast, 101 BY-2 plastid homologues were detected in envelope membrane studies and only 13 were detected in thylakoid lumen proteome analyses. Eighty-four proteins were common to rice etioplasts and BY-2 plastids, both are heterotrophic plastid types (Figure 3.22). Forty-eight BY-2 plastid proteins have no homoloues in either Arabidopsis chloroplasts or rice 91 Results etioplasts and therefore are candidates to indicate the distinct functions of BY2 plastids (Supplementary Table 2). A number of proteins characteristic for BY-2 plastids were identified as well. Protein products from two large plastid encoded open reading frames ycf1 and ycf2 were identified only in BY-2 plastid proteome analsysis. This shows that BY-2 plastids are undifferentiated plastid types and that these two open reading frames perform an important function in early plastid development and differentiation. Their exact function is currently unknown, but the data suggest that they are indispensable for plastid development (Drescher et al., 2000). Arabidopsis chloroplasts Rice etioplast BY-2 Plastid Complete CP [240] Kleffmann et al., 2004 [173] [636] {118} (48) {84} Envelope Ferro et al., 2003 Froehlich et al.,2003 [403] {101} Lumen Peltier et al., 2002 Schubert et al., 2002 {13} [298] Figure 3.22: A comparison of proteins identified in BY-2 plastids to other plastid types (Arabidopsis chloroplast and rice etioplasts) on the basis of BLAST searches. An e-value of e20 was used as a cutoff. The number in the intersection represented those proteins that were found in different plastid types. In the third BLAST search set, 149 proteins of C. annuum chromoplasts were compared to 1337 experimentally identified Arabidopsis chloroplast proteins from previously published chloroplast proteome studies (Peltier et al., 2002; Schubert et al., 2002; Ferro et al., 2003; Froehlich et al., 2003; Kleffmann et al., 2004) and 240 proteins from rice etioplasts (von Zychlinski et al., 2005) [Figure 3.23]. On the basis of BLAST analysis, 109 C. annuum chromoplasts proteins were homologues to complete chloroplast, 95 were homologues to the envelope membrane proteome and 104 C. annuum chromoplasts proteins were found in thylakoid lumen proteome analyses. 92 Results Fortynine proteins were common to both etioplasts and chromoplasts. Only fourteen proteins have no homologue in either Arabidopsis chloroplasts or rice etioplasts, and these 13 (9%) identified proteins appeared to have a higher relative abundance in C. annuum chromoplasts as compared to other plastid types. Most of these 13 proteins were unknown or hypothetical (7) and currently have no predicted functions. Therefore, these proteins might reveal chromoplast specific functions in a plant cell. The remaining proteins, like UDP-glucose pyrophosphorylase, is involved in carbohydrate metabolism and beta-carotene hydroxylase is involved in the carotenoid biosynthesis pathway. Putative glucosyltransferase, maturase (matK), senescence-inducible chloroplast stay-green protein 1 and ARA; GTP binding are involved in other functional aspects. Arabidopsis chloroplast Rice etioplast C. annuum chromoplast Complete CP [240] Kleffmann et al., 2004 [149] [636] {109} (13) {49} envelope Ferro et al., 2003 Froehlich et al.,2003 [403] {95} Lumen Peltier et al., 2002 Schubert et al., 2002 {104} [298] Figure 3.23: Proteome comparison between chromoplast and two other plastid types (Arabidopsis chloroplast and Rice etioplasts) on the basis of BLAST searches. An e-value of e-20 was used as cutoff. The number in the intersection represents those proteins that were found in different plastid types.13 proteins are unique to the C. annuum chromoplast proteome. In the last BLAST search set, four different plastid types were compared with each other. In this data set, 1337 Arabidopsis chloroplast proteins from previously published chloroplast proteome studies (Peltier et al., 2002; Schubert et al., 2002; Ferro et al., 2003; Froehlich et al., 2003; Kleffmann et al., 2004), 240 proteins from rice etioplasts (von Zychlinski et al., 93 Results 2005), 173 proteins from BY-2 plastid (Own research) and 149 proteins from C. annuum chromoplast (own research) were compared [Figure 3.24]. Rice etioplast BY-2 plastid [240] [173] (78) [1337] Arabidopsis chloroplast [149] C. annuum chromoplast Figure 3.24: Identification of proteins that are prevalent in different plastid types. A comparison of proteins identified in different plastid types (BY-2 plastids, Rice etioplasts, Arabidopsis chloroplast and C. annuum chromoplast) on the basis of BLAST searches. An evalue of e-20 was used as cutoff. The number in the intersection represents those proteins that were found in different plastid types. In this BLAST-analysis, 78 proteins have homologues in all plastid types.The majority of proteins detected in all plastid types suggests that they represent a housekeeping “core proteome” that is common to all plastid types. The majority of all proteins in this “core proteome” have a function in carbohydrate and amino acid metabolism. These two essential functions of plastids are important for the plant cell (reviewed in Tetlow et al., 2004). The comparison of protein profiles from different plastid types is useful to assess prevalent enzymatic activities and plastid specific metabolic functions. All proteomics data from the different plastid types are available in the plprot database, which is accessible at http://www.plprot.ethz.ch/. 94 Discussion 4. Discussion Today proteomics analyses are widely used in science for the identification of hitherto unknown proteins and first proteomics analyses were done 30 years ago. The field is growing very fast due to the availability of publicly accessible genome and protein databases. The development of database search engines, introduction of high sensitivity and easy to use mass spectrometry technique and advancement in other proteomics related techniques like 2DPAGE, labeling techniques (ICAT and iTRAQ) paved the way for modern proteomics research (reviewed in Han and Lee, 2006). Using these advancements in proteomics is of importance to analyse organelle proteomes and better understand plastid development and differentiation. Plastids are classified as either autotrophic chloroplasts or nongreen (heterotrophic) plastids (e.g. proplastids, amyloplasts, leucoplasts, or chromoplasts) according to morphological and biochemical differences. They perform many specialized functions such as photosynthesis, nitrogen assimilation, the synthesis of amino acids and fatty acids as well as storage of carbohydrates and lipids. In plastid proteomics, the chloroplast proteome was extensively investigated in recent years (reviewed in Baginsky and Gruissem, 2004; 2006). Chloroplast proteome analyses have reached in the meantime their saturation level and further research is unlikely to increase knowledge about the proteome of this plastid type. Due to this fact, other plastid types like BY-2 plastids and C. annuum chromoplasts were used in my research described here. There are several reports suggesting that BY-2 plastids have functional and morphological properties similar to those of undifferentiated proplastids from meristematic cells (Sakai, 2001). C. annuum chromoplasts are responsible for pigment synthesis and storage and this plastid has an advantage in terms of isolation compared to other chromoplasts (reviewed in Camara et al., 1995). A detailed analysis of their proteomes and functional categories of their proteins is the topic of my PhD thesis. 95 Discussion 4.1 Characterization of plastids 4.1.1 BY-2 plastid BY-2 plastids were isolated by sucrose density-gradient centrifugation and different analyses were done to check their characteristics and intactness. Electron microscopy analysis showed that band B (interphase between 70%50% sucrose density-gradient) contained proplastid like organelles with a size of ~ 1µm and lacking any prolamella body (Figure 3.2). The BY-2 plastid does not accumulate protochlorophyllide A, which is last intermediate of the tetrapyrrole pathway in the dark, even after ALA feeding (Figure 3.3). This is in contrast to Rice etioplasts that accumulate protochlorophyllide A and have a prolamellar body (von Zchlinski et al., 2005). These observations suggested that BY-2 plastids are undifferentiated and they resemble proplastids. Much research conducted on BY-2 plastid showed their resemblance to undifferentiated proplastids. As an example, the transcription system of BY-2 plastids shows that plastid encoded genes are transcribed from NEP consensus promoter elements (Fan and Sugiura, 1995; Sakai et al., 1992). The NEP RNA polymerases is the predominant transcription enzyme in proplastids and is responsible for the activation of the plastid encoded transcription machinery (Hajdukiewicz et al., 1997; Liere and Maliga, 1999). The BY-2 plastid proteome analysis supports this hypothesis as all genes for plastid encoded proteins identified in the shotgun-approach have a NEP promoter element (Table 3.3). Several peptides from the two proteins ycf1 and ycf2 (gi|231660 and gi|3915961) were identified (Table 3.3) and they are indispensable for the plastid development (Drescher et al., 2000). Although the molecular function is unknown, it is conceivable that these two hypothetical proteins play an important role in the plastid differentiation from proplastids (Drescher et al., 2000). 