Abstract: EMBO Member's Review10 September 2009Open Access The making of a chloroplast Mark T Waters Mark T Waters Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK Search for more papers by this author Jane A Langdale Corresponding Author Jane A Langdale Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK Search for more papers by this author Mark T Waters Mark T Waters Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK Search for more papers by this author Jane A Langdale Corresponding Author Jane A Langdale Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK Search for more papers by this author Author Information Mark T Waters1 and Jane A Langdale 1 1Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK *Corresponding author. Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK. Tel.: +44 1865 275099; Fax: +44 1865 275074; E-mail: [email protected] The EMBO Journal (2009)28:2861-2873https://doi.org/10.1038/emboj.2009.264 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Since its endosymbiotic beginning, the chloroplast has become fully integrated into the biology of the host eukaryotic cell. The exchange of genetic information from the chloroplast to the nucleus has resulted in considerable co-ordination in the activities of these two organelles during all stages of plant development. Here, we give an overview of the mechanisms of light perception and the subsequent regulation of nuclear gene expression in the model plant Arabidopsis thaliana, and we cover the main events that take place when proplastids differentiate into chloroplasts. We also consider recent findings regarding signalling networks between the chloroplast and the nucleus during seedling development, and how these signals are modulated by light. In addition, we discuss the mechanisms through which chloroplasts develop in different cell types, namely cotyledons and the dimorphic chloroplasts of the C4 plant maize. Finally, we discuss recent data that suggest the specific regulation of the light-dependent phases of photosynthesis, providing a means to optimize photosynthesis to varying light regimes. Introduction As a defining feature of plants, the chloroplast represents a marvel of evolution. Since its origin as a cyanobacterial symbiont about 1 to 1.5 billion years ago (Douzery et al, 2004; Yoon et al, 2004), this organelle has become fully integrated into the life cycle of photosynthetic eukaryotes and has essentially underpinned global ecosystems. Photosynthesis comprises two conceptually distinct phases that occur entirely within the chloroplast. The light-dependent reactions take place on the thylakoid membrane, in which light energy drives electron transport between a series of multi-subunit protein complexes. In two of these complexes, photosystem I (PSI) and photosystem II (PSII), protein-bound chlorophyll pigments are excited by light and initiate electron flow, so generating ATP and reducing equivalents. This chemical energy is then used in the light-independent reactions that take place in the chloroplast stroma, in which CO2 is fixed by Rubisco to generate sugars. Subsequently, this carbohydrate is either immediately exported to the cytosol or is stored within the chloroplast as starch. Beyond photosynthesis, the chloroplast is also the site of fatty acid biosynthesis, nitrate assimilation and amino-acid biosynthesis. Given the importance of plant products to human beings, photosynthetic development and the biogenesis of chloroplasts have received intense scrutiny. In seed plants, chloroplasts develop from a non-photosynthetic form called the proplastid, which is transmitted between generations through the ovule and is maintained in meristematic stem cells. How does a chloroplast develop from a proplastid? How is photosynthetic competence reached and sustained? These are certainly complex and open questions, but two central themes emerge. First, the co-ordination and integration of multiple parallel processes, none of which operates in isolation, are absolutely necessary. This theme is most clearly shown by the fact that mutations in single chloroplast components can have major ramifications beyond the immediate process in question. Second, constant interorganellar crosstalk occurs both during the initial construction of the chloroplast and to maintain form and function in mature tissues. Coupled with the need to respond to a constantly variable environment, this crosstalk reflects the existence of two genomes and the need to regulate dynamically the relative input from each towards constituent parts of the chloroplast. This review covers some of the major cellular and developmental aspects of chloroplast biogenesis that encompass the above themes. Light signalling during photomorphogenesis In seed plants, light is a prerequisite for the synthesis of chlorophyll, and chloroplasts do not develop in the dark. Photomorphogenesis