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Proteomics of the chloroplast:
experimentation and prediction
Klaas Jan van Wijk
New technologies, in combination with increasing amounts of plant genome sequence data,
have opened up incredible experimental possibilities to identify the total set of chloroplast
proteins (the chloroplast proteome) as well as their expression levels and post-translational
modifications in a global manner. This is summarized under the term ‘proteomics’ and typically involves two-dimensional electrophoresis or chromatography, mass spectrometry and
bioinformatics. Complemented with nucleotide-based global techniques, proteomics is
expected to provide many new insights into chloroplast biogenesis, adaptation and function.
hloroplasts are chlorophyll-containing plastids and originate from proplastids, which are generally maternally
inherited via the embryo. Although the study of the chloroplast is a classic field in plant biology, there is no good overview
of the total set of chloroplast proteins (the chloroplast proteome).
Improvements in two-dimensional electrophoresis (2-DE) and
mass spectrometry have, in combination with increasing amounts
of sequence data from Arabidopsis, rice, maize and other plant
species, opened up fantastic experimental possibilities enabling
the chloroplast proteins as well as their expression levels and
post-translational modifications to be identified rapidly. This
is summarized under the term ‘proteomics’. Proteomics typically
involves biochemical purification techniques such as 2-DE,
chromatography or affinity purification, mass spectrometry and
bioinformatics1,2. Complemented with other functional genomics
techniques such as cDNA or oligonucleotide microarrrays (Box 1)
and reverse genetics, a better understanding of chloroplast biogenesis, adaptation to the environment, signal transduction and
metabolic pathways can be obtained.
Here, we discuss in detail such an experimental approach to the
characterization of the chloroplast proteome. A complete characterization includes not only the identification of proteins but also
studies of their expression levels, post-translational modifications,
protein–protein interactions and apparent discrepancies between
the identified proteins and their predicted protein sequence from
nucleotide sequencing data. We also comment on possibilities and
limitations for the theoretical prediction of the chloroplast proteome based on targeting or presequence information.
C
Experimental characterization of the chloroplast proteome by
2-DE and mass spectrometry
The improvement of 2-DE through the development of immobilized pH gradients3 and optimization of solubilization techniques4,5 now allows the reproducible separation of more than
2000 proteins on a single 2-DE gel. Such gel-separated proteins
can be identified by mass spectrometry if genomic information is
available1,2,6 (Box 2). In addition, mass spectrometry is a powerful
tool for analyzing isoforms, secondary modifications of proteins
(e.g. glycosylation and phosphorylation) and proteolysis using
low amounts (picomoles to attomoles) of proteins7–9. Thus, a proteomics approach allows us to bridge the gap between genomic
sequence information and the actual protein population in a cell.
Proteomics is already an important tool in medical research and
the analysis of yeast and prokaryotes. With the rapid progress of
the sequencing of the Arabidopsis genome and ongoing EST and
genomic sequencing of many agricultural crops, proteomics is
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bound to become another important tool for plant biology. Several
plant proteomics studies have been published in recent years10.
However, in these studies, protein identification was achieved
through Edman sequencing, which necessarily limited the identification of proteins in terms of cost, speed and sensitivity. Mass
spectrometry will allow identification at a much higher speed and
with 100–1000 times less protein. Two plant proteomics studies
using mass spectrometry have been published recently, one concerning anoxia tolerance in maize root tips11 and the other on pea
thylakoid proteins12. An explosion of plant proteomics initiatives
can be expected in the coming years.
Compartmenting the chloroplast proteome
From a biochemical point of view, the chloroplast can be divided
into several compartments, with each compartment having its own
specific subset of proteins, or subproteome. To characterize the
chloroplast proteome fully, either experimentally or by prediction,
it is useful (and probably essential) to subdivide the chloroplast
proteome into such subproteomes13. Only then can we devise optimal experimental strategies to identify and characterize most proteins, including those that are hydrophobic5, of low abundance or
transiently expressed14. For each of the chloroplast compartments,
we briefly review current knowledge of each corresponding subproteome and discuss possible experimental and theoretical strategies for further characterization.