4.1.2 C. annuum chromoplast Several studies were conducted on chromoplasts from different plant species and these studies were mostly on specific proteins or metabolic pathways e.g., the carotenoid biosynthesis pathway. So far no chromoplast proteome study has been conducted, with one exception where the protein profiling of 96 Discussion plastoglobules in chromoplasts were reported (Ytterberg et al., 2006). For the chromoplast proteome analysis, Capsicum annuum L. was selected due to their availability and high stablility during Percoll density gradient centrifugation (Hadjeb et al., 1988; reviewed in Camara et al., 1995). The fluorescence microscopy observation shows that Band D (interphase between 30%-40% of the Percoll density gradient) contained most chromoplast-like organelles (Figure 3.15, III). In chromoplasts, there is an increased accumulation of carotenoids as compared to the other plastid types (reviewed in Camara et al., 1995). In plants, carotenoids are synthesized within the plastids and this pathway has been extensively studied because of dramatic color changes that occur during chromoplast development. In the C. annuum chromoplast proteome analysis nine enzymes from this pathway were identified, which provides 40% coverage of this pathway. Seven enzymes were identified by the shotgunapproach and two were detected by MS-BLAST (Figure 3.19; Table 3.8 and Table 3.9). All the identified enzymes have well known functions in the carotenoid biosynthesis pathway (Hirschberg, 2001). A detailed explanation of these enzymes and their precise functions are mentioned in the following section. 4.2 Proteome coverage: encouraging results different approaches and 4.2.1 BY-2 plastid In the BY-2 plastid proteome study, two main approaches were applied for complete proteome analysis (Shotgun and 2D-PAGE) and functional proteome analysis (BN-PAGE and NP-40 insoluble fraction). In total, 428 proteins were detected from BY-2 plastids, 197 of which are unique (Table 3.7). The aim of the shotgun proteome study as reported here is the identification of all metabolic and regulatory pathways that are active in an organelle. Since shotgun proteomics is biased toward abundant proteins and thus provides some limited quantitative information, it is assumed that the identified proteins belong to the most abundant proteins of the organelle (Greenbaum et al., 2003; Baginsky et al., 2005). By inference, the detection of 97 Discussion many proteins of a metabolic pathway is indicative of a most prevalent pathway activity (Figure 3.12). Expanding this conclusion to pathway cross talk, the identification of the prevalent enzymes present in BY-2 plastids reflects the heterotrophic growth of the cell culture and its elevated amino acid biosynthetic activity. In the BY-2 plastids glutamine synthetase, glutamate synthase and glutamate dehydrogenase are abundant enzymes. These enzymes are active in the primary nitrogen assimilation (ammonia fixation) (Last and Coruzzi, 2000). The identification of many peptides from the oxoglutarate/malate transporter suggests that BY-2 plastids import oxoglutarate, the precursor of ammonia fixation at a high rate. It is conceivable that a high plastidic concentration of oxoglutarate is required when primary nitrogen assimilation is highly active (Neuhaus and Emes, 2000; Neuhaus and Wagner, 2000; Weber and Flügge, 2002). Furthermore, the identified ATP/ADP translocator and the glucose 6-phosphate/phosphate translocator (GPT) are responsible for the import of metabolites that are generally required for the energy metabolism of heterotrophic plastids. This energy is necessary for the biosynthesis of several compounds, among them amino acids and starch (Neuhaus and Emes, 2000; Weber and Flügge, 2002, reviewed in Tetlow et al., 2004). The shotgun-approach gives the number of proteins from the respective organelle. For a quantative analysis, 2D-PAGE is most appropriate and widely used in the proteome studies (Görg et al., 20004). In the 2D-PAGE analysis a total number of 72 proteins were identified, 16 out of them are new plastid hits, which were not detected in the shotgun-approach (Table 3.7). Amino acid metabolism and carbohydrate metabolism are also the major functional categories in 2D-PAGE technique (Figure 3.11). A detailed explanation of a few new protein identifications that are specific to 2D-PAGE is follows: aspartate semialdehyde dehydrogenase (ASDH) [F10B6.22] belongs to amino acid biosynthesis (threonine / methionine biosynthesis). In plants and microorganisms, ASDH produces the branch point intermediate between lysine and threonine/methionine pathways and it is a 36 kDa homodimeric enzyme (Paris et al., 2002). The ATP phosphoribosyltransferase regulatory subunit is also involved in amino acid metabolism (L-histidine 98 Discussion biosynthesis). It is a 220 kDa heteromultimer and is composed of hisG and hisZ subunits. This enzyme is required for the first step of histidine biosynthesis and it may allow for the feedback regulation of ATP phosphoribosyltransferase activity by histidine (Vega et al., 2005). In carbohydrate metabolism, one interesting hit was identified; 6phosphogluconate dehydrogenase is a key enzyme of the oxidative pentose phosphate pathway (OPPP) that is a homodimer with subunits of 50 kDa. The relative staining intensity of 0.187 suggests that it is a rather abundant enzyme. The oxidative pentose phosphate pathway provides NADPH for reductive biosyntheses and for protection against oxidative stress. It also provides pentoses for synthesis of nucleotides and sugar phosphates for the shikimate pathway (Schnarrenberger et al., 1995). The first two reactions of the pathway, glucose-6-phosphate dehydrogenase (G6PDH) and 6phosphogluconate dehydrogenase (6PGDH) occur in the chloroplast and in the cytosol of green leaves of higher plants and in non-photosynthetic tissues such as etiolated radish cotyledons and castor bean endosperm (Nishimura and Beevers, 1981; Schnarrenberger et al., 1995). A specific translocator that transports cytosolic ribulose-5-phosphate into chloroplasts has been identified (Schnarrenberger et al., 1995). BN-PAGE was performed for native protein complexes. BN-PAGE is the most versatile and successful approach to separate soluble and membrane protein complexes with different physicochemical properties. In BN-PAGE, pH 7.5 is in a physiological range in intracellular compartments and allows the migration of protein assemblies, which are susceptible to acidic and/or basic conditions that lead to dissociation of subunits and/or loss of enzymatical activities (Schägger et al., 1994). In this approach, 50 proteins were identified; seven are new plastid hits (Table 3.7). Two new hits like ATP phosphoribosyltransferase and 6-phosphogluconate dehydrogenase were also identified in the 2D-PAGE analysis (discussed on the previous section). In order to verify the results, I checked the native complex associations for some proteins through literature search (section 3.1.4.1 Blue Native BNPAGE). The chaperonin family of molecular chaperones is an essential class of proteins that is required for the folding of proteins within the cells. Rubisco 99 Discussion subunit binding-protein alpha subunit (CPN-60α) and Rubisco subunit bindingprotein beta subunit (CPN-60β) of the CPN60 complex family are the most abundant proteins in the BN-PAGE (Table 3.5). They have homology to the E. coli GroEL and share the ability to interact with many target polypeptides to influence their folding into functional proteins (Martin et al., 1991). In the Arabidopsis proteome, the CPN60 complex was identified (806 kDa) with it’s α and β subunits in a 1:1 ratio (Peltier et al., 2005). The oligomeric state of this complex is a homo or heterooligomeric tetradecamer (Baneyx et al., 1995; Peltier et al., 2005). The CPN60 complex was also found at 500-700 kDa, which reflects the above observation about its molecular size (~800 kDa) and is in agreement with the data available in the literature. With this approach CPN60 complex was also found at 400-500 kDa but the number of peptides was not as high as in the upper band (Figure 3.13; table 3.5). This might be due to overlapping of peptides during MS-analysis. It can be concluded that the BN-PAGE can resolve protein complexes in their native form and detection of CNP60 (MW range ~500-700 kDa) is uniform with data avaibale in the literature. Leucine aminopeptidase (LAP) 1 and Leucine aminopeptidase 2 (LAPN) are new hits which are unique to BN-PAGE. These proteins were found at MW range ~ 400-450 kDa (Table 3.5), they are presumably involved in the processing and regular turnover of intracellular proteins. LAPs belong to the peptidase M17 family and are ubiquitously found in animals, plants, and prokaryotic cells. These hexameric metallopeptidases catalyze the release of the N-terminal residues from proteins and peptides. In most plants, three classes of LAP-related polypeptides are detected including the 66- and 77-kD LAP-like proteins and the 55-kD neutral LAP (Chao et al., 2000). The Nterminus of tomato LAP-N contains several features similar to plastid transit peptides including a high percentage of Ser, Thr and positively charged residues and absence of acidic residues (reviewed in de Boer and Weisbeek, 1991). On the other hand, the Arabidopsis LAP1 has no N-terminal targeting sequence, suggesting a cytosolic location. In contrast, all other plant LAPs have putative transit peptides suggesting localization within the plastid stroma (Gu et al., 1996a). In plants, animals and prokaryotes, the