describes the developmental programme undertaken by seedlings exposed to light, and is typified by the inhibition of hypocotyl growth, the development of chloroplasts and the opening of cotyledons (in eudicotyledonous species). Light is perceived by a suite of wavelength-specific photoreceptor proteins that undergo conformational changes to allow interaction with downstream signalling partners. The phytochromes, which perceive red and far-red light, and the cryptochromes, which respond to blue and UVA light, are the two varieties of photoreceptor responsible for photomorphogenesis (Jiao et al, 2007). In Arabidopsis, there are five phytochromes (encoded by PHYA to PHYE), of which primarily phyA and phyB act during seedling photomorphogenesis (Quail, 2002; Tepperman et al, 2006). Phytochromes exist in the cytosol in an inactive Pr form that is activated by light and is converted into the biologically active Pfr form, which translocates into the nucleus to initiate signalling (Quail, 2002). The cryptochromes are represented by three proteins: cry1, which translocates from the nucleus to the cytosol on light activation; cry2, which is constitutively nuclear localized and cry3, which seems to be dual targeted to mitochondria and plastids (Kleine et al, 2003; Lin and Shalitin, 2003). A great deal of effort has been invested in clarifying the signalling and transcriptional networks that follow the perception of light, and the field has recently been reviewed thoroughly elsewhere (Jiao et al, 2007). Here, we offer a brief overview of the light signalling pathways that lead to the biogenesis of chloroplasts to provide a basis for introducing recent findings regarding signalling mechanisms. A series of genetic screens to uncover regulators of light-dependent development revealed a class of loci that, when mutated, confer a partially constitutively photomorphogenic (cop) or de-etiolated (det) phenotype (reviewed by von Arnim and Deng, 1996). Collectively, these mutants define the COP/DET/FUS class of loci. When grown in the dark, these mutants resemble light-grown seedlings in many respects, typically with a short hypocotyl, open, expanded cotyledons and enhanced levels of photosynthetic gene expression. They do not show complete chloroplast development in the dark, because chlorophyll synthesis requires light, and photosystems cannot assemble without chlorophyll; however, plastids in dark-grown cop1 and cop9 seedlings, for example, contain a partially formed thylakoid network instead of normal etioplasts (see below) (Deng and Quail, 1992; Wei and Deng, 1992). Furthermore, cop1 and det1 are hyper-responsive to light, exhibiting ectopic chloroplast development in the roots (Chory and Peto, 1990; Deng and Quail, 1992). The recessive nature of these mutants suggests that the COP/DET/FUS proteins are light-inactivatable repressors of photomorphogenesis in the dark, and that they also have a function in suppressing chloroplast development in non-photosynthetic tissues. It is now known that several of these loci encode subunits of the COP9 signalosome (CSN), a nuclear-localized protein complex that functions as part of the ubiquitin-proteosome pathway, which regulates E3 ubiquitin ligases (Wei et al, 2008). COP1 encodes such a ligase (Seo et al, 2003). COP1 activity is regulated in part at the level of nucleo-cytoplasmic partitioning: in the dark, it is preferentially localized to the nucleus, and it transfers to the cytoplasm in the light (von Arnim and Deng, 1994). COP1 functions together with three other components, COP10, DET1 and DNA damage-binding protein 1B (DDB1), to target specific proteins such as HY5 for proteasomal destruction by the CSN (Osterlund et al, 2000; Yanagawa et al, 2004). HY5 is a positive regulator of photomorphogenesis under a broad spectrum of light, suggesting that it acts downstream of phyA, phyB and the cryptochromes (Chory, 1992). It encodes a bZIP transcription factor that binds to a conserved G-box motif, CACGTG, in the promoters of many light-regulated genes including those related to photosynthesis (Oyama et al, 1997; Lee et al, 2007). HY5-binding targets account for some 60% of those genes regulated by phytochromes within 1 h of light exposure (Lee et al, 2007), suggesting that HY5 acts high up in the hierarchy of photomorphogenic regulation. Both phy- and cry-dependent signalling lead to an increase in HY5 levels (Osterlund et al, 2000), and while the phy-dependent mechanism for this observation is not fully understood, photo-activated cry1 inhibits COP1 in the nucleus, thus preventing HY5 degradation; this may be brought about through the translocation of COP1 into the cytoplasm (Figure 1) (Yang et al, 2001). Thus, cryptochrome-mediated chloroplast development is at least partly mediated through HY5. Phytochrome signalling, meanwhile, makes extensive use of a basic helix-loop-helix family of transcription