Natural Variation in Sensitivity to a Loss of Chloroplast Translation in Arabidopsis

Mutations that eliminate chloroplast translation in Arabidopsis (Arabidopsis thaliana) result in embryo lethality. The stage of embryo arrest, however, can be influenced by genetic background. To identify genes responsible for improved growth in the absence of chloroplast translation, we examined seedling responses of different Arabidopsis accessions on spectinomycin, an inhibitor of chloroplast translation, and crossed the most tolerant accessions with embryo-defective mutants disrupted in chloroplast ribosomal proteins generated in a sensitive background. The results indicate that tolerance is mediated by ACC2, a duplicated nuclear gene that targets homomeric acetyl-coenzyme A carboxylase to plastids, where the multidomain protein can participate in fatty acid biosynthesis. In the presence of functional ACC2, tolerance is enhanced by a second locus that maps to chromosome 5 and heightened by additional genetic modifiers present in the most tolerant accessions. Notably, some of the most sensitive accessions contain nonsense mutations in ACC2, including the “Nossen” line used to generate several of the mutants studied here. Functional ACC2 protein is therefore not required for survival in natural environments, where heteromeric acetyl-coenzyme A carboxylase encoded in part by the chloroplast genome can function instead. This work highlights an interesting example of a tandem gene duplication in Arabidopsis, helps to explain the range of embryo phenotypes found in Arabidopsis mutants disrupted in essential chloroplast functions, addresses the nature of essential proteins encoded by the chloroplast genome, and underscores the value of using natural variation to study the relationship between chloroplast translation, plant metabolism, protein import, and plant development.Embryo development in Arabidopsis (Arabidopsis thaliana) requires the coordinated expression of a large number of essential genes (Muralla et al., 2011). Recessive mutations that disrupt these nuclear genes result in an embryo-defective (emb) mutant phenotype (Meinke, 2013). Many EMB genes of Arabidopsis encode chloroplast-localized proteins involved in basic metabolism, protein import, and chloroplast gene expression (Hsu et al., 2010; Bryant et al., 2011; Savage et al., 2013). Functional plastids are therefore required for embryo development in Arabidopsis. Mutations that disrupt photosynthesis alone interfere with embryo and seedling pigmentation, not embryo development. Multiple examples of EMB genes that encode chloroplast-localized aminoacyl-tRNA synthetases, RNA-binding proteins, translation factors, and ribosomal proteins have been described in the literature (Berg et al., 2005; Bryant et al., 2011; Muralla et al., 2011; Romani et al., 2012; Tiller and Bock, 2014). Translation of some chloroplast-encoded mRNAs is therefore essential for seed development. This raises a basic question: which chloroplast genes are required? In this report, we used natural variation and genetic analysis to evaluate the model (Bryant et al., 2011) that a single chloroplast gene, acetyl-coenzyme A carboxylase D (accD), needed for the initial stages of fatty acid biosynthesis, underlies the requirement for chloroplast translation during heterotrophic growth and embryo development in Arabidopsis.Targeted gene disruptions in tobacco (Nicotiana tabacum) have identified four chloroplast genes with essential functions that extend beyond photosynthesis: accD, caseinolytic protease P1 (clpP1), hypothetical chloroplast open reading frame1 (ycf1), and ycf2 (Drescher et al., 2000; Kuroda and Maliga, 2003; Kode et al., 2005). Comparative genomics have shown that all four genes are retained in the plastid genomes of most angiosperms, including chlorophyll-deficient, parasitic species (dePamphilis and Palmer, 1990; Funk et al., 2007; Jansen et al., 2007). Several examples of essential chloroplast genes that relocated to the nucleus have also been described (Magee et al., 2010; Rousseau-Gueutin et al., 2013). The absence of ycf1 and ycf2 in grasses (Jansen et al., 2007) and the replacement of accD with a nuclear gene that targets functional protein back to the chloroplast (Konishi and Sasaki, 1994; Chalupska et al., 2008) remain to be explained.The accD gene in Arabidopsis (AtCg00500) encodes one subunit of the chloroplast-localized heteromeric acetyl-coenzyme A carboxylase (ACCase), an essential enzyme in fatty acid biosynthesis that converts acetyl-CoA to malonyl-CoA. Three other subunits are encoded by nuclear genes, one of which is also known to be required for embryo development (Li et al., 2011). Disruptions of three additional genes (At3g25860, At1g34430, and At2g30200) associated with the reactions that precede and follow the step catalyzed by heteromeric ACCase also result in embryo lethality (Lin et al., 2003; Bryant et al., 2011; Muralla et al., 2011). Embryo lethality is also encountered in auxotrophic mutants unable to produce biotin, an essential vitamin required for ACCase function (Schneider et al., 1989; Patton et al., 1998; Muralla et al., 2008). The conversion of acetyl-CoA to malonyl-CoA during fatty acid biosynthesis within the plastid is therefore required for embryo development in Arabidopsis.In addition to the chloroplast-localized, heteromeric ACCase found in most angiosperms, there is also a cytosolic, homomeric ACCase involved in later stages of fatty acid biosynthesis. In both Arabidopsis and Brassica napus, the gene that encodes this homomeric enzyme is duplicated (Yanai et al., 1995; Schulte et al., 1997). One copy (ACC1; At1g36160) encodes an essential protein localized to the cytosol. Disruption of this gene in Arabidopsis (EMB22, GURKE, and PASTICCINO3 [PAS3]) results in an embryo-defective phenotype distinct from that seen following a loss of chloroplast translation (Meinke, 1985; Baud et al., 2004). Weak alleles exhibit cold sensitivity and glossy inflorescence stems resulting from changes in cuticular wax composition (Lü et al., 2011; Amid et al., 2012). The adjacent copy (ACC2; At1g36180) is expressed at low levels and is predicted to encode a chloroplast-localized protein (Yanai et al., 1995; Baud et al., 2003; Babiychuk et al., 2011). Knockouts of this gene exhibit no obvious phenotype under normal growth conditions (Babiychuk et al., 2011).In Brassica spp., plants with albino leaves devoid of chloroplast ribosomes have been produced by germinating seeds on spectinomycin, an inhibitor of chloroplast translation, and then transplanting the young seedlings to basal medium (Zubko and Day, 1998). This experimental approach was initially described as a promising system for generating stable albinism without mutagenesis. However, different results were obtained with tobacco and Arabidopsis seedlings, which were much more sensitive to spectinomycin. In light of this reported variation in seedling responses to spectinomycin and the known duplication of ACC1 in the Brassicaceae, we decided to explore whether natural accessions of Arabidopsis differed in their ability to tolerate a loss of chloroplast translation and whether genetic analysis in Arabidopsis could uncover some of the genes involved. The results described here confirm the value of this approach, provide insights into the phenotypes of mutants defective in essential chloroplast functions, and help to explain the requirement of chloroplast translation for plant growth and development.     

Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the Liverwort Marchantia polymorpha

Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark. Moreover, in silico and Southern-blot analyses revealed that the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.Light is not only an energy source for photosynthesis but it is also a signal that regulates numerous physiological responses for plants. Because chloroplasts are the important organelle for photosynthesis, most plant species possess a light-dependent mechanism to regulate the intracellular position of chloroplasts (chloroplast photorelocation movement). Intensive studies on chloroplast photorelocation movement have been performed since the 19th century (Böhm, 1856). Senn (1908) described the chloroplast distribution patterns under different light conditions in various plant species, including algae, liverworts, mosses, ferns, and seed plants, and revealed the general responses of chloroplasts to intensity and direction of light. Under low-light conditions, chloroplasts are positioned along the cell walls perpendicular to the direction of incident light (i.e. periclinal cell walls) to efficiently capture light for photosynthesis (the accumulation response). By contrast, under high-light conditions, chloroplasts are stacked along the cell walls parallel to the direction of incident light (i.e. anticlinal cell walls) to minimize total light absorption and to avoid photooxidative damage (the avoidance response). These chloroplast movements are induced primarily by blue light in most plant species (Suetsugu and Wada, 2007a). In some plant species, such as several ferns including Adiantum capillus-veneris, the moss Physcomitrella patens, and some charophycean green algae (Mougeotia scalaris and Mesotaenium caldariorum), red light is also effective to induce chloroplast movement (Suetsugu and Wada, 2007b). Analyses of chloroplast movement in response to irradiation with polarized light and/or a microbeam suggest that the photoreceptor for chloroplast movement is localized on or close to the plasma membrane (Haupt and Scheuerlein, 1990; Wada et al., 1993). In addition, chloroplasts assume their specific positions in the dark (dark positioning), although the patterns vary among plant species (Senn, 1908). For example, the chloroplasts are localized at the bottom of the cell in palisade cells of Arabidopsis (Arabidopsis thaliana; Suetsugu et al., 2005a) and on the anticlinal walls bordering neighboring cells in the prothallial cells of A. capillus-veneris (Kagawa and Wada, 1993; Tsuboi et al., 2007).Molecular mechanisms for chloroplast photorelocation movements have been revealed through molecular genetic analyses using Arabidopsis (Suetsugu and Wada, 2012). The light-activated kinase phototropin was identified as the blue-light receptor (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropin consists of two functional regions: a photosensory domain at the N terminus and a Ser/Thr kinase domain at the C terminus (Christie, 2007). The N-terminal photosensory domain contains two light, oxygen, or voltage (LOV) domains, which belong to the Per/ARNT/Sim domain superfamily. Each LOV domain binds to one FMN and functions as a blue-light sensor (Christie et al., 1999). The LOV2 domain is essential for blue-light-dependent regulation of the activation of the C-terminal kinase domain (Christie et al., 2002; Harper et al., 2003).Arabidopsis has two phototropins: phot1 and phot2 (Christie, 2007). Besides chloroplast photorelocation movement, phototropin controls other photoresponses to optimize the photosynthetic efficiency in plants and improves growth responses such as phototropism, stomatal opening, and leaf flattening (Christie, 2007). Both phot1 and phot2 redundantly regulate the chloroplast accumulation response (Sakai et al., 2001), hypocotyl phototropism (Huala