biological role is not completey understood, it may be complex and species specific. The role of 100 Discussion plant LAP-N and LAP-A may be different, shown by the differences in their distribution in the plant kingdom and their responses to stress (Chao et al., 2003). Although the role of LAP-N is not yet understood, it is likely that LAP-N has a role in the protein turnover required for cell maintenance in vegetative and reproductive organs. LAP-N may act on general proteins or peptides or may facilitate the turnover of specific polypeptides (Callis and Vierstra, 2000). The identification of these proteins is also supported by the identification of many chaperones and heat-shock related proteins, which have a function in protein folding. Besides these proteins, CLP-protease subunits were identified, which are involved in high protein turnover (Adam, 2000). I found these enzymes at ~400-450 kDa that is not consistent with the literature. The possible explanation for this is some variance in the measured native molecular weight. Plastid DNA is organized into a particular structure called nucleoid, which consists of various DNA-binding proteins, plastid DNA and uncharacterized RNA (Sakai, 2001). In the past, various groups developed methods to isolate the BY-2 plastid nucleoids without any contaminations like mitochondria and cell nuclei. They compared the molecular architecture of the isolated nucleoids to chloroplast nucleoids (Nemoto et al., 1988; Nemoto et al., 1990; Sakai, 2001; Sakai et al., 2004). The DNA-binding proteins in BY-2 plastids (undifferentiated proplastid) were replaced with a different set of DNAbinding proteins during the differentiation of plastids from proplastids to chloroplasts. During this differentiation process, plastid DNA did not change at all (Nemoto et al., 1990). There are some specific proteins which are related to plastid nucleoids of plants, like sulfite reductase, DNA gyrase, plastid envelope DNA-binding protein (PEND) and CND41 (DNA-binding protease) [Sato et al., 2001]. In order to analyse the protein constituents of BY-plastid nucleoids, I analyzed NP-40 insoluble protein fractions. In this approach 101 proteins were identified, 25 are new plastid hits and unique to this approach. Like sulfite reductase (SiR) and DNA gyrase were found. SiR’s primary role is in the sulfur assimilation for cysteine and methionine synthesis. SiR is found to be one of the major DNA-binding proteins. It is able to compact DNA as well as isolated nucleoids in vitro (Sato et al., 2001). SIR reversibly regulates 101 Discussion the global transcription activity of plastid nucleoids through changes in DNA compaction (Sekine et al., 2002). DNA gyrase plastid function is still unknown but it might be critical for nucleoid division (Itoh et al., 1997). Most of the new hits (20) are ribosomal proteins and nine ribosomal proteins have a plastidic origin. There are two hypotheses about the identification of ribosomes; the first one is that intact ribosomes co-sediment with nucleoids due to their large mass rather than via association with nucleoids (Phinney and Thelen, 2004). The second one is that ribosomes may be associated with nucleoids and this is based upon the identification of a 28 kDa ribosomal-like protein as part of nucleoid preparation from pea chloroplasts (Oleskina et al., 1999). This approach did not give satisfactory results in terms of transcriptionally active proteins and it could be that higher abundance proteins such as ribosomal subunits masked transcriptionally active proteins (Wagner and Pfannschmidt, 2006). Based on these assumptions, some ribosomes are nucleoids or plastid related and others are not true constituents of nucleoids. 4.2.2 C. annuum chromoplast In the past a significant amount of research has been devoted to chromoplast development (reviewed in Camara et al., 1995), but so far no proteome analysis of chromoplasts was reported. There was one publication on protein profiling of plastoglobules (PG) in chloroplasts and chromoplasts, with the conclusion that PGs in chromoplast are the active site for carotenoid conversions (Ytterberg et al., 2006). In contrast to chloroplasts, most studies with chromoplasts were conducted on the carotenoid biosynthesis in ripening tomato fruit (Hirschberg, 2001). Thus, tomato fruits have become a model system for chromoplast studies. The isolation of intact chromoplasts from C. annuum with Percoll density gradient centrifugation seems to be a more stable model system as compared to tomato fruit (Hadjeb et al., 1988). Therefore, C. annuum was my choice for chromoplast proteome analysis. In C. annuumm chromoplast proteome analysis, two approaches were conducted to increase the proteome coverage. One approach was shotgunproteomics and the other was MS-BLAST. In the shotgun-approach most of the identified proteins belong to a “core proteome”, with proteins that are found in all plastid types although there are some proteins which are specific 102 Discussion to chromoplasts like those of the carotenoid biosynthesis pathway. I found nine enzymes of the carotenoid biosynthesis pathway which gives 40% coverage of this pathway (Figure 3.19). Chloroplasts also contain β-carotene and various xanthophylls from carotenoid pathway but in chromoplasts, carotenoid accumulation is more abundant. Capsanthin/capsorubin synthase (CSS) was found to be the most abundant enzyme with detected 293 peptides (Table 3.7). It is a bifunctional enzyme of the last step of the carotenoid biosynthetic pathway in C. annuum fruits. It converts antheraxanthin into capsanthin and violaxanthin into capsorubin (reviewed in Camara et al., 1995; Hirschberg, 2001). A chromoplast-specific lycopene beta-cyclase (Q9FV32) and neoxanthin synthase (Q9M424) were detected in the MS-BLAST approach (Figure 3.19). Lycopene β-cyclase (LCY-B/CRTL-B) catalyzes a two-step reaction, which creates one β-ionone ring at each end of the lycopene molecule to produce β-carotene, which is a precursor for the synthesis of capsanthin and capsorubin. Whereas lycopene-ε-cyclase (LCYE/CRTL-E) creates one ε-ring to give δ-carotene. It is therefore likely that lycopene β-cyclase is present in the chromoplast extracts. I did not detect lycopene ε-cyclase that is predominant in chloroplasts but not in chromoplasts (Galpaz et al., 2006). Neoxanthin synthase (NXS) catalyzes the conversion of the di-epoxidated precursor violaxanthin into a xanthophyll, named neoxanthin, which represents the classical final step in the plant xanthophyll formation (Hirschberg, 2001). In vivo, the xanthophyll is a precursor for the formation of the plant hormone abscisic acid (ABA) [Al-babili et al. 2000]. NXS was first cloned in potato (Al-Babili et al. 2000) and tomato (Bouvier et al., 2000). In tomato, NXS is 99% identical to lycopene β-cyclase and it is supposed that this enzymes is a bi-functional enzyme; converting lycopene to β-carotene and violaxanthin to xanthophylls (Bouvier et al., 2000). In this case, another NXS must exist in tomato and this is currently the subject of further work. It was also postulated that NXS, CCS and lycopene cyclase (LCY) have significant similarity and they have a common origin because CCS and NXS are derived from LCY (Al-babili et al. 2000; Bouvier et al., 2000). Another abundant protein in our study is plastoglobules associated protein (giІ1279231), which is related to the plant fibrillins family (Deruere et al., 1994). Plastoglobules are common in plastids and are localized within the 103 Discussion plastid stroma. When chromoplasts are developing from chloroplasts, plastoglobules are the compartment where carotenoids accumulate or they are subsidiary sites where crystals or fibrils are developed (reviewed in Camara et al., 1995). Fibrillin (Plastoglobules associated protein) is a nuclearencoded protein with a transit peptide for chromoplast targeting (Deruere et al., 1994). Fibrillin is involved in the packaging and organization of excess carotenoids. Its role is largely structural and it is responsible for the linear arrangement of lipochromes into fibrils (Deruere et al., 1994). It was also suggested that there is some coordination between fibrillin and carotenoid biosynthetic genes during chromoplast differentiation but the mechanism by which these events are regulated is unknown (reviewed in Camara et al., 1995). 