Chloroplasts are the organelles that define plants, and they are responsible for photosynthesis as well as numerous other functions. They are the ancestral members of a family of organelles known as plastids. Plastids are remarkably dynamic, existing in strikingly different forms that interconvert in response to developmental or environmental cues. The genetic system of this organelle and its coordination with the nucleocytosolic system, the import and routing of nucleus-encoded proteins, as well as organellar division all contribute to the biogenesis and homeostasis of plastids. They are controlled by the ubiquitin-proteasome system, which is part of a network of regulatory mechanisms that integrate plastid development into broader programmes of cellular and organismal development.

Elaborate mechanisms have evolved for the translocation of nucleus-encoded proteins across the plastid envelope membrane. Although putative components of the import apparatus have been identified biochemically, their role in import remains to be proven in vivo. An Arabidopsis mutant lacking a new component of the import machinery [translocon at the outer envelope membrane of chloroplasts (Toc33), a 33-kilodalton protein] has been isolated. The functional similarity of Toc33 to another translocon component (Toc34) implies that multiple different translocon complexes are present in plastids. Processes that are mediated by Toc33 operate during the early stages of plastid and leaf development. The data demonstrate the in vivo role of a translocon component in plastid protein import.

Development of chloroplasts and other plastids depends on the import of thousands of nucleus-encoded proteins from the cytosol. Import is initiated by TOC (translocon at the outer envelope of chloroplasts) complexes in the plastid outer membrane that incorporate multiple, client-specific receptors. Modulation of import is thought to control the plastid's proteome, developmental fate, and functions. Using forward genetics, we identified Arabidopsis SP1, which encodes a RING-type ubiquitin E3 ligase of the chloroplast outer membrane. The SP1 protein associated with TOC complexes and mediated ubiquitination of TOC components, promoting their degradation. Mutant sp1 plants performed developmental transitions that involve plastid proteome changes inefficiently, indicating a requirement for reorganization of the TOC machinery. Thus, the ubiquitin-proteasome system acts on plastids to control their development.

ReviewB l a c k w e l l P u b l i s h i n g L t d O x f o r d , U K N P H N e w P h y t o l o g i s t 0 0 2 8 -6 4 6 X 1 4 6 9 -8 1 3 7 © T h e A u t h o r s ( 2 0 0 8 ) . J o u r n a l c o m p i l a t i o n © N e w P h y t o l o g i s t ( 2 0 0 8 ) 10.1111/j. 1469-8137.2008 SummaryMost chloroplast proteins are encoded in the nucleus and synthesized on free, cytosolic ribosomes in precursor form. Each precursor has an amino-terminal extension called a transit peptide, which directs the protein through a post-translational targeting pathway and is removed upon arrival inside the organelle. This 'protein import' process is mediated by the coordinate action of two multiprotein complexes, one in each of the envelope membranes: the TOC and TIC (Translocon at the Outer/ Inner envelope membrane of Chloroplasts) machines. Many components of these complexes have been identified biochemically in pea; these include transit peptide receptors, channel proteins, and molecular chaperones. Intriguingly, the Arabidopsis genome encodes multiple, homologous genes for receptor components of the TOC complex. Careful analysis indicated that the different receptor isoforms operate in different import pathways with distinct precursor recognition specificities. These 'substrate-specific' import pathways might play a role in the differentiation of different plastid types, and/or act to prevent deleterious competition effects between abundant and nonabundant precursors. Until recently, all proteins destined for internal chloroplast compartments were thought to possess a cleavable transit peptide, and to engage the TOC/TIC machinery. New studies using proteomics and other approaches have revealed that this is far from true. Remarkably, a significant number of chloroplast proteins are transported via a pathway that involves the endoplasmic Plastids are a diverse group of organelles found ubiquitously in plant cells (Whatley, 1978), as well as in apicomplexan parasites (Foth & McFadden, 2003). The most prominent and wellstudied members of this plastid family are the chloroplasts. These contain the green pigment chlorophyll and carry out the light-driven carbon fixation reactions of photosynthesis, as well as important steps in the biosynthesis of many essential primary and secondary metabolites (Nelson & Ben-Shem, 2004;Lopez-Juez & Pyke, 2005). Other plastid family members are the amyloplasts, which contain large amounts of starch and play important roles in energy storage and gravitropism, and the chromoplasts, which accumulate red, orange or yellow carotenoid pigments and act as attractants in flowers and fruits (Neuhaus & Emes, 2000;Lopez-Juez & Pyke, 2005). Like mitochondria, plastids entered the eukaryotic lineage through endosymbiosis. It is thought that they evolved monophyletically from an ancient photosynthetic prokaryote similar to extant cyanobacteria (Larkum et al., 2007; ReyesPrieto et al., 2007). While plastids retain a functional genetic system of their own, the organellar genome is greatly reduced and typically encodes just ...

