Update on Chloroplast Research: New Tools, New Topics, and New Trends
Ute Armbruster
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Paolo Pesaresi
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Mathias Pribil
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Alexander Hertle
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Dario Leister
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a Lehrstuhl fu r Molekularbiologie der Pflanzen (Botanik), Department Biologie I, Ludwig-Maximilians-Universita t Mu nchen
,
Grohaderner Str. 2, D-82152 Planegg-Martinsried
,
Germany b Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` degli studi di Milano
,
I-20133 Milano
,
Italy
Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional characterization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowledge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire complement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants.
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INTRODUCTION
The chloroplast (cp), the characteristic organelle of plants and
green algae, harbors its own tiny genome and is responsible
for various essential functions, including photosynthesis, lipid
metabolism, starch and amino acid biosynthesis (Finkemeier
and Leister, 2010). Chloroplasts are descended from an ancient
cyanobacterial endosymbiont and many of its functions have
been conserved. However, most of the genes it brought with it
have been transferred to the host nucleus during the
subsequent evolution of the organelle (Timmis et al., 2004).
Early functional studies of chloroplasts depended largely on
the use of biochemical and biophysical approaches. During the
1980s and 1990s, methods were developed for transforming
chloroplasts by homologous recombination and for
systematically disrupting nuclear genes by inserting transposons or
T-DNAs. These advances markedly enhanced the utility of
genetic approaches to the study of cp function. With the
sequencing of entire genomes and the establishment of
high-throughput tools for the analysis of their expression,
cp research also entered the era of genomics (Leister, 2003).
Functional genomicsthe analysis of transcriptomes,
proteomes, and metabolomesopens immense possibilities for
the elucidation of cp functions, serving both to characterize
available mutants and to identify candidate loci for targeted
mutagenesis.
At present, the green alga Chlamydomonas reinhardtii and
the flowering plants Zea mays and Arabidopsis thaliana serve
as the main workhorses in cp research. In this review, we focus
on the impact of novel technologies and discuss some selected
highlights and emerging trends in cp research, particularly in
A. thaliana.
Forward and Reverse Genetics
Forward geneticsthe isolation of mutants with specific
phenotypes followed by the identification and analysis of the
relevant geneshas long been the method of choice for
identifying novel components that underlie plant functions of
interest. Forward genetics is still an important tool in cp
research, as evidenced by recent classical primary and suppressor
mutant screens (Table 1), as well as screens based on the
altered activity of a reporter gene in a wild-type or mutated
genetic background (e.g. Baruah et al., 2009).
However, sequencing of the complete genomes of several
photosynthetic organisms, generation of large collections of
insertion mutants in A. thaliana (T-DNA insertion mutants)
and Z. mays (endogenous transposons), and the advent of
Name Mutant phenotype Molecular function(s) Reference(s)
Classical forward genetics
Low PSII Accumulation (LPA) Reduced PSII
accumulation
Non- Photochemical
Quenching (NPQ)/Proton
Gradient Regulation (PGR)
Chlororespiratory Reduction No NDH activity
(CRR)
High Chlorophyll
Fluorescence (HCF)
High level of Chl a
fluorescence
LPA1# D1 membrane integration
LPA2 Efficient PSII assembly
LPA3 Efficient PSII assembly
LPA19 D1 precursor processing
LPA66* psbF editing
NPQ1/VPE Xanthophyll cycle
NPQ2/ABA1/ZEP Xanthophyll cycle
NPQ4/PSBS Subunit of PSII
PGR1/PETC Subunit of Cyt b6/f complex
PGR3* cp gene expression
PGR5 Cyclic electron flow around PSI
CRR1 NDH assembly or stability
CRR2* Expression of ndhB
CRR3 Subunit of NDH complex
CRR4* Site recognition factor in ndhD
editing
CRR6 NDH assembly
CRR7 NDH assembly
CRR23 L subunit of NDH
HCF101 [4Fe4S] cluster assembly
Lennartz et al., 2001; Motohashi
and Hisabori, 2006
Suppressor screens
Executer/Singlet
Oxygen
Linked Death Activator (SOLDAT)
# TPR protein.
* PPR protein.
Suppression of singlet EXECUTER1
oxygen-mediated EXECUTER2
rmeusptaonntsses in flu SOLDAT8
Unknown SIGMA6 factor of the plastid encoded RNA polymerase Plastid gene expression
Meskauskiene et al., 2009
RNA interferen (...truncated)