1-Deoxy-D-xylulose-5-phosphate synthase (CapTKT2) was also detected in this shot-gun proteome analysis. CapTKT2 is involved in the synthesis of isoprenoids in plastids via the non-mevalonate pathway. CapTKT2 converts pyruvate and glyceraldehyde-3-phosphate into 1-deoxyxylulose-5-phosphate, which is the precursor of isopentenyl diphosphate (IPP) [Duvold et al., 1997; Bouvier et al., 1998]. Amino acid metabolism and carbohydrate metabolism are major functional categories in the C. annuum chromoplast proteome (Table 3.8). It is postulated that nitrogen assimilation is partially inactive or subject to strict control during chromoplast development (reviewed in Camara et al., 1995). Glutamine synthetase (GS) which is a key enzyme for the ammonia fixation could be the main target for regulation during plastid differentiation. In tomato, this enzyme has two isoforms GSI corresponding to cytosolic and GSII corresponding to chloroplastidic forms. GSI has an important role in the nitrogen metabolism of immature cells (Gallardo et al., 1988) while GSII is involved in both primary and photorespiratory ammonia fixation (nitrogen assimilation) [Gallardo et al., 1988]. It may be assumed that GSII activity is declining to undetectable levels (Gallardo et al., 1988; reviewed in Camara et al., 1995). In the C. annuum chromoplast proteome, there was only one protein detected from this pathway namely glutamate-ammonia ligase (GS2) [gi|1084412], which catalyzes glutamine biosynthesis. This might support the hypothesis that during chloroplast-chromoplast differentiation process GSII activity disappears (Gallardo et al., 1988; reviewed in Camara et al., 1995). In 104 Discussion another study with ripe tomato, it was concluded that ferredoxin-dependent glutamate synthase (Fd-GOGAT) is a predominant form in both green and ripe fruits (Gallardo et al., 1993). This enzyme belongs to the glutamate synthase family and it is involved in the assimilation of ammonia. It was also concluded that Fd-GOGAT is also responsible for reassimilation of nitrogen for glutamate synthesis and other amino acids during fruit ripening (Gallardo et al., 1993). However, during chloroplast-chromoplast transition the role of this enzyme is currently unknown but the high abundance in chromoplasts (64 detected peptides, Table 3.8) points its functional importance (Gallardo et al., 1993). The largest group of identified proteins (25 proteins and 580 detected peptides) is involved in carbohydrate metabolism (Figure 3.18, Table 3.8). I have identified transketolase, transaldolase, chloroplast phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase with many peptides signifying their high abundance (Table 3.8). Isolated chromoplasts possess high hexose kinase activity, which could indicate that chromoplast carbohydrate metabolism depends on the import of hexoses from the cytosol (Camara et al., 1995). The data substantiate this hypothesis because I detected fructokinase and a hexose transporter in the chromoplast preparation (Table 3.8). In addition to metabolic pathways, several proteins were detected in the protease categories (Table 3.8). It is postulated that the loss of chlorophyll or photosynthesis-related proteins from leucoplasts and chromoplasts has to be under strict tissue-specific control or under developmental regulation of the protein import capacity during chromoplast differentiation (reviewed in Camara et al., 1995). Clp, an ATP-dependent protease system originally described in E. coli has the potential to degrade components of the thylakoid membranes in a highly controlled manner (Malek et al., 1984). The Clp protease is composed of ClpP, a 20 kDa plastid encoded catalytic subunit (Gottesman et al., 1990) and ClpA or ClpB two closely related 50-kDa subunits that are ATPdependent and encoded in the nucleus (Seol et al., 1994). For this protease activity, there are two hypotheses; the first one postulates that the chloroplast possesses ClpP and low concentration of ClpA, which results in a slow turnover of chloroplast proteins, but as tissue starts become non-green, ClpB 105 Discussion is expressed and the active protease activity degrades the thylakoid membranes of chloroplast. The second hypothesis is that the expression of these protease families ClpA or ClpB are tissue specific therefore, ClpA is expressed only in flowers and fruits and ClpB only in senescing leaves (Price et al., 1993). Ribulose 1, 5-bisphosphate carboxylase large subunit (gi|7240236) and Ribulose bisphosphate carboxylase small chain (gi|59800169) are abundant proteins also in C. annuum chromoplast proteome analysis (Table 3.8). Ribulose 1, 5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the first step in the photosynthetic CO2 assimilation and the photorespiratory pathway (reviewed in Spreitzer, 2003). Rubisco is composed of two types of subunits a 55-kDa large subunit and a 15-kDa small subunit prevalent in most photosynthetic organisms (reviewed in Spreitzer and Salvucci, 2002).These subunits are required to catalyze the reactions of the enzyme, for substrate binding and product formation (reviewed in Spreitzer and Salvucci, 2002). It was shown that the small subunit of ribulose-1, 5bisphosphate carboxylase is present in the chromoplast containing bell pepper leaves but they disappeared in the fruit (Kuntz et al., 1989). On the other hand, the large subunit of ribulose-1, 5-bisphosphate carboxylase was detected during the complete chloroplast to chromoplast differentiation process (Kuntz et al., 1989). This was confirmed with the detection of 169 tryptic of large subunit of ribulose-1, 5-bisphosphate carboxylase in the shotgun proteome approach (Table 3.8). No ribosomal proteins were detected in our proteome study. This might be interpreted as a poor development of thylakoid and no photosynthetic activity in the chromoplasts that diminishes the need for protein translation in the organelle (Carde et al., 1988). On the other hand, the plastid DNA is retained through chromoplast differentiation. Due to biochemical and structural changes, the transcription activity of plastid DNA is decreased (Carde et al., 1988). This also effected the additional production of mRNA of nuclear origin (Carde et al., 1988). Kuntz and colleagues did not find any significant changes in the overall transcriptional activity in chloroplast and chromoplast but found dramatic changes in translational activity that indicate 106 Discussion the prevalence of translational control of plastid genes expressed in the chromoplast differentiation process (Kuntz et al., 1989). The other approach to increase the C. annuum chromoplast proteome coverage was MS-BLAST. The intention of applying this technique was to find the proteins that were missed in the first approach (shotgun-approach) due to database constraints (Figure 3.20). This approach yielded 26 new proteins, which are 17% of the total chromoplast proteome (Table 3.9). 4.3 Plastid comparisons The proteomes of different plastid types were compared to get deeper insights into plastid development and function. A comparison of proteins identified from BY-2 plastids with C. annuum chromoplasts revealed significant differences between these two plastid types (Figure 3.21). This suggests functional adaptations of the plastid proteome during plastid differentiation. The comparison between two plastid types allows to distinguish them and provides insights into plastid metabolic functions. In BY-2 plastids, amino acid metabolism is the major functional catogery and in C. annuum chromoplast carotenoid biosynthesis is a significant constituent of the proteome. The comparison between BY-2 plastids and other plastid types (chloroplast and etioplast) reveals that 101 BY-2 plastid proteins have a homologue in one of the envelope proteome studies and 118 BY-2 plastid proteins have a homologue in the proteome of complete chloroplast (Figure 3.22). These proteins have house keeping functions, e.g., metabolite exchange between the plastid and cytosol, carbohydrate and amino acid metabolism. Thus, these proteins are essential for the viability of plants regardless of the plastid type. The BY-2 plastid has 84 proteins, which have a homologue in etioplasts. Most of the shared proteins belong to the amino acid metabolism, which is the key function in the BY-2 plastids. In contrast, rice etioplast has abundance proteins involving in gene expression, carbohydrate and amino acid metabolism (von Zychlinski et al., 2005). A similar comparison was done between the C. annuum chromoplast, the chloroplast, and the etioplast proteome (Figure 3.23). Only 9% of the 107 Discussion proteins have no homology with protein from other plastid types. Most of the proteins are unknown or hypothetical. They might have a specific function in the chromoplast like beta carotene hydroxylase (gi|19071768). This protein is also not detected in the other two plastid types but has a key role in the carotenoid biosynthesis pathway (Figure 3.19) [Hirschberg, 2001]. In the last comparison, all four plastid proteomes (BY-2 plastid, etioplast, chloroplast and chromoplast) were compared (Figure 3.24) and 78 proteins had a homology to proteins in all plastid types. These proteins perform house keeping functions and are present in all plastid types. Specific functions that are common in all plastid types are carbohydrate metabolism, amino acid metabolism, transporter-functions and chaperones. These comparisons allow for better insight into specific enzymatic activities between different plastid types. However, it is difficult to conclude which proteins is plastid specific e.g., transketolase (TK) is present in all plastid types. TK catalyzes the reversible transfer of two-carbon units from ketose-phosphate to aldose phosphate (reviewed in Schenk et al., 1998). In heterotrophic plastids, it provides a link between glycolysis and the pentose phosphate, which provide precursors for nucleotide, aromatic amino acids and vitamin biosynthesis. In addition to this role, it plays a central role in the Calvin cycle (Neuhaus and Wagner, 2000). Thirteen chromoplast proteins were not identified in other plastid proteome analysis. Among these proteins is the plastid-encoded maturase K (gi|2281235) that is involved in RNA splicing of class II introns. The expressed protein has RNA binding activity and both gel-shift and UV-crosslinking experiments revealed preferential binding to the trnK precursor transcript (Liere and Link, 1995). Senescence-inducible chloroplast stay-green protein 1 (gi|68510418) was also not detected in other plastid proteome studies. This protein is senescence-inducible and potentially involved in chlorophyll catabolism or protein degradation. Its exact role however, is unknown. Seven proteins were unknown or hypothetical protins and their exact role in chromoplast is not clear. 