factors called phytochrome-interacting factors or PIFs (Castillon et al, 2007). PIFs control distinct but overlapping sets of responses—again by binding to the G-box motif—and are mainly considered to be negative regulators of photomorphogenesis that act by blocking transcription (Castillon et al, 2007). The founding member of the PIF family, PIF3, has been characterized in some detail. On light exposure, phyB moves into the nucleus and binds to PIF3, triggering its phosphorylation and rendering it susceptible to degradation; however, this degradation is not mediated by COP1 (Bauer et al, 2004). Transcription from photomorphogenesis-related genes is then able to proceed (Figure 1). Recent evidence has shown that, in the dark, PIF3 negatively regulates the expression of HEMA1 and GUN5, genes encoding two key regulatory enzymes in the chlorophyll biosynthetic pathway, and of LHCA1 and PsaE1, two genes encoding PSI components (Shin et al, 2009). Consistent with this, dark-grown pif3 mutants accumulate double the wild-type level of protochlorophyllide (Pchlide), a late chlorophyll intermediate, in the dark (Shin et al, 2009). PIF1 has also recently been shown to control chlorophyll biosynthesis, partly through direct interaction with the promoter of PORC, a gene that encodes Pchlide oxidoreductase (Moon et al, 2008). Significantly, pif1 pif3 pif4 pif5 quadruple mutants are constitutively photomorphogenic with short hypocotyls and open cotyledons (Shin et al, 2009), revealing that this family of transcription factors strongly represses a suite of photomorphogenic attributes, especially chloroplast development. Figure 1.A simplified model of light signalling during photomorphogenesis. (A) In darkness, phytochrome dimers are in the inactive Pr state in the cytoplasm, and inactive CRY1 dimers are bound to COP1 in the nucleus. CSN, COP1 and the COP10/DET1/ DDB1 (CDD) complexes co-operate to promote the ubiquitination of photomorphogenesis-promoting transcription factors such as HY5. The CSN stabilizes the CDD complex and may regulate the activity of COP1. HY5 interacts with the WD40 repeat domain of COP1 and is ubiquitinated by the ubiquitin E3 ligase activity of COP1. Polyubiquitinated HY5 is subsequently degraded, presumably by the 26S proteasome. HY5 is mostly phosphorylated in the dark, a form that interacts poorly with target promoters; in addition, COP1 preferentially interacts with the unphosphorylated form of HY5, further suppressing levels of biologically active HY5 (Hardtke et al, 2000). In parallel, PIF3 is bound to G-box sequences in target promoters, inhibiting transcription of photomorphogenesis-related genes. (B) Blue light exposure triggers the photoactivation of CRY1, which leads to the exit of COP1 from the nucleus and thus allows HY5 levels to increase. HY5 is dephosphorylated, increasing its biological activity and further reducing its affinity for COP1; more HY5 is then available to bind to G-box motifs and promote transcription of genes such as light-harvesting chlorophyll-binding1 (Lhcb1/CAB1), a major antenna protein of PSII. Note that HY5 can also negatively regulate transcription of target genes and is necessary, but insufficient to regulate transcription alone (Lee et al, 2007). Meanwhile, Pr is converted into the biologically active Pfr form by red light, which translocates into the nucleus and binds PIFs (such as PIF3). Phy-bound PIF3 is phosphorylated, rendering it susceptible to ubiquitination and subsequent degradation. As a result, transcription of genes such as those involved in chlorophyll biosynthesis can proceed. Phy-dependent repression of COP/DET/FUS proteins (revealed by epistasis) is depicted by a dashed arrow. Note that PIF3-regulated genes are not necessarily HY5 regulated, even though both transcription factors bind DNA through the G-box. In addition, there is some evidence that phyB may interact with COP1 (Yang et al, 2001). For abbreviations, see text. Download figure Download PowerPoint Transition from proplastid to chloroplast Once the seedling has become photoautotrophic, the next key stage in photomorphogenesis is activation of the shoot apical meristem (SAM) to produce leaves and chloroplasts therein. The hy1 mutant is unable to synthesize phytochromobilin, the chromophore of phytochromes, and, therefore, lacks all phytochrome activity (Muramoto et al, 1999). Triple hy1 cry1 cry2 mutants are highly defective in the release of SAM arrest, showing that phytochromes and cryptochromes act redundantly to initiate leaf production after emergence from the dark (Lopez-Juez et al, 2008). A careful transcriptome analysis of the SAM immediately after light exposure has revealed that the release of SAM arrest is accompanied by the upregulation of cytokinin and giberellin responses, and the expression of genes involved in ribosome production, protein translation and cell proliferation before