Update on Chloroplast Research: New Tools, New Topics, and New Trends

Molecular Plant, Jan 2011

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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Update on Chloroplast Research: New Tools, New Topics, and New Trends

Ute Armbruster 0 Paolo Pesaresi 0 Mathias Pribil 0 Alexander Hertle 0 Dario Leister 0 0 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. - 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)


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Ute Armbruster, Paolo Pesaresi, Mathias Pribil, Alexander Hertle, Dario Leister. Update on Chloroplast Research: New Tools, New Topics, and New Trends, Molecular Plant, 2011, pp. 1-16, 4/1, DOI: 10.1093/mp/ssq060