108 Discussion 4.4 Current status of plastid proteomics At the present, the limited number of proteome analyses with different plastid types from the same source makes a cross species comparison of proteomes from different plastid types necessary. Nevertheless, as more proteome studies become available, comparative protein profiling will become a valuable strategy to obtain deeper insights into the plastid development and function. In all plastid comparisons more than 2000 proteins from different plastid types were compared and only 78 proteins have homology in all plastid types (Figure 3.24). In this comparison, the chloroplast proteome made the largest contribution (1337 proteins) due to its extensive proteome data and availability of Arabidopsis genome information (van Wijk, 2004; Baginsky and Gruissem, 2004, 2006). However, many protein identifications are redundant in all plastid types and approximately only 1000 proteins are unique (reviewed in Rossignol et al., 2006). In the current plastid proteome study different plastid types were analysed. For this purpose two non-model organisms BY-2 cell culture and C. annuum were used. There were different constraints to achieve high proteome coverage from these plastid types because genome or complete protein database are unavailable for these non-model organisms. In the last few years, there was massive improvement in the MS-field especially concerning software tools, which help the MS/MS data analysis. We have used a collection of these tools to circumvent the database constraint and by during so, could expand the proteome coverage. 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Proteome analysis of the rice etioplast: metabolic and 128 References regulatory networks and novel protein functions. Mol Cell Proteomics 4: 10721084. 129 Curriculum vitae Curriculum vitae _____________________________________________________________________ Personal Information Name Muhammad Asim Siddique 31st December 1973 Date of Birth Present Address Forsterstrasse-80 CH-8044 Zurich, Switzerland Tel: + 41 44 255 37 29 Fax: + 41 44 252 26 26 Email: [email protected] ___________________________________________________________________________ Education 1999 M.Sc (Hons). (Master of Science, Agronomy) University of Agriculture Faisalabad (UAF), Pakistan. 1997 B.Sc. (Hons). (Bachelor of Science, Agriculture; Major Agronomy) University of Agriculture Faisalabad (UAF), Pakistan. ________________________________________________________________ Post Graduate Training: Research 2001-Present Academic guest and PhD student at the Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Zurich. 1997-1999 Agronomics performance of different genotypes of Mung bean (Vigna radiata L.) M.Sc Thesis 1997 To assess the allelopathic effects of lentil water extract on the germination and Seeding growth of wheat, maize and linseed. B.Sc Semester work ___________________________________________________________________________ Research Articles Baginsky S, Siddique A and Gruissem W. (2004). Proteome analysis of tobacco bright yellow-2 (BY-2) cell culture plastids as a model for undifferentiated heterotrophic plastids. J Proteome Res 3: 1128-1137. Siddique MA, Gruissem W and Baginsky S. (2006). Proteomics of Tobacco Bright Yellow-2 (BY-2) cell culture plastids. Biotechnology in Agriculture and Forestry “Tobacco BY-2 Cells”: From cellular dynamics to Omics. Vol. 58. A new treatise. Springer Verlag, Heidelberg, Germany. Siddique A, Gruissem W and Baginsky. (2006). Proteome analysis of Bell Pepper (Capsicum annuum L.) chromoplast. Plant Cell Physiol 47: 1663-1673 a Acknowledgements Acknowledgements I would like to thank ALLAH Almighty, who gave me health and knowledge to finish this work. I am very grateful to the Holy Prophet Muhammad (Peace be upon him) for his teaching, which are helpful and inspiring for my daily life. There are a lot of people whom I would like to thank, first of all Prof. Wilhelm Gruissem who gave me opportunity to do PhD in his laboratory and to Dr. Sacha Baginsky for his supervision, ideas, criticism and continuous support for this research. Many thanks go to all former and present lab members, especially to Jonas Grossman for his support in bioinformatics and computer related stuff and Dr. Johannes Fütterer for helping to arrange the BY-2 cell culture. I am also grateful to the members at the Functional Genomics Center Zurich (FGCZ) for their technical support. I also would like to thank Dr. Silvia Hofer to review my thesis and to give suggestions concerning the English grammar. Thanks also to the EMDO foundation for their financial help in early years at the ETH and the VELUX foundation to financing my PhD research. In all these years I cannot forget the support and encouragement from all my family members, I especially prayers for my success from my mother Sajida Siddique and my wife Samra Asim. Last but not the least I am very thankful to Prof. Walter Siegenthaler for his highly motivating support and for everything in the success of my life. b Supplementarty Table 1 Supplementarty Table 1 List of proteins and spot intensities from all five 2D-PAGE. a a Only those proteins which have significant MASCOT score (Material and methods, section 2.2.7.2 MASCOT search engine ) were taken into account. Provided is spot identification by the Proteome weaver software (“Spot ID”), the identifier (“Identifer” the SWISS prot accession number), protein name (“Protein”), spot intensities from all five replicates and average gel (“Spot intensities”) and spot frequencies in all gels (Spot frequencies”). Spot ID Identifier Protein Spot Intesnsities Gel I Gel 2 Gel 3 Gel 4 Gel 5 Avg.Gel 141290 O78327 transketolase 1. 0.144 N.A. 0.236 0.225 0.084 0.159 Spot Frq. 4 141200 Q7TV03 ATP phosphoribosyltransferase regulatory subunit 0.697 0.556 0.244 0.533 0.291 0.43 5 141766 Q9LJL3 zinc metalloprotease 0.194 0.115 N.A. N.A. N.A. 0.15 2 141685 Q9SE20 zeta-carotene desaturase 0.476 0.252 N.A. N.A. N.A. 0.346 2 141865 P29515 tubulin beta-7 chain. 0.202 N.A. N.A. N.A. N.A. 0.202 1 141828 Q43594 tubulin beta chain. 0.264 0.296 N.A. N.A. N.A. 0.279 2 141260 Q96460 tubulin alpha-2 chain. 0.236 0.287 0.125 0.164 0.237 0.201 5 141432 141031 141373 P46225 Q851Y8 Q851Y8 triosephosphate isomerase translational elongation factor Tu. translational elongation factor Tu. 0.164 0.099 0.199 N.A. 0.094 0.177 0.162 0.147 0.242 0.204 0.1 N.A. 0.141 0.147 0.282 0.166 0.115 0.222 4 5 4 141062 O78327 transketolase 1. 0.363 0.221 0.177 0.216 0.185 0.224 5 141417 141426 O78327 O78327 transketolase 1. transketolase 1 0.385 0.64 0.356 0.573 0.228 0.43 0.16 0.469 N.A. N.A. 0.266 0.521 4 4 141058 O78327 transketolase 1. 0.12 0.11 0.135 0.102 0.075 0.106 5 141171 O78327 transketolase 1. 0.824 0.76 0.493 0.727 0.352 0.602 5 c Supplementarty Table 1 141321 O78327 transketolase 1. 0.307 0.174 0.187 0.153 N.A. 0.198 4 141312 Q941R1 transaldolase. 0.16 0.217 0.086 0.15 0.105 0.136 5 141075 Q941R1 transaldolase 0.262 0.229 0.199 0.254 0.277 0.242 5 141380 Q7XJh9 transaldolase 0.131 0.208 0.191 0.138 N.A. 0.164 4 141224 Q8RVF8 thioredoxin peroxidase. N.A. 0.518 0.554 0.457 0.424 0.486 4 141233 Q8RVF8 thioredoxin peroxidase. N.A. 0.487 0.315 0.408 0.546 0.43 4 141248 141311 Q8RVF8 Q84LB6 thioredoxin peroxidase. succinyl Coa ligase beta subunit N.A. 0.129 0.286 N.A. 0.166 0.177 0.253 0.117 0.192 0.167 0.219 0.145 4 4 141294 Q02028 stromal 70 kDa heat shock-related protein 0.31 0.332 0.396 N.A. 0.297 0.332 4 141941 P21240 rubisco subunit binding-protein beta subunit N.A. 0.378 N.A. N.A. N.A. 0.378 1 141334 P21240 rubisco subunit binding-protein beta subunit 1.042 0.633 0.356 0.48 0.338 0.52 5 141188 P21239 rubisco subunit binding-protein alpha subunit 0.686 0.525 0.365 0.424 0.402 0.468 5 141057 P21239 rubisco subunit binding-protein alpha subunit 0.241 0.215 0.131 0.204 0.151 0.184 5 141158 P21239 rubisco subunit binding-protein alpha subunit 0.27 0.251 0.206 0.187 0.259 0.232 5 141164 Q9C6Z3 pyruvate dehydrogenase E1 beta subunit, putative 0.11 0.09 0.112 0.131 0.156 0.118 5 141147 Q9C6Z3 pyruvate dehydrogenase E1 beta subunit, putative 0.185 0.254 0.255 0.275 0.238 0.239 5 141806 Q8Sa22 putative pyruvate kinase . 0.291 0.191 N.A. N.A. N.A. 0.236 2 141584 Q94CE2 putative monodehydroascorbate reductase. 0.405 0.293 N.A. N.A. 0.157 0.265 3 141055 Q8L5C8 putative mitochondrial NAD-dependent malate dehydrogenase. 0.118 0.106 0.15 0.113 0.207 0.134 5 141083 141037 Q949X7 Q9LQU9 putative diaminopimelate decarboxylase. F10B6.22. 0.18 0.51 0.221 0.417 0.126 0.39 0.205 0.509 0.165 0.442 0.176 0.451 5 5 141254 Q8L6J9 putative carbamoyl phosphate synthase large subunit. 0.242 0.176 0.118 0.156 0.059 0.135 5 141866 Q8L6J9 putative carbamoyl phosphate synthase large subunit. 0.361 N.A. N.A. N.A. N.A. 0.361 1 141375 Q82MT5 putative acyl-Coa thioesterase. 0.159 0.135 N.a. 0.269 0.123 0.163 4 d Supplementarty Table 1 141060 Q8GXh6 putative 3-isopropylmalate dehydrogenase. 0.179 0.136 0.11 0.184 0.091 0.135 5 141063 Q9FS26 plastidic cysteine synthase 1. 0.164 0.186 0.138 0.137 0.126 0.149 5 141084 Q9SXX5 plastidic aldolase. 0.244 0.19 0.151 0.142 0.134 0.168 5 141167 Q9FPL3 phosphoribosylaminoimidazolecarboxamide formyltransferase 0.203 0.308 0.171 0.266 0.237 0.232 5 141322 Q7XUX0 OSJNBa0027P08.15 protein. 