visible leaf emergence (Lopez-Juez et al, 2008). Genes involved in chloroplast biogenesis—primarily photosynthesis genes—are expressed subsequently, 6 to 24 h after light exposure (Lopez-Juez et al, 2008). Within the leaf primordium, phytochromes and cryptochromes bring about a myriad of changes that initiate chloroplast biogenesis, and a series of subsequent molecular events must occur in parallel to complete the process successfully. Obvious activities include the import of nuclear-encoded proteins, the ramping up of chlorophyll levels and the establishment of a thylakoid network complete with photosynthetic electron transport (PET) complexes. Table I and Figure 2 summarize the main functional processes that occur in making a chloroplast, along with examples of chloroplast components that perform those processes. Below, we discuss some aspects of this process in more detail. Figure 2.Early events during the transition from proplastid to chloroplast. (1) Import of nuclear-encoded proteins through the Tic/Toc complex. Stromal proteins fold directly in the stroma with the assistance of chaperone proteins. Some thylakoid-targeted proteins, such as Lhc, are recognized by the stromal chloroplast signal recognition particle (cpSRP43/54), which mediates insertion of the protein into the inner envelope (IE) membrane (Amin et al, 1999; Klimyuk et al, 1999). Complete insertion of Lhc requires the membrane-resident protein ALB3 (Bellafiore et al, 2002), and the binding of chlorophyll and carotenoids that are synthesized on the IE membrane. Note that the targeting of proteins to the thylakoid membrane is highly simplified here; the cpSRP- and ALB3-dependent route is only true for certain thylakoid-resident proteins such as Lhc, which may also insert directly into the thylakoid network, bypassing the IE membrane. (2) The thylakoid network is generated from Lhc/chlorophyll-laden vesicles derived from the IE membrane in a budding process dependent on factors such as VIPP1. GTPases such as FZL may perform further remodelling of thylakoid membranes into a reticulate network. (3) Concurrently, light activates PGE through nuclear-encoded sigma factors (σ), resulting in the synthesis of core proteins of the photosystem reaction centres, such as PsbD. Extensive additional regulation takes place at the levels of RNA processing and ribosome assembly. (4) Assembly of the photosystems and other electron transport components leads to further elaboration of the thylakoid network, forming stacked regions (grana) and unstacked stromal lamellae. (5) PDV involves the assembly of an inner PDV ring, consisting of FtsZ proteins, and an outer PDV ring that is partly comprised of DRP5B, which is recruited and anchored to the outer envelope membrane by the PDV proteins. The division rings form around the middle of the chloroplast, yielding two chloroplasts through binary fission. Download figure Download PowerPoint Table 1. Examples of nuclear-encoded, chloroplast-localized components necessary for chloroplast biogenesis, grouped by functional class Protein Molecular function Mutant phenotypea Remarks Reference Protein import and suborganellar targeting AtTOC33 Protein translocation across outer envelope Pale green, especially juvenile plants (ppi1) Involved in import of photosynthetic proteins Kubis et al (2003) cpSRP43 Subunit of stromal signal recognition particle Pale green with reduced levels of thylakoid protein complexes (chaos) Mediates insertion of proteins into thylakoid membrane Klimyuk et al (1999), Amin et al (1999) RNA processing PPR4 Splicing of plastid rps12 transcript Embryo lethal (ppr4) PPR family member required for plastid ribosome biogenesis Schmitz-Linneweber et al (2006) CRR2 PPR-like protein; regulates RNA splicing between rps7 and ndhB transcripts Impaired accumulation of NDH complex (crr2-1 and crr2-2) NDH complex is involved in cyclic electron flow around PSI Hashimoto et al (2003) SVR1 Pseudouridine synthase, RNA editing Yellow-green; reduced stature (svr1-2) svr1 is also a suppressor of var2 Yu et al (2008) Protein maturation and degradation BSD2 DnaJ-like protein chaperone Pale green due to abnormal BS cell chloroplasts (Zea mays) Required for post- transcriptional regulation of Rubisco large subunit (LSU) Brutnell et al (1999) FtsH2 (VAR2) ATP-dependent metalloprotease Variegated yellow-green leaves; cotyledons normal (var2) Likely function in D1 protein turnover in photodamaged PSII Chen et al (2000), Lindahl et al (2000) ClpP6 Stromal ATP-dependent Clp protease RNAi lines exhibit chlorosis of younger leaves Degrades a variety of stromal proteins Sjögren et al (2006) Plastid gene expression SIG6 Sigma factor conferring promoter specificity to RNA polymerase Delayed greening in cotyledons (sig6-1) One of many sigma factors required for plastid gene transcription Ishizaki et al (2005) FUG1 Plastid translation initiation factor fug1-2 