Stromules, the chloroplast envelope and vesicles
Starting from the cytosolic side of the chloroplast, the first compartment is the chloroplast outer and inner envelope. The envelope is the site of transport of metabolites, proteins and
messengers between plastids and the cytosol15,16. The inner envelope membrane is also a site for the biosynthesis of several products (e.g. lipids and pigments15,17,18) and has also been implicated
in DNA replication and transcription of the chloroplast genome19.
Several protein complexes involved in the translocation of
nucleus-encoded chloroplast proteins have been characterized in
great detail20,21. At least 100 protein bands can be resolved on onedimensional electrophoresis (1-DE) silver- or Coomassie-stained
gels of purified inner and outer membranes. However, 1-DE gels
do not have sufficient resolution to obtain a global overview of the
envelope proteome or to study post-translational modifications or
changes in protein expression.
No successful systematic analysis of the envelope proteome has
been carried out to date, which is partly related to the hydrophobic
nature of this subproteome. The 2-DE of envelope membrane
proteins was reported to be unsuccessful in the recovery of
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01737-4
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Box 1. Functional genomics tools to understand
cellular processes
Transcriptomics
Definition
Type of information
Strong points
Weak points
Proteomics
Definition
Type of information
Strong points
Weak points
• The systematic analysis of accumulated
transcripts in the cell or tissue
• The accumulation of transcripts, indicating
the level of gene expression
• Extremely sensitive
• Many different transcripts can be monitored
simultaneously
• mRNA levels often do not correspond to
protein accumulation
• Cannot study post-translational modifications
• Location of mRNA does not provide
information about the location of the gene
product
• The systematic analysis of the proteins
(the proteome) of a cell, tissue, organelle
or membrane
• The identification and expression level
of the proteome
• Post-translational modifications and protein–
protein interactions (protein complexes)
• Relatively fast and sensitive (femtomole
range) identification of proteins
• Protein–protein interactions can be studied
• Monitors the protein directly, rather than
monitoring the mRNA
• With current technology, the number of
proteins that can be simultaneously followed
is not as high as with transcriptomics
• Protein separation steps are still timeconsuming
• Monitoring the expression levels of
membrane proteins is technically demanding
envelope proteins22, a phenomenon that has been reported for
membrane proteins in general5. However, it is at least possible to
resolve smaller membrane proteins (1– 4 transmembrane domains)
on 2-DE gels if soluble proteins as well as lipids are removed23. A
similar strategy, using extraction with organic solvents, but with
1-DE, has been used to identify several hydrophobic chloroplast
envelope proteins24. In this way, the most hydrophobic proteins
from chloroplast envelope preparations could be enriched while
excluding more hydrophilic proteins, thereby reducing the complexity of the protein mixture. Using this method, a few integral
envelope membrane proteins were identified, and ~5–10% of the
total envelope protein content were estimated to be hydrophobic
proteins, representing at least 15–20 different proteins24.
Intriguing tube-like structures protruding out of the chloroplast
envelope were detected in chloroplasts and chlorophyll-free proplastids and plastids, using transgenic plants in which green fluorescent protein (GFP) was targeted into the chloroplast25,26. This
confirmed less detailed observations reported between 1960 and
the 1980s (Ref. 27). These dynamic structures were named stromules (for stroma filled tubules) and sometimes interconnected
different plastids with the surrounding nuclei and mitochondria.
Analysis of the proteome of these stromules could provide an
insight into their specific function.
Microscopic and radiolabeling studies have revealed that vesicles can form at the inner chloroplast envelope and are involved in
Box 2. Mass spectrometry for the analysis of proteins
and peptides
Mass spectrometry is the preferred method in the study of protein
identification. Mass spectrometers with ‘soft’ ionization techniques
allow the rapid identification of proteins provided that genomic or
cDNA sequence is available. Protein identification and characterization generally involves two types of mass spectrometers:
• Matrix-assisted laser desorption–ionization time-of-flight
(MALDI-TOF) mass spectrometers – accurately measure the
masses of a protein (mixture) or of proteolytic digests of gel
separated or otherwise purified proteins. A selected protein spot
is digested with a site-specific protease such as trypsin, resulting
in a set of peptides. The masses of the peptides are then measured by MALDI-TOF MS, resulting in a list of peptide
masses. For each entry in the nucleotide and protein databases,
the masses of the predicted tryptic peptides are calculated and
compared (within the experimental mass accuracy) with the
list of measured peptide masses using web-based search
engines (e.g. Protein Prospector, Mascot, Profound). The correct protein will have many ‘matching’ peptides. Proteins can be
identified even when they consist of a mixture of two or three
proteins. This method relies on the mass accuracy (5–15 ppm)
and sensitivity (femtomole range) of the latest generation of
MALDI-TOF MS instruments.