0.164 0.112 N.A. 0.765 0.147 0.213 4 141507 Q40360 NADH-dependent glutamate synthase. N.A. 0.332 0.284 N.A. 0.226 0.277 3 141093 Q9LV03 NADH-dependent glutamate synthase. 0.163 0.133 0.276 0.092 0.135 0.149 5 141082 Q93WZ7 NADH glutamate synthase isoform 2 0.128 0.083 0.128 0.07 0.122 0.103 5 141414 Q41445 mitochondrial processing peptidase. 0.218 0.144 0.221 0.124 N.A. 0.171 4 141076 Q41440 mitochondrial processing peptidase. 0.32 0.25 0.203 0.296 0.372 0.282 5 141111 142052 141338 141174 Q9aXQ2 Q41440 Q8RX97 Q03685 mitochondrial processing peptidase beta subunit. mitochondrial processing peptidase. ferritin 1 78 kDa glucose-regulated protein homolog 5 0.257 N.A. 0.357 0.307 0.391 N.A. 0.186 0.251 0.287 0.138 0.157 0.13 0.302 N.A. 0.311 0.146 0.157 N.A. 0.205 0.2 0.268 0.138 0.231 0.196 5 1 5 5 141855 O78327 transketolase 1. 0.422 0.309 N.A. N.A. N.A. 0.361 2 141173 141256 141735 Q9SNY8 Q851Y8 O65852 branched-chain amino acid aminotransferase translational elongation factor Tu. isocitrate dehydrogenase 0.315 0.065 0.142 0.161 0.098 0.297 0.149 0.202 N.A. 0.192 0.055 N.A. 0.243 0.127 N.A. 0.204 0.098 0.205 5 5 2 141910 141844 O82030 Q43638 histidinol-phosphate aminotransferase precursor heat-shock protein precursor. 0.27 0.126 N.A. 0.186 N.A. N.A. N.A. N.A. N.A. N.A. 0.27 0.153 1 2 141187 141179 Q84QJ3 O50036 heat shock protein 70. heat shock protein 70. 0.3 0.288 0.22 0.199 0.162 0.162 0.154 0.167 0.14 0.108 0.187 0.176 5 5 141732 141545 Q84QJ3 P09189 heat shock protein 70. heat shock cognate 70 kDa protein. N.A. 0.155 N.A. 0.095 0.114 0.087 0.121 0.097 N.A. N.A. 0.118 0.105 2 4 141296 O50036 heat shock 70 protein. 0.812 0.615 0.445 0.373 0.45 0.518 5 e Supplementarty Table 1 141289 Q01899 heat shock 70 kDa protein, mitochondrial 0.583 0.493 0.576 N.A. 0.361 0.494 4 141092 Q01899 heat shock 70 kDa protein, mitochondrial 0.232 0.147 0.263 0.118 0.219 0.187 5 141183 Q01899 heat shock 70 kDa protein, mitochondrial 0.436 0.309 0.463 0.22 0.365 0.347 5 141244 Q9SUU0 glycine hydroxymethyltransferase 0.501 0.365 N.A. 0.433 0.412 0.425 4 141781 Q9SUU0 glycine hydroxymethyltransferase 0.379 0.33 N.A. N.A. N.A. 0.354 2 141506 Q944T4 glyceraldehyde 3-phosphate dehydrogenase 1 0.211 0.548 N.A. N.A. 0.654 0.423 3 141721 Q944T4 glyceraldehyde 3-phosphate dehydrogenase 1 0.232 0.426 N.A. N.A. N.A. 0.314 2 141461 Q944T4 glyceraldehyde 3-phosphate dehydrogenase 1 1.06 0.702 N.A. N.A. 0.747 0.822 3 141101 Q944T4 glyceraldehyde 3-phosphate dehydrogenase 1 0.097 0.261 0.195 0.122 0.217 0.167 5 141059 Q43127 glutamine synthetase 0.272 0.305 0.258 0.359 0.282 0.293 5 141448 Q9LEC8 glutamate dehydrogenase B 0.206 0.172 N.A. N.A. 0.317 0.224 3 141879 O82058 glucose-6-phosphate isomerase precursor 0.333 N.A. N.A. N.A. N.a. 0.333 1 141104 O82058 glucose-6-phosphate isomerase precursor 0.169 0.146 0.107 0.15 0.159 0.145 5 141419 Q9LQU9 F10B6.22. 0.25 0.186 0.17 N.A. 0.208 0.202 4 141162 Q40467 eukaryotic initiation factor 4a-14 0.204 0.123 0.26 0.208 0.272 0.206 5 141856 141052 P68158 P41342 elongation factor Tu, chloroplast precursor elongation factor Tu, chloroplast precursor N.A. 0.149 0.136 0.113 0.299 0.15 N.A. 0.103 N.A. 0.174 0.202 0.135 2 5 141871 P41342 elongation factor Tu, chloroplast precursor 0.206 N.A. N.A. N.A. N.A. 0.206 1 141435 Q9LIR4 dihydroxy-acid dehydratase 0.198 0.468 0.175 0.25 N.A. 0.252 4 141108 Q9LFG2 diaminopimelate epimerase-like protein 0.222 0.232 0.133 0.192 0.145 0.18 5 141120 Q9hJB6 DNA gyrase subunit a 0.127 0.143 0.221 0.192 0.376 0.196 5 141423 141434 Q9MT29 O98447 cystathionine gamma-synthase isoform 2 ClpC protease. 0.107 0.117 0.162 0.253 0.135 0.191 0.178 N.a. N.A. 0.143 0.143 0.169 4 4 f Supplementarty Table 1 141208 Q42884 chorismate synthase 1 0.3 0.234 N.A. 0.322 0.303 0.288 4 141022 Q40450 chloroplast elongation factor TuA 0.431 0.448 0.5 0.45 0.477 0.461 5 141307 Q40450 chloroplast elongation factor TuA 0.184 0.109 N.a. 0.121 0.153 0.139 4 141105 Q40450 chloroplast elongation factor TuA 0.108 0.167 0.204 0.094 0.065 0.118 5 141441 Q40450 chloroplast elongation factor TuA N.A. 0.295 0.283 0.149 0.135 0.203 4 141177 Q40450 chloroplast elongation factor TuA 0.417 0.368 0.167 0.422 0.1 0.255 5 141309 P93570 chaperonin-60 beta subunit precursor. 0.202 N.A. 0.211 0.187 0.118 0.175 4 141708 P29197 chaperonin HSP60, mitochondrial precursor 0.223 N.A. N.A. 0.229 N.A. 0.226 2 141300 141201 141040 141169 Q05046 Q05046 Q05046 Q05045 chaperonin CPN60-2, mitochondrial precursor chaperonin CPN60-2, mitochondrial precursor chaperonin CPN60-2, mitochondrial precursor chaperonin CPN60-1, mitochondrial precursor 0.317 0.71 0.143 0.157 0.282 0.462 0.097 0.119 0.644 0.731 0.18 0.218 N.A. 0.425 0.104 0.1 0.423 0.847 0.198 0.134 0.395 0.613 0.139 0.14 4 5 5 5 141863 Q05045 chaperonin CPN60-1, mitochondrial precursor 0.094 N.A. 0.134 N.A. N.A. 0.112 2 141228 141227 P29197 P29197 chaperonin CPN60, mitochondrial precursor . chaperonin CPN60, mitochondrial precursor 0.311 0.16 0.235 N.A. 0.442 0.194 0.252 0.1 N.A. 0.205 0.301 0.159 4 4 141406 Q9M5a8 chaperonin 21 precursor N.A. 0.311 0.184 0.305 0.3 0.269 4 141072 141034 141558 Q40475 Q40475 Q40475 biotin carboxylase subunit. biotin carboxylase subunit. biotin carboxylase subunit 0.253 0.206 0.264 0.234 0.184 0.248 0.229 0.182 0.244 0.253 0.192 N.A. 0.16 0.111 N.A. 0.223 0.171 0.252 5 5 3 141551 O25716 aspartate carbamoyltransferase 0.126 N.A. 0.1 N.A. 0.435 0.176 3 141335 141485 Q8VXC9 Q9FYW9 Alpha-tubulin. adenylosuccinate synthetase 0.338 0.124 0.285 N.A. 0.099 N.A. N.A. 0.229 0.248 0.108 0.221 0.145 4 3 141274 Q9FYW9 adenylosuccinate synthetase 0.151 0.206 0.116 0.126 0.502 0.187 5 141288 Q88aE0 acyltransferase family protein. 0.294 0.191 0.198 0.193 N.A. 0.215 4 141116 Q43593 acyl-[acyl-carrier protein] desaturase 0.206 0.324 0.294 0.254 0.115 0.225 5 g Supplementarty Table 1 141089 141675 141242 Q9ZSD7 Q84NI5 P09114 actin. aconitase acetolactate synthase II 0.121 N.A. 0.293 0.245 N.A. 0.211 0.258 0.099 0.184 0.112 N.A. 0.221 0.119 0.105 N.A. 0.159 0.102 0.224 5 2 4 141144 P09342 acetolactate synthase I 0.165 0.097 0.092 0.106 0.055 0.097 5 141313 141497 Q9SIF2 Q9SDP4 putative heat shock protein ATP-sulfurylAse. 0.352 0.374 0.172 0.165 0.27 N.A. 0.209 N.A. 0.366 0.301 0.263 0.265 5 3 141199 P31542 ATP-dependent Clp protease ATP-binding subunit clp 0.153 0.083 0.136 0.14 0.088 0.116 5 141568 P31542 ATP-dependent Clp protease ATP-binding subunit clp 0.358 0.21 N.A. 0.248 N.A. 0.265 3 141557 P17614 ATP synthAse betA chain, mitochondrial precursor N.A. 0.115 0.155 0.05 0.126 0.103 4 141305 P17614 ATP synthAse betA chain, mitochondrial precursor 0.198 0.11 0.143 N.A. 0.135 0.143 4 141291 P17614 ATP synthAse betA chain, mitochondrial precursor 0.469 0.4 0.507 N.A. 0.448 0.454 4 141096 P17614 ATP synthAse betA chain, mitochondrial precursor 0.379 0.308 0.427 0.229 0.389 0.338 5 141043 141126 P17614 P05495 ATP synthAse betA chain, mitochondrial precursor ATP synthAse Alpha chain, mitochondrial 0.272 0.211 0.188 0.105 0.293 0.242 0.172 0.074 0.3 0.227 0.239 0.155 5 5 141086 P05495 ATP synthAse Alpha achain, mitochondrial 0.124 0.118 0.195 0.127 0.135 0.137 5 141027 P05495 ATP synthAse Alpha chain, mitochondrial 0.842 0.531 0.815 0.545 0.931 0.714 5 141071 141926 P05495 P05495 ATP synthAse Alpha chain, mitochondrial ATP synthAse Alpha chain, mitochondrial 0.176 0.714 0.318 N.A. 0.412 N.A. 0.217 N.A. 0.25 N.A. 0.263 0.714 5 1 141336 Q9Sh69 6-phosphogluconate dehydrogenase 0.119 0.181 0.309 N.A. 0.182 0.187 4 141586 141356 Q9ZTS5 Q89MR7 6-phosphogluconate dehydrogenase 5'-phosphoribosyl-5-aminoimidazole synthetase. 0.282 0.159 0.16 N.a. N.A. 0.088 N.A. 0.106 0.142 0.109 0.186 0.113 3 4 141987 Q8DaR4 3-phosphoshikimate 1-carboxyvinyltransferase. N.A. 0.267 N.A. N.A. N.A. 0.267 1 141122 Q9C550 2-isopropylmalate synthase 0.608 0.387 0.375 0.215 0.344 0.365 5 141436 Q96460 tubulin alpha-2 chain 0.235 0.21 0.163 N.A. 0.164 0.191 4 h Supplementarty Table 1 141959 Q8L6J9 putative carbamoyl phosphate synthase large subunit. N.A. 0.255 N.A. N.A. N.A. 0.255 1 141883 P29677 mitochondrial processing peptidase alpha subunit, 0.204 N.A. N.A. N.A. N.A. 0.204 1 141253 Q43593 acyl-[acyl-carrier protein] desaturase 0.237 0.268 0.154 N.A. 0.255 0.223 4 141555 P09342 acetolactate synthase I N.A. 0.224 0.175 N.A. 0.118 0.166 3 i Supplementary Table 2 Supplementary Table 2 List of proteins identified from first Percoll gradient centrifugation for isolation and purificationa a only peptides which have significant score (i.e., Xcorr >2.5, dCn >0.1) in SEQUEST database search were taken in this approach. Proteins hits were accepted only if two peptides were detected from this protein. Localization prediction was performed with ChloroP (Emanuelsson et al., 1999). Provided is the identifier (“Identifier”), the NCBI database accession number (“acc. no”), the organism (“organism”), the number of identified tryptic peptides (“no. of peptides”), protein localization [“Localization” Y: transit peptide by ChloroP (Emanuelsson et al., 1999), RP: reported chloroplast function or Arabidopsis orthologue with a transit peptide, PE: plastid encoded Mit: mitochondria, Nu; nucleus, Ot: other localization, b SP: Secretary pathway]. prediction by the ChloroP program. Identifier No of peptides Protein localization Band A Plastid gi|3559814 gi|12643508 gi|1076575 gi|16973465 gi|10441272 gi|15220397 gi|134642 gi|1351271 gi|38679329 gi|14719331 gi|29839371 gi|12322892 gi|1177314 gi|15797694 gi|27807824 gi|7431781 gi|1168411 gi|47169163 gi|14423502 