is embryo lethal fug1 alleles suppress var2 Miura et al (2007) Thylakoid biogenesis and lipid biosynthesis AtTerC Unknown; required for early thylakoid biogenesis Seedling lethal on light exposure Similar to bacterial tellurite resistance proteins Kwon and Cho (2008) FZL Dynamin-like GTPase; membrane fusion Pale green; disorganized granal thylakoids May be involved in thylakoid remodelling Gao et al (2006) MGDG synthase Catalyses final step in MGDG biosynthesis Sucrose required for germination; albino; frequent inner envelope invaginations Mutant phenotype supports budding hypothesis for thylakoid biogenesis Kobayashi et al (2007) VIPP1 Possible function in membrane budding from inner chloroplast envelope Viable with exogenous sucrose Protein located on inner envelope and thylakoid membrane Kroll et al (2001), Aseeva et al (2007) Chlorophyll biosynthesis GUN4 Enhances Mg-cheletase activity Pale green (gun4-1, weak); yellow-white (gun4-2, null) Essential under normal growth conditions Larkin et al (2003) CHLM Mg-protoporphyrin methyltransferase chlm null mutants are albino and lack thylakoid protein complexes Essential under normal growth conditions Pontier et al (2007) Metabolite transport CUE1 (AtPPT1) Imports phosphoenolpyruvate (PEP) into chloroplast stroma Reticulate pale green leaves with dark green BS cells; perturbed M cell differentiation PEP is required for fatty acid, amino acid and isoprenoid biosynthesis through the shikimate pathway Li et al (1995), Streatfield et al (1999) Photosystem assembly LPA2 Required for stability/ assembly of PSII core Pale green (lpa2); reduced PSII levels Intrinsic thylakoid protein Ma et al (2007) PPR, pentatricopeptide repeat protein; NDH, nicotinamide dinucleotide (phosphate) dehydrogenase; MGDG, monogalactosyldiacylglycerol, a non-phosphorous glycolipid of thylakoid membranes. a Arabidopsis unless otherwise specified. Protein import The biogenesis of chloroplasts requires substantial protein import from the cytosol. Most chloroplast proteins are imported through the Toc/Tic complex, which both recognizes and transports nascent proteins across both envelope membranes (for a review, see Soll and Schleiff, 2004). Major components of the Toc/Tic complex are upregulated by light and even provide substrate specificity. For example, the Arabidopsis Toc33 knockout mutant, ppi1, is defective in the import and accumulation of photosynthetic proteins, but not of most non-photosynthetic proteins, and AtTOC33 is most strongly expressed in young, light-grown seedlings (Kubis et al, 2003). Toc159, a GTP-dependent molecular motor that drives translocation, is also required for precursor protein recognition. The Toc159 subunits are encoded by four genes in Arabidopsis: AtTOC159, AtTOC132, AtTOC120 and AtTOC90. The atToc159 mutant is albino and does not survive past the cotyledon stage, implying that the other Toc159 family members cannot compensate for this defect (Bauer et al, 2000). Furthermore, overexpression of AtTOC159 is unable to complement the pale green atToc132 atToc120 phenotype (Kubis et al, 2004). Together, these findings imply that each Toc159 isoform exhibits substrate selectivity. Expression of such different isoforms may provide an efficient strategy for enhancing the rate of photosynthetic protein import over that of non-photosynthetic proteins during early chloroplast development. Thylakoid biogenesis Thylakoid membranes are rich in galactolipids, which are synthesized in the chloroplast envelope membranes (Kelly and Dörmann, 2004), and galactolipid biosynthesis is essential for thylakoid formation (Kobayashi et al, 2007). Proplastids contain a limited amount of internal membranes, called prothylakoids, which form the starting point for the biogenesis of bona fide thylakoids. Many of the enzymes in the later stages of carotenoid and chlorophyll biosynthesis are also present on the plastid envelope, as these lipid-soluble pigments must be incorporated into light-harvesting chlorophyll (Lhc)-binding proteins that are being inserted into the inner envelope membrane as a continuation of the protein import process (Hoober et al, 2007). These hydrophobic components must reach the prothylakoids by crossing the aqueous stroma. Several lines of evidence suggest that vesicles bud from the inner envelope membrane, most likely carrying a cargo of chlorophyll, enzymes and photosynthetic proteins, and migrate across the stroma to fuse with the developing thylakoids. First, when leaves are cooled to 12°C, vesicle-like structures contiguous with the inner envelope membrane accumulate in the chloroplast stroma (Morre et al, 1991). Second, direct connections between the inner envelope and thylakoid membranes have been reported, implying that the two compartments represent a partly contiguous, dynamic continuum (Shimoni et al, 2005). Third, the vipp1 mutant is defective in thylakoid formation and