• Tandem mass spectrometer – often coupled to liquid chromatography, using electrospray ionization with a collision cell for
induced fragmentation (the technique is thus abbreviated to
ESI-MS/MS). When a protein cannot be positively identified
by MALDI-TOF MS, peptide sequence tags are obtained by
ESI-MS/MS. The individual peptides are ‘screened’ in the first
section of the tandem mass spectrometer and selected peptides
are subsequently further fragmented along the protein backbone
by collision with argon or nitrogen molecules. This is termed
collision-induced dissociation. These peptide fragments are
then separated by a second analyzer to provide amino acid
sequence information. The peptide sequence tags and measured ion masses are used to search for proteins in the database
using specialized software.
These two types of instruments together allow the rapid identification of
proteins, the accurate determination of partial amino acid sequences
and the elucidation of post-translational protein modifications.
lipid transfer from the inner envelope to the thylakoids15,17,28. The
concept of active vesicle formation within the chloroplast was further supported in other studies29,30, and such vesicles might contain their own subproteome.
A low-density chloroplast membrane fraction with an acyl lipid
composition similar to inner envelopes and thylakoid membranes
has been isolated from the green alga Chlamydomonas reinhardtii31. Several chloroplast mRNA binding proteins were found
to be strongly enriched, suggesting that these membranes could be
a site of chloroplast gene expression31. To elucidate the role of
these intriguing membranes in biogenesis, a more systematic
analysis of their proteins is urgently needed.
Chloroplast stroma
The chloroplast stroma is a compartment with a high protein content and many well known enzymes involved in carbon assimilation, as well as many biosynthetic pathways. Although best
known for their role in photosynthesis, chloroplasts synthesize
many essential compounds, such as plant hormones, fatty acids
and lipids, amino acids, vitamins (B1, K1 and E), purine and
pyrimidine nucleotides, and secondary metabolites such as
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alkaloids or isoprenoids. Moreover, chloroplasts are also required
for nitrogen and sulfur assimilation. In addition to these enzymes,
the stroma also contains the transcriptional and translation
machinery. No systematic analysis of the stromal proteome has
been presented in the literature.
Clearly, a large-scale protein analysis during chloroplast development, environmental changes and of (chloroplast) mutants
could reveal connections between the many biosynthetic pathways and provide insight into chloroplast functions and signaling.
However, it will be a challenge to resolve the proteins of lower
abundance, given the presence of several abundant proteins.
Removal of these dominant proteins (or protein complexes) by
affinity purification or other separation techniques will probably
be crucial to get a good overview of the stromal proteome.
Nucleoids and plastoglobules
The chloroplast genome is organized in so-called nucleoids,
which are associated with the inner envelope. Nucleoids contain
at least 15–20 proteins, which are poorly characterized19,32. Identification of these proteins might give us an insight into gene activation, genome organization and, possibly, DNA replication
during chloroplast division. Other interesting structures in the
chloroplast include the plastoglobules, which are lipid-containing
particles in the stroma that are thought to serve as lipid reservoirs
for thylakoid membranes. To date, two proteins (PG1 and fibrillins or PAP) have been identified33,34.
Thylakoid membrane system
The thylakoid membrane system contains four abundant multisubunit protein complexes (Photosystems I and II, the ATP synthase, and the cytochrome b6f complex). Together, they contain
~70 proteins and carry out the photosynthetic reactions. The thylakoid membrane might contain many other proteins that are
involved in the biogenesis and regulation of these complexes35,36.
This includes processes such as biosynthesis and ligation of cofactors, and the insertion, folding or degradation of proteins. Based
on preliminary information and postulated functions, at least 100
proteins (many of low abundance) are expected to be present.