gi|11387206 gi|50878369 gi|4457219 gi|27805483 gi|51535721 gi|9757593 gi|28261725 gi|18378982 gi|28261724 gi|2129926 gi|38231570 gi|1129145 gi|42564093 7 14 27 7 5 4 14 10 4 2 3 7 1 2 2 4 3 2 2 2 2 2 2 2 2 2 6 2 8 3 transketolase 1 capsanthin/capsorubin synthase plant fibrillin precursor lethal leaf spot 1-like protein transaldolase lactoylglutathione lyase, putative / glyoxalase I, putative superoxide dismutase [Fe] triosephosphate isomerase (TIM) harpin binding protein 1 putative 3-beta hydroxysteroid dehydrogenase/isomerase protein ferritin 1 putative 2-cys peroxiredoxin BAS1 glyoxalase-I unnamed protein product nucleoside diphosphate kinase glutamate synthase (ferredoxin) fructose-bisphosphate aldolase a chain A, crystal structure snalysis sf The ferredoxin-NADP+ reductase unknown protein putative L-ascorbate peroxidase unknown protein acyl carrier protein 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase putative thioredoxin peroxidase 1 maturase ribulose 1,5-bisphosphate carboxylase/oxygenase large chain ATP-dependent Clp protease proteolytic subunit (ClpP1) ATP synthase CF1 beta chain wound-induced protein Sn-1, vacuolar membrane PII-like protein Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y PE RP Y 4 4 Mitochondria 3-ketoacyl-CoA thiolase B; acetyl-CoA C-acyltransferase basic beta-1,3-glucanase Mit Mit j Supplementary Table 2 Nucleus gi|23506611 gi|15232146 gi|47026912 gi|7387727 gi|15226944 gi|1084419 gi|119143 gi|15234867 gi|9294568 gi|11268170 gi|22326737 gi|12230867 2 13 30 25 44 9 histone H1D histone H3 histone H2A histone H2B histone H4 histone H1 Nu Nu Nu Nu Nu Nu 9 2 3 2 2 2 Other elongation factor 1-alpha (EF-1-alpha) expressed protein unnamed protein product malate synthase-like protein protein kinase family protein 14-3-3-like protein Ot Ot Ot Ot Ot Ot Secretory pathway b gi|1171503 gi|15419836 gi|1709500 gi|100309 4 2 5 6 gamma-thionin thaumatin-like protein osmotin precursor chitinase SP SP SP SP 62 25 23 13 11 15 4 2 4 2 4 10 6 4 4 3 2 4 4 32 8 2 4 Plastid capsanthin/capsorubin synthase plant fibrillin precursor ferredoxin-dependent glutamate synthase 1 (Fd-GOGAT 1) alpha-1,4 glucan phosphorylase, L-1 isozyme lethal leaf spot 1-like protein heat shock protein, 70K transaldolase superoxide dismutase [Fe] cysteine synthase peroxiredoxin unnamed protein product chaperonin, putative pruvate kinase isozyme A rptide methionine sulfoxide reductase rubisco subunit binding-protein alpha subunit glyceraldehyde-3-phosphate dehydrogenase B harpin binding protein 1 1,4-alpha-glucan branching enzyme putative heat-shock protein transketolase endopeptidase Clp ATP-binding chain CD4A beta carotene hydroxylase wound-induced protein Sn-1, vacuolar membrane Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y RP Band B gi|12643508 gi|1076575 gi|12643970 gi|130173 gi|16973465 gi|7441856 gi|10441272 gi|134642 gi|322740 gi|11558242 gi|15797694 gi|30696748 gi|2497541 gi|12230349 gi|15226314 gi|20455491 gi|38679337 gi|1169912 gi|50947075 gi|3559814 gi|100189 gi|19071768 gi|2129926 Nucleus gi|15232146 gi|15226944 2 3 histone H3 histone H4 Nu Nu k Supplementary Table 2 Secretory pathway b gi|20339364 4 UOS1 SP Band C gi|12643508 gi|7433617 gi|1076575 gi|7431781 gi|10441272 gi|11131628 gi|130173 gi|16973465 gi|13431953 gi|40644130 gi|134642 gi|15234797 gi|4457219 gi|30696748 gi|15228431 gi|7546556 27 15 15 15 3 4 8 3 3 2 2 2 2 2 2 3 gi|14030711 gi|2129926 3 4 Plastid capsanthin/capsorubin synthase transketolase precursor plant fibrillin precursor glutamate synthase (ferredoxin) transaldolase cysteine synthase alpha-1,4 glucan phosphorylase, L-1 isozyme lethal leaf spot 1-like protein triosephosphate isomerase allene oxide cyclase superoxide dismutase [Fe] expressed protein acyl carrier protein chaperonin, putative ferritin, putative S chain S, crystal structure of unactivated tobacco rubisco with bound phosphate ions AT3g20390/MQC12_15 wound-induced protein Sn-1, vacuolar membrane gi|15226943 gi|15232146 gi|15226944 2 3 7 histone H2B, putative histone H3 histone H4 Nu Nu Nu 49 67 67 12 3 3 27 14 8 41 3 5 3 7 2 7 12 14 5 5 4 Plastid capsanthin/capsorubin synthase transketolase plant fibrillin precursor lethal leaf spot 1-like protein allene oxide cyclase chloroplast latex aldolase-like protein alpha-1,4 glucan phosphorylase, L-1 isozyme chaperonin, putative cysteine synthase transaldolase peroxiredoxin pyruvate kinase isozyme A GcpE ferredoxin-dependent glutamate synthase (Fd-GOGAT) starch associated protein R1 1,4-alpha-glucan branching enzyme (Q-enzyme) malate dehydrogenase stromal 70 kDa heat shock-related protein clpB heat shock protein-like chloroplast protease plastidial phosphoglucomutase Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y RP Nucleus Band D gi|12643508 gi|7433617 gi|1076575 gi|16973465 gi|40644130 gi|56122688 gi|130173 gi|30696748 gi|11131628 gi|2078350 gi|11558242 gi|2497541 gi|27462474 gi|12644435 gi|13124867 gi|1169912 gi|7431231 gi|399942 gi|11265210 gi|3808101 gi|6272283 l Supplementary Table 2 gi|7443855 gi|51340062 gi|2492953 3 3 3 2 2 2 11 4 4 2 3 2 3 5 15 probable chaperonin 60 beta chain precursor glucose-6-phosphate isomerase chorismate synthase 2(5-enolpyruvylshikimate-3-phosphate phospholyase 2) aminolevulinate dehydratase rubisco subunit binding-protein alpha subunit putative UDP-sulfoquinovose synthase FtsH protease (VAR2) zeta-carotene desaturase(Carotene 7,8-desaturase) fructose-bisphosphate aldolase, putative phosphoglycerate kinase homologous to plastidic aldolases glyceraldehyde-3-phosphate dehydrogenase B iron superoxide dismutase harpin binding protein 1 lactoylglutathione lyase, putative / glyoxalase I, putative triosephosphate isomerase (TIM) unnamed protein product ferritin, putative putative peptidylprolyl isomerase AT3g26060/MPE11_21 putative thioredoxin peroxidase 1 peptide methionine sulfoxide reductase, putative adenine phosphoribosyltransferase, putative beta carotene hydroxylase AT3g20390/MQC12_15 S chain S, crystal structure of unactivated tobacco rubisco with bound phosphate ions Clp protease 2 proteolytic subunit ATP-dependent Clp protease proteolytic subunit (ClpP1) ribulose 1,5 bisphosphate carboxylase large subunit ATP-dependent clp protease ATP-binding subunit clpA homolog CD4B ribulose bisphosphate carboxylase/oxygenase activase ascorbate peroxidase cytochrome f acyl-[acyl-carrier-protein] desaturase predicted P0710F09.129 gene product wound-induced protein Sn-1, vacuolar membrane biotin carboxylase ATP synthase CF1 alpha chain gi|1097877 gi|15226314 gi|54287592 gi|30684767 gi|17367814 gi|18399660 gi|2499497 gi|1781348 gi|15217555 gi|27543371 gi|38679319 gi|15220397 gi|13431953 gi|15797694 gi|15228431 gi|46359893 gi|15081743 gi|51535721 gi|18424492 gi|15235709 gi|19071768 gi|14030711 gi|7546556 5 3 3 4 2 6 9 11 4 11 6 10 9 7 3 3 2 2 2 2 2 2 5 gi|15485610 gi|18378982 gi|27528299 gi|399213 gi|10720247 gi|21039134 gi|28261730 gi|3915035 gi|51963380 gi|2129926 gi|1582354 gi|28261702 gi|18391115 gi|42564093 gi|7431444 gi|24940264 Y Y Y Y Y Y Y Y Y Y Y Y RP Y PE 4 2 2 2 Mitochondria expressed protein basic beta-1,3-glucanase aldehyde dehydrogenase (NAD) mitochondrial ATP synthase beta subunit Mit Mit Mit Mit Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Nucleus gi|15226944 3 histone H4 Nu gi|15233207 gi|2129924 gi|119143 gi|6715212 2 13 5 2 Other receptor-like protein kinase-related ATPase elongation factor 1-alpha phytochrome A Un Un Un Mit m Supplementary Table 2 gi|1705805 gi|2506467 gi|56787638 5 2 2 Secretory pathway b endochitinase 1 precursor lichenase precursor (Endo-beta-1,3-1,4 glucanase) osmotin-like protein sp sp sp Band E 3 5 Plastid capsanthin/capsorubin synthase plant fibrillin precursor transketolase fructose-bisphosphate aldolase (ALDP) transaldolase phosphoglycerate kinase ferredoxin-dependent glutamate synthase (Fd-GOGAT) glycogen phosphorylase B; starch phosphorylase chromoplast-specific lycopene beta-cyclase heat shock 70 protein superoxide dismutase [Fe] chain S, crystal structure of unactivated tobacco rubisco with bound phosphate ions AT3g20390/MQC12_15 wound-induced protein Sn-1, vacuolar membrane Y RP gi|11131023 2 Mitochondria ADP-ribosylation factor 1 Mit gi|15232146 gi|15226944 2 9 histone H3 histone H4 Nu Nu 8 Other elongation factor 1-alpha (EF-1-alpha) Un 2 Secretory pathway b beta(1,3)-glucanase regulator SP 19 53 32 8 7 10 3 7 4 4 9 2 7 4 Plastid capsanthin/capsorubin synthase plant fibrillin precursor transketolase 1 transaldolase lethal leaf spot 1-like protein heat shock 70 protein ferredoxin-dependent glutamate synthase(Fd-GOGAT) fructose-bisphosphate aldolase chaperonin, putative cell division protein ftsH homolog phosphoglycerate kinase pyruvate kinase isozyme A probable chaperonin 60 beta chain precursor cysteine synthase gi|12643508 gi|1076575 gi|7433617 gi|3913018 gi|10441272 gi|2499497 gi|7431781 gi|11994512 gi|10644119 gi|2654208 gi|134642 gi|7546556 9 13 6 2 2 3 3 2 2 3 2 4 gi|14030711 gi|2129926 Y Y Y Y Y Y Y Y Y Y Y Y Nucleus gi|119143 gi|170243 Band F gi|12643508 gi|1076575 gi|3559814 gi|10441272 gi|16973465 gi|2654208 gi|12644435 gi|1168411 gi|30696748 gi|17865457 gi|2499497 gi|2497541 gi|7443855 gi|399333 Y Y Y Y Y Y Y Y Y Y Y Y Y Y n Supplementary Table 2 gi|15229231 gi|135061 gi|42408569 gi|10720247 gi|18423214 gi|28261702 gi|2129926 3 2 2 2 2 3 11 glyceraldehyde-3-phosphate dehydrogenase sucrose synthase (Sucrose-UDP glucosyltransferase) putative glucose-6-phosphate isomerase precursor ribulose bisphosphate carboxylase/oxygenase