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

A multisubunit translocon of the inner envelope membrane, termed Tic, mediates the late stages of protein import into chloroplasts. Membrane proteins, Tic110 and Tic40, and a stromal chaperone, Hsp93, have been proposed to function together within the Tic complex. In Arabidopsis, single genes, atTIC110 and atTIC40, encode the Tic proteins, and two homologous genes, atHSP93-V and atHSP93-III, encode Hsp93. These four genes exhibited relatively uniform patterns of expression, suggesting important roles for plastid biogenesis throughout development and in all tissues. To investigate the roles played by these proteins in vivo, we conducted a comparative study of T-DNA knockout mutants for each Tic gene, and for the most abundantly expressed Hsp93 gene, atHSP93-V. In the homozygous state, the tic110 mutation caused embryo lethality, implying an essential role for atTic110 during plastid biogenesis. Homozygous tic110 embryos exhibited retarded growth, developmental arrest at the globular stage and a 'raspberry-like' embryo-proper phenotype. Heterozygous tic110 plants, and plants homozygous for the tic40 and hsp93-V mutations, exhibited chlorosis, aberrant chloroplast biogenesis, and inefficient chloroplast-import of both photosynthetic and non-photosynthetic preproteins. Non-additive interactions amongst the mutations occurred in double mutants, suggesting that the three components may cooperate during chloroplast protein import.

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34 . Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1 . Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1 . Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

The lipid monogalactosyl diacylglycerol (MGD) is a major structural component of photosynthetic membranes in chloroplasts. Its formation is catalyzed by the enzyme MGD synthase. In many plants, MGD derives from two different biosynthetic pathways: the prokaryotic pathway, which operates entirely within the plastid, and the eukaryotic pathway, which involves steps in the endoplasmic reticulum. Here, we describe the identification and characterization of an Arabidopsis mutant with a defective MGD synthase gene (MGD1). The mutant was identified in a screen of T-DNA lines for individuals with defects in chloroplast biogenesis. It has a yellowgreen phenotype that correlates with a Ϸ50% deficiency in total chlorophyll per plant. A single T-DNA insertion is located adjacent to the transcription initiation site of the MGD1 gene, and the abundance of MGD1 mRNA is reduced by 75% compared with wild type. Correlation between steady-state MGD1 transcript levels and MGD synthase activity (also reduced by 75% in mgd1) suggests that MGD1 is the most important MGD synthase in green tissues. The amount of MGD in mutant leaves is reduced by 42% compared with wild type. MGD from the mutant contains 23% less 16:3 fatty acid and 10% more 18:3 fatty acid. Because 16:3 is a characteristic feature of MGD from the prokaryotic pathway, it is possible that MGD1 operates with some preference in the prokaryotic pathway. Finally, the MGD-deficiency of mgd1 is correlated with striking defects in chloroplast ultrastructure, strongly suggesting a unique role for MGD in the structural organization of plastidic membranes.G alactose-containing lipids are the predominant nonproteinaceous components of photosynthetic membranes in plants, algae, and a variety of bacteria. The two most common galactolipids are monogalactosyl diacylglycerol (MGD) and digalactosyl diacylglycerol (DGD). In plants, MGD and DGD occur exclusively in plastidic membranes, where they account for about 50 and 20 mol% of the lipid matrix, respectively (1). Up to 80% of all lipids in plants are associated with photosynthetic membranes, and MGD is widely considered to be the most abundant membrane lipid on earth. Most vegetables and fruits in human and animal diets are rich in galactolipids, and their breakdown products represent an important dietary source of galactose and polyunsaturated fatty acids (2, 3).Galactolipids play an important role in the organization of photosynthetic membranes. The abundance and physical properties of MGD make it particularly important in this respect. Its small galactose head group and large unsaturated fatty acid chains give it a cone-like molecular shape and a consequent predisposition to form nonlamellar, hexagonal-phase aggregates (4). The molecular shape of MGD may be important for the structural organization of thylakoid membranes. Because much of the photosynthetic apparatus is embedded within thylakoids, the lipids that make up these membranes are of profound importance. Evidence also suggests that MGD is more directly involved in certain ph...