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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
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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. Further advancements in these
technologies will help to increase the proteome coverage from non-model
organisms, which is important for a better understanding of biological
systems.
109
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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