Recently, we have initiated a systematic analysis of thylakoid
proteins from pea12 (Fig. 1). We constructed high-resolution 2-DE
maps of lumenal and peripheral proteins of the thylakoid membrane system purified from intact pea chloroplasts. After correction for possible isoforms and post-translational modifications, at
least 200–230 different lumenal and peripheral proteins were calculated to be present. 61 proteins were identified by mass spectrometry and Edman sequencing12, of which 33 had a clear
function or functional domain, whereas no function could be
assigned for ten proteins. For the other 18 proteins, no corresponding ESTs or full-length genes could be identified at the time
of publication, in spite of experimental determination of a significant amount of amino acid sequence. Ongoing genome sequencing is expected to identify these genes, and they can then be
analyzed for functional domains.
Although pea is a good model system for biochemical work,
there is one important drawback to using it for proteomics studies:
only a limited amount of DNA and protein sequences are available for pea, and protein identification must therefore be carried
out based on the homology of pea proteins to well sequenced plant
species, such as Arabidopsis thaliana. Homology-based identification with mass spectrometry data is possible12 but generally
requires a larger amount of experimentally determined protein
sequence tags or peptide mass fingerprints. Therefore, the use of
Arabidopsis, rice or other plant species that will be completely
sequenced is highly favorable.
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(a)
Isolation of chloroplasts
Purification of chloroplast subproteomes
pI
3.0
10.0
(b)
MW
(c)
Pick spots
in gel digestion
extraction of peptides
(d)
Identification by mass spectrometry
MALDI-TOF
ESI-MS/MS
Peptide masses
Sequence tags
(e)
Bioinformatics
(f)
Verification of identified proteins
– analysis of targeting signals
– comparison experimentally determined and
– predicted MW and pI
Fig. 1. Possible strategy for identifying chloroplast proteins using
two-dimensional gel electrophoresis, mass spectrometry and targeting analysis. (a) Purification of intact chloroplasts followed by
fractionation of different chloroplast compartments, such as thylakoids, stroma and envelope membranes. Further separation and
delipidation is required for the identification of membrane proteins.
(b) After isolation, chloroplast subproteomes are separated according to their isoelectric point (pI) and then according to their molecular weight (MW), resulting in a two-dimensional gel. The spots
are then visualized by Coomassie blue or silver staining. (c)
Individual protein spots are selected, excised from the gel and
digested with a site-specific protease, resulting in a set of peptides.
(d) Extracted peptides from each gel spot are measured by matrixassisted laser desorption–ionization time-of-flight (MALDI-TOF)
mass spectrometry. For further identification and characterization,
selected peptides are analyzed by electrospray tandem mass spectrometry (ESI-MS/MS), resulting in the generation of amino acid
sequence tags. (e) The set of peptide masses measured by MALDITOF mass spectrometry are compared with the masses of the predicted peptides for each entry in the sequence databases, possibly
identifying the protein. The sequence tags obtained by ESI-MS/MS
are used for further confirmation of the identified protein or to analyze post-translational modifications. (f) The identified proteins are
further analyzed for positive identification by analyzing targeting
information, such as chloroplast transit peptide or lumenal transit
peptide. In addition, the experimentally determined molecular
mass and isoelectric point are compared with the predicted values,
after removal of predicted (cleavable) transit peptides.
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The construction of high resolution 2-DE maps of Arabidopsis
thylakoids has therefore been initiated and is well under way, with
more than 1000 protein spots analyzed by matrix-assisted laser
desorption–ionization time-of-flight mass spectrometry (J.B.
Peltier et al., unpublished). Analysis of the integral thylakoid proteins is also in progress. These 2-DE maps can be used as reference maps to study post-translational modifications or the
expression of thylakoid proteins under varying environmental
conditions and during chloroplast development, or to evaluate the
effect of specific gene deletions. In addition, these newly identified proteins will provide a useful database to more rationally
design strategies aimed at understanding thylakoid biogenesis.
Prediction of the chloroplast proteome through analysis of
targeting signals
Attempts have been made to predict the size of the chloroplast
proteome based on location prediction programs and relationships
to cyanobacterial ancestors, and estimates vary from 1950 to 2500
(Refs 37,38). Several programs are available on the Internet to
predict the cellular location of a protein. For chloroplasts, two
programs [PSORT and ChloroP (Box 3)] have been available for
several years. Both programs were used on a set of identified
nuclear encoded thylakoid proteins to test the prediction for
chloroplast localization and the expected cleavage site by stromal
processing peptidase(s)12. Ninety four percent of this set was correctly predicted to be located in the chloroplast by ChloroP,
whereas PSORT predicted chloroplast localization for 52%.