does not form cold-induced vesicles (Kroll et al, 2001; Aseeva et al, 2007). Another mutant, thf1, exhibits a variegated phenotype, and affected chloroplasts contain profuse vesicles with no thylakoid membrane (Wang et al, 2004). VIPP1 is associated with both the thylakoids and inner envelope, whereas THF1 is found in the stroma and thylakoids; the presence of two suborganellar locations is consistent with a trafficking function for these proteins. Finally, chloroplast bioinformatics has revealed the presence of homologues of small GTPases with putative membrane fusion functions similar to those in the eukaryotic secretory pathway, such as ARF1 and Sar1 (Andersson and Sandelius, 2004). Recently, a dynamin-like GTPase called FZL has been identified that specifically affects thylakoid membrane structure in Arabidopsis. Again, FZL is localized to both the inner envelope and the thylakoid membranes (Gao et al, 2006). Although fzl mutant plants are not deficient in thylakoid formation per se, fzl chloroplasts are large and unusually shaped, they contain abnormal proportions of stromal and granal lamellae and they frequently accumulate small vesicles (Gao et al, 2006). These findings imply that FZL is a membrane-remodelling factor that is required for maintaining a dynamic thylakoid network, but the basis for abnormal chloroplast division is unclear. Chloroplast division Once chloroplast biogenesis is underway, the chloroplasts must proliferate to match cell division and expansion: Arabidopsis mesophyll (M) cells can contain over 100 individual chloroplasts and the final count is tightly correlated with cell size (Pyke and Leech, 1994). The molecular nature of chloroplast division has been covered extensively in recent reviews (Maple and Moller, 2007; Yang et al, 2008), but one particular development is worth discussing here. As leaf development progresses, chloroplasts become progressively larger and dumb-bell-shaped plastids become less common, suggesting that division occurs early in chloroplast biogenesis (Pyke, 1999; Okazaki et al, 2009). Chloroplasts divide by binary fission, driven by two contractile protein rings that form on each side of the chloroplast envelope. The inner division ring forms first and is composed of the FtsZ1 and FtsZ2 proteins, which are homologous to bacterial fission proteins (Osteryoung and McAndrew, 2001). The constituents of the outer ring are not fully known, but the plastid division1 (PDV1) and PDV2 proteins in the outer envelope membrane recruit a cytosolic dynamin-like component, DRP5B, around the chloroplast exterior in alignment with the inner ring (Miyagishima et al, 2006). It has recently been shown that PDV1 and PDV2 are determinants of the rate and extent of chloroplast division, a question that has remained open for some time (Okazaki et al, 2009). pdv1 and pdv2 mutants had earlier been shown to contain large, deformed chloroplasts (Miyagishima et al, 2006), but when both PDV1 and PDV2 are overexpressed together, Arabidopsis M cells contain small chloroplasts that are twice as numerous as in wild type (Okazaki et al, 2009). PDV promoter activity is highest around the meristem, in which proplastids are differentiating into chloroplasts. Crucially, the levels of PDV protein decrease in concert with the rates of chloroplast division as leaves aged, but FtsZ2 and DRPB5 levels remain at similar levels throughout development (Okazaki et al, 2009), tying in neatly with observed developmental patterns of chloroplast division and size. Constitutive expression of the cytokinin responsive transcription factor CRF2 and application of exogenous cytokinins both increase the activity of PDV2, linking cell division and chloroplast division and implying that the PDV proteins are primary mechanistic components in determining the cell's chloroplast complement. This PDV-dependent mechanism seems to be evolutionarily conserved, holding true in the moss Physcomitrella patens, in common with other components of the PDV machinery such as FtsZ (Okazaki et al, 2009). It is clear that molecular-genetic approaches have been incredibly powerful tools in establishing what events are critical for chloroplast biogenesis. A notable point is that mutations in genes required for any one particular molecular process, such as chloroplast RNA processing, severely hamper the establishment of photosynthetic competence in general, as many mutants are pale green, albino or even embryo lethal (Table I). As such, many chloroplast processes are in some way interdependent: for example, the light-harvesting complex of PSII (LHCII) is comprised of several Lhc-binding proteins, which are only imported into the chloroplast and properly folded in the presence of chlorophyll synthesized on the inner envelope membrane (Espineda et al, 1999; Reinbothe et al, 2006). Similarly, defects in lipid biosynthesis severely compromise chlorophyll lev