activase ATP-dependent Clp protease ATP-binding subunit / ClpC ATP synthase CF1 alpha chain wound-induced protein Sn-1, vacuolar membrane Y Y Y Y Y PE RP gi|15225798 3 gi|114420 gi|15228319 3 2 Mitochondria acetyl-CoA C-acyltransferase, putative / 3-ketoacyl-CoA thiolase, putative ATP synthase beta chain, mitochondrial precursor aldehyde dehydrogenase (ALDH2) gi|15226944 6 histone H4 Nu gi|50901852 gi|7708327 gi|37953301 gi|17865469 gi|119143 gi|58013197 3 4 4 4 6 4 Other B1097D05.21 ATP synthase beta subunit alanine aminotransferase catalase (CaCat1) elongation factor 1-alpha (EF-1-alpha) actin Un Un Un Un Un Un 2 3 4 Secretory pathway b lichenase precursor (Endo-beta-1,3-1,4 glucanase) chitinase beta(1,3)-glucanase regulator SP SP SP Mit Mit Mit Nucleus gi|2506467 gi|100309 gi|170243 o Supplementary Table 3 Supplementary Table 3 MS-BLAST output which previously identified in the SEQUEST database search. a a MS-BLAST score >62 for multiple hits, >65 for single hits took in this section. Provider is the identifier (“Identifer”, the Swiss prot accession number), the organism (“Organism”), protein name (“Protein”), the number of identified tryptic peptides (“No. of peptides”), significant score (“MS-BLAST score”), PepNovo sequence output (“Query”) and MS-BLAST result (“Subject”). Swiss port ID organism Protein name No of peptides Q9XGM0 Brassica napus [acyl-carrier protein] S-malonyltransferase 2 O04864 Solanum tuberosum 1,4-alpha-glucan branching enzyme 4 Q9FRX7 Oryza sativa aldehyde dehydrogenase 2 DQ386163 Lycopersicon esculentum ATP synthase CF1 alpha subunit 2 Q42435 Capsicum annuum capsanthin/capsorubin synthase 5 Q5PYQ2 Manihot esculenta chloroplast latex aldolase-like protein 4 Q53N80 Oryza sativa rieske [2Fe-2S] domain, putative 2 MSBLAST score 80 62 96 79 73 64 92 89 71 69 108 66 62 92 72 103 82 85 64 100 Query Subject BXXXASEVTSPVQWET EAMQDAADAA BLPDVDSNPALPHNSR BXXTSSTDEDNQVVVFER BXXTSSTDEDNQVVVFER LPDVDSNPALPHNSR BXXNVLANVAEGDAEDLNR LANVAEGDAEDLNR BKFTEEAEALLK KKILARQVTSPVQWET EAMQAAADAA RIPDVDSKPVIPHNSR KQIVSSMDDDNKVVVFER KQIVSSTNEEDKVIVFER IPDSGGNPAIHHNSR RTGELIAHVAEGDAEDINR IAHVAEGDAEDINR KTFTEEAEALLK FTEEAEALLK LVDASGYASDMLEYDQPR BXXXXXEEEQCVLTM WHGFLSSR FTEEAEAILK IVDASGYASDFIEYDKPR KVRSVLEEEKCVITM WHGFLSSR KVKHEEFESSIVCDDGR BXXHEEMESSLVCEDGR HEEMESSLVCEDG BATPQQVADYTLNLL BXXXQQVADYTLNLL PQQVADYTLNLL ASSGTYVTEV BSPAEGAYSEGLLNAK HQEFESSIVCDDG RATPQQVADYTLNLL RATPQQVADYTLNLL PQQVADYTLNLL ATSGTYVTEV RSPAEGAYTEGLLNAK p Supplementary Table 3 Q40450 Nicotiana sylvestris elongation factor TuA (EF-TuA) 3 Q9T0P4 Arabidopsis thaliana ferredoxin-dependent glutamate synthase 2 6 2 Q7M242 Nicotiana tabacum glutamate synthase (Ferredoxin) 2 Q69RJ0 Oryza sativa putative ferredoxin-dependent glutamate synthase 2 Q84P91 Oryza sativa fibrillin-like protein 3 Q9SVJ6 arabidopsis thalaniana putative fructose-bisphosphate aldolase 4 P93565 Solanum tuberosum homologous to plastidic aldolases 3 Q8LL68 Hevea brasiliensis latex plastidic aldolase-like 4 Q9SXX5 Nicotiana paniculata plastidic aldolase 6 74 78 76 89 76 68 64 98 72 64 BXXPGVTATDVNVDEVR NTATVEYETED TATVEYETESR LNTATVEYETESR PVDSSVVGYYAK FNNEGLEVLGWR GGLTLDELAR HTNNCPVGVASQR FNNEGLEVLGWR 81 80 73 GGLTLDELAR BLLGEANDYVGK BMPSVTLEQAQK BMPSVTLEQAQ 75 86 65 65 84 73 71 75 82 85 75 83 67 65 64 65 83 67 FNNEGLEVLGWR BNPTPAPTEALSLL BXXXVELLTQLESK BRQLTDSMYGTDR BATPQQVADYTLNLLR PLVEPELLNNGDH BXXLNMDESNATCGK AMDESNATCAK BXXXXQQVADYTLNLL PQQVADYTLNLL AMDESNATCAK BXXLNMDESNATCGK PLVEPELLNNGDH WSGRPENVK BASANSLAQLGK BXXXAMDESNAT BXXLNMDESNATCGK PLVEPELLNNGDH KAQPGFTASDVNIEEVR NTATVEYETEN TATVEYETENR INTATVEYETENR PVNTSVVGYYAK FAQEGIEVIGWR GGLTLDELAR HTNNCPVGVASQR FEKEGLEVLGWR GGLTLDELAR RLIGEANDYVGK KMPTVTIEQAQK KMPTVTIEQAQ FTDEGLEVLGWR RNPTPAPTEALTLL RAEIVELITQLEAK KRSLADSLYGTDR RATPEQVAAYTLKLLR PIVEPEILLDGDH RGILAIDESNATCGK AMDESNATCGK RQGTPQQVADYTLNLL PQQVADYTLNLL AMDESNATCGK RGILAMDESNATCGK PIVEPEILLDGEH WAGRPENVK RAKANSLAQLGK RGILAMDESNAT RGILAMDESNATCGK PIVEPEILLDGEH q Supplementary Table 3 Q69K00 Oryza sativa putative triosephosphate isomerase 2 Q9SKP6 Arabidopsis thaliana triosephosphate isomerase 7 P21820 Oryza sativa triosephosphate isomerase, cytosolic 5 Q8LGH5 Arabidopsis thaliana GTP binding protein 4 Q9LX13 Arabidopsis thaliana 1 P46253 Solanum tuberosum (3R)-hydroxymyristoyl-[acyl carrier protein]dehydratase acyl-[acyl-carrier-protein] desaturase 64 83 68 92 69 66 65 71 73 81 73 94 71 86 87 78 89 75 66 83 77 96 66 90 68 72 105 69 68 68 1 76 Q42493 Capsicum annuum plastoglobules associated protein precursor 5 3 BASANSLAQLGK BXXLNMDESNATCGK BASANSLAQLGK BNPTPAPTEALSLL TDSFYGTNR TPAPTEALSL AVVEEEPPQE BXXXVELLTQLESK BXXVPQLAGTSNQGTAK ETLEQTDNALVEK VETLEQTDNALV ATPEQAQEVHVAVR BHLLGENDEFLGK BXXYGGSVNGSNSSDLAK ATPEQAQEVHVAVR BHLLGENDEFLGK ATPEQAQEVHVAVR BHLLGENDEFLGK BXXYGGSVNGSNS PEQAQEVHVAVR BXXYGGSVNGSNSSDLA ATPEQAQEVHVAVR BXXYGGSVNGSNS PEQAQEVHVAVR BXXYGGSVNGSNS GTNVEQAFQCV BSHVVQQSLGQAVGDLR BXXPLPYMLSSMLQSR BXXXSSDEPLAAQVLYR BYPAMPTVMDLNQL RAKANSLAQLGK RGILAMDESNATCGK RANANSLAQLGK KNPTPAPTEALSLL TDSFYGTNR TPAPTEALSL AVVEEEPPKE RAEIVELITQLESK RQKLPQLAGTSIEGTAK ETLEKTDNALVEK VETLEKTDNALV ATPEQAQEVHAAVR RHVIGEDDQFIGK RIIYGGSVNGGNSAELAK ASPEQAQEVHAAVR RHVIGEDDEFIGK ASPQQAQEVHVAVR RHVIGEKDEFIGK RIIYGGSVNGGNS PQQAQEVHVAVR RIIYGGSVNGANSKELA ASPEQAQEVHVAVR RIIYGGSVNGGNS PEQAQEVHVAVR RIIYGGSVNGANS GTNVEEAFQCI RSHIVQQAIGQAVGDLR RAPPLPYLLSWLLQSR RLGFTTEESIAAQVLYR RYEAFPTVMDINKI VDPGSVLLALGDMMR VDPDGAVLAIGDMMR r Supplementary Table 3 Q9FR04 Q9SIE3 Q8L5U2 Perilla frutescens Arabidopsis thaliana Arabidopsis thaliana Q9SS98 Q8W5A3 Arabidopsis thaliana Lycopersicon esculentum Sorghum bicolor Arabidopsis thaliana Glycine max Yucca schidigera Q8LST4 P46248 O65027 Q4FGH1 Q9M5A8 Q8L784 DQ459071 Q9XIS4 Q8LHF0 Q01517 AC128643 Q9ATJ1 Q9M8D3 Lycopersicon esculentum Nicotiana tabacum Oryza sativa Lycopersicon esculentum arabidopsis thalaniana Sorghum bicolor Phaseolus vulgaris Oryza sativa Pisum sativum Oryza sativa Dunaliella salina Arabidopsis thaliana Q84VE2 Q9TN65 Oryza sativa Peliosanthes violacea Q9FVH1 Lycopersicon esculentum Solanum tuberosum Q8RVF8 Q6H7E4 Q7YK44 Q9LLE0 malonyl-CoA:ACP transacylase putative beta-hydroxyacyl-ACP dehydratase putative malonyl-CoA:Acyl carrier protein transacylase putative oleosin lethal leaf spot 1-like 1 1 1 73 92 81 EAMQDAADAA BYPAMPTVMDLNQLR BXXXXSQVTSPVQWET EAMQDAADAA KFPAFPTVMDINQIR KKILARQVTSPVQWET 1 1 66 70 BLADYAEYVGQR VEDLDPSLPT RLADMAEYVGQR VEDLDPSLPT mitochondrial aldehyde dehydrogenase aspartate aminotransferase aspartokinase-homoserine dehydrogenase ATP-depedent Clp protease proteolytic 1 1 1 1 99 76 83 66 BXXNVLANVAEGDAEDLNR NEEYLPLEGLSAMNK DLLDGDNLASFLCK BGPGEANPLWVDVN RTGEVIAHVAEGDAEDINR NKEYLPIEGLAAFNK DILDGDNLASFLSK chaperonin 21 1 75 DFQGADGSDYLTL RSPGEEDAVWVDVN EFKGADGSDYITL thioredoxin putative Thioredoxin M-type superoxide dismutase 1 1 1 65 79 69 BASSEVPLVGN BXXXDESPSLATQFGLR DYLSLFMEK RASSELPLVGN KLNTDENPDIATQFGIR DYISIFMEK cytoplasmic aconitate hydratase putative aconitate hydratase 1 starch branching enzyme NADH-dependent glutamate synthase fructose-bisphosphate aldolase 2 fructose-bisphosphate aldolase class-I fructose-bisphosphate aldolase isoenzyme 1 probable phosphoribosylformylglycinamidine synthase coproporphyrinogen III oxidase ribulose-1,5-bisphosphate carboxylase large subunit transaldolase 1 1 1 1 1 1 1 1 83 85 97 69 69 64 65 71 DADTLGLTGHER BAVSDADTLGLTGHER BLPDVDSNPALPHNSR HTNNCPVGVASQ PENVKAAQEGVL BASANSLAQLGK BXXXALDESNATAGK EASTLGVWAAH DADTLGLTGHER KAGEDADTLGLTGHER KIPDVDGNPAIPHSSR HTNTCPVGIATQ PENVKAAQEALL RAKANSLAQLGK RGILAMDESNATVGK EGSTLGVWAAH 1 1 86 85 ETDAPNDAPGAPR BXXYLNATAGTCESM ETDAPKDAPGAPR KGHYLNATAGTCEEM 1 87 BLADDTEGTLEAAK RLADDTEGTVEAAK hexose transporter 1 86 BASGEDLEDAAPLK RAAGEDIEDAAPLK s Supplementary Table 3 Putative contaminants Q6XLQ1 Capsicum annuum dehydrin-like protein 6 Q96318 Arabidopsis thaliana 12S cruciferin seed storage 5 P15458 Arabidopsis thaliana 2S seed storage protein 2 5 P33522 Brassica napus cruciferin cru4 3 P15456 Q41909 Q7M1P1 P05151 Arabidopsis thaliana Arabidopsis thaliana Brassica napus Triticum aestivum 12S seed storage protein CRB 2S seed storage protein 1 napin cytochrome f New putative contaminants Q9ZR68 Nicotiana tabacum Q9FJW1 Arabidopsis thaliana Q56Z11 Arabidopsis thaliana 1 1 1 1 64 76 72 90 80 95 73 69 71 73 69 81 64 78 63 73 68 66 74 69 72 66 84 ETTTGANVESTDR BPSWEETTTGANVE EETTTGANVE BXXTEETTTGANVESSER BPWSEETTTGANVES DETTTGANVESTDR EGQGQQGQEGR BDHENLDDPAR FGGSQQQQEKK EGQGQQGQEGR BDHENLDDPAR LQGQHGPFQSR RQEEPVCV BAVSLQGQHGPMQ QGQHGPMQSR QEEPVCVCAKLK HENLDDPAR NNAQVNTLAGR QLQLVNDNGDR NAQVNTLAGR BAVSLQGQHGPMQGS QEEPVCVCAKLK LFAQQGYENPR EETVGANVEATDR KPSVEETTTGANVE EETTTGANVE KPSVEETTTGANVESTDR KPSVEETTTGANVES EETTTGANVESTDR EGQGQQGQQGR RSHENIDDPAR FGGSQQQQEQK EGQGQQGQQGR RSHENIDDPAR LQGQHGPFQSR RQEEPVCV RAVSLQGQHGPFQ QGQHGPFQSR QEEPVCVCPTLK HENIDDPAR DNAQINTLAGR QIQVVNDNGDR NAQVNTLAGR KAVRLQGQHQPMQVS QEEPLCVCPTLK IFAQQGYENPR aquaporin 1 arabidopsis thaliana genomic DNA, chromosome 5, TAC clone:K9I9 1 2 68 64 QLAMGSHEELR NPNADPNPN QIAVGSHEELR NPNSDPNPN legumin-like protein(At5g44120) 2 66 73 KDSDSLEDEDE QLQLVNDNGDR KDSDGLEDEDD QIQIVNDNGNR t Supplementary Table 3 Q9ZSK3 Q40576 Q9LP07 Q9LGY7 Arabidopsis thaliana Nicotiana tabacum Arabidopsis thaliana Oryza sativa actin depolymerizing factor 4 orf protein putative auxin response factor 23 putative SDL-1 protein 1 1 1 1 69 75 72 66 65 AQLQLVNDNG NSASGMAVHDD PAPEVDSQSSEPTH DETYLEVTLM MVDAQADAMP AQIQIVNDNG NAASGMAVHDD PSSEVDAQSSEPIH DETYVEITLM MVDAQEEAMP u