Unfortunately, ChloroP also predicts a significant number of false
positives, which are mostly mitochondrial proteins: out of a test
set of 715 Arabidopsis entries in the SWISS-PROT database, 11%
were falsely predicted as chloroplast proteins39.
However, the program TargetP has recently become available
(Box 3). This is basically a retrained ChloroP integrated with a
new mitochondrial predictor and a retrained version of SignalP
(Box 3). TargetP is reported to be about three times better than
ChloroP at discriminating between chloroplast and mitochondrial
signal sequences and should therefore result in fewer false positives for chloroplast prediction40. Using N-terminal sequence
information, it discriminates between proteins destined for the
mitochondrion, the chloroplast, the secretory pathway and ‘other’
locations with a reported success rate of 85% on redundancyreduced test sets of mitochondrial, chloroplast, secretory, nuclear
and cytosolic plant proteins. However, these test sets excluded
other organelles such as vacuoles and peroxisomes.
In a TargetP analysis of the complete Arabidopsis chromosomes II and IV, the number of estimated chloroplast proteins
was 171 (2.2% of 7798 assigned genes; Ref. 40) if using only
the highest level of confidence (corresponding to no false positives on the test sets). Allowing lower levels of confidence (or
Box 3. Programs to predict the cellular location of
a protein
ChloroP, TargetP and SignalP
http://www.cbs.dtu.dk/services/
PSORT
http://psort.nibb.ac.jp/
Chloroplast and mitochondrial predictor
http://www.inra.fr/Internet/Produits/Predotar/
higher sensitivity) 10% of the proteins encoded by the assigned
genes on chromosomes II and IV were estimated to be located
in mitochondria and 14% to be located in the chloroplast.
Cleavage site prediction for identified chloroplast proteins was
40% correct when allowing an error of 62 amino acids. This
prediction can only be improved after generating more experimental data.
Thylakoid proteins destined for the lumen have an additional
transit peptide that shows strong similarities to bacterial signal
peptides21,41. In the aforementioned thylakoid proteomics study,
the lumenal transit peptides of a set of 26 non-redundant proteins
(16 known and 10 newly discovered) were compared by aligning
the sequences according to their experimentally determined
cleavage sites. As expected, the presequence and cleavage site had
similar features to signal peptides in bacteria. However, the lumenal transit peptides also showed a strong presence of prolines at
the end of the hydrophobic domain, a nearly complete conservation (25 out of 26) of the alanine at the 21 position and an abundance of glutamic acid at the 12 and 14 positions. The prediction
program SignalP, which was originally developed to predict
cleavable signal peptides of secretory proteins in bacteria and
eukaryotes, also predicted lumenal transit peptides and their
cleavage sites, but with mixed success12. However, if SignalP
could be adapted to lumenal signal peptides by using the specific
features of lumenal proteins as mentioned above, its prediction of
lumenal proteins might be significantly improved.
Recently, a new chloroplast and mitochondrial predictor has
been developed (Box 3). This program is still in development but
it is reported to be good at discriminating between proteins localized in the chloroplasts and mitochondria, and at recognizing proteins targeted to both organelles (I. Small, pers. commun.).
What does this mean for the use of these programs to identify
(or confirm) chloroplast proteins and their location within the
chloroplast? When the complete annotated genome of Arabidopsis or other higher plants becomes available, it is expected that
many chloroplast-localized proteins will be tentatively identified
based on presequence information. In particular, the lumenal proteins should be predicted with high confidence and sensitivity by
initial screening with TargetP set at a low confidence level (or
high sensitivity) followed by analysis with SignalP in Gram-negative mode. Recognition of outer envelope proteins based on primary sequence information is difficult or impossible because
most lack obvious chloroplast targeting signals42. Several tRNA
synthetases have been reported to be targeted to both the mitochondria and chloroplasts43, but they are not easily recognized by
prediction as chloroplast proteins. Several more dual targeted proteins are likely to be present.
Other chloroplast proteins that might escape positive prediction
are those targeted not via information in the N terminus but by
alternative mechanisms. This could include targeting via the
C terminus, as observed for plant vacuoles44 and yeast mitochondria45, or targeting via the so-called ‘hitch-hiker’ mechanism that
has been observed in bacteria46, or targeting as polyproteins47.
Predicting whether chloroplast membrane proteins are destined
for the thylakoid membrane or the (inner) envelope is currently
not possible because there are no obvious targeting signals. Thus,
the most confident location prediction within the chloroplast is
currently for the lumenal proteins.
It is important to realize that, for these programs to work correctly, the assignment of the start methionine needs to be correct.
Several cases were observed in which incorrect assignment
resulted in a negative chloroplast (incorrect) prediction12. In addition, chloroplast location prediction programs cannot determine
actual protein accumulation. Finally, as a word of caution, the
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currently available chloroplast predictors cannot identify chloroplast proteins of the unicellular alga C. reinhardtii, which is an
important model organism in the study of chloroplast biogenesis35,48.
Post-translational modifications, isoforms and mRNA editing
Characterization of the chloroplast proteome involves the identification not only of proteins but also of their post-translational modifications. Many post-translational modifications of chloroplast
proteins have been reported, including methylation (for RbcS)49,
carbamylation (for RbcS, RbcL)50, glycosylation (for CF)51 and
palmitoylation (for D1)52. However, no global analysis of posttranslational modifications has been carried out to date. With
the recent developments of mass spectrometry and 2-DE, such
analysis is now feasible and could lead to many interesting and
unexpected observations. For instance, systematic mapping of
phosphorylation of chloroplast proteins during light–dark transitions is likely to reveal unexpected kinetics and substrates, possibly leading to more insight into redox-driven signal transduction
chains and (de)activation mechanisms in the chloroplast.
Proteomics approaches can also provide a global overview of
splicing, mRNA editing and the expression of isoforms. Several
cases of mRNA editing53 and splicing54 have been described for
chloroplast proteins, and multigene families have been reported
in pea and spinach. These phenomena can, of course, also be
detected from a careful analysis of the mRNA (or cDNA), but proteomics is clearly a strong alternative or complementary way to
elucidate such events.
Functional proteomics of the chloroplast
Protein reference maps of subfractions of various organisms constructed by 2-DE are expected to become a central tool for organizing and understanding proteomes. A few web sites with small
organized 2-DE databases are already available (e.g. yeast,
E. coli, plant plasma membranes and chloroplasts; Box 4). Reference 2-DE maps will be used to follow differential protein expression and post-translational modifications. A combination of
proteomics with other large-scale techniques such as DNA
microarrays will be needed because they are expected to be highly
complementary (Box 1). DNA- and RNA-based technologies are
more sensitive tools, which can handle much larger numbers of
genes simultaneously. By contrast, proteomics determines the
presence of the gene product directly (rather than at the mRNA
level) and can also monitor post-translational modifications,
including proteolysis.
Only after the experimental determination of further chloroplast proteins and the cleavage site of their targeting signals can
the prediction of chloroplast localization and cleavage sites be
improved. To identify the functional role of newly discovered
proteins, additional approaches (e.g. reverse genetics) will be
needed in combination with proteomics. Systematic analysis of
protein–protein interactions using non-denaturing biochemical
Box 4. 2-DE databases
Yeast, E. coli and others
http://www.expasy.ch/ch2d/
Plant plasma membranes
http://sphinx.rug.ac.be:8080/ppmdb/index.html
Chloroplasts
http://www.biokemi.su.se/chloroplast/
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October 2000, Vol. 5, No. 10
techniques (such as affinity purification and gel filtration) followed by mass spectrometry or yeast two-hybrid systems will be
essential to unravel biochemical functions at a molecular level.
Proteomics studies on chloroplast biogenesis mutants obtained by
phenotypic screening, chlorophyll fluorescence screens55, other
phenotypic criteria or reverse genetics will help to pinpoint the
function of the gene products involved in chloroplast biogenesis
and function.
Acknowledgements
I gratefully acknowledge members of my laboratory and Olof
Emanuelsson for numerous stimulating discussions and critically
reading the manuscript. My funding was provided by the Swedish
National Research Council (NFR), the Swedish Agricultural
Research Council (SJFR), the Swedish Strategic Funds (SSF) and
the Carl Trygger Foundation.
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Klaas Jan van Wijk is at the Dept of Biochemistry, Arrhenius
Laboratories, Stockholm University, S-10691 Stockholm, Sweden
(tel 146 8 162420; fax 146 8 153679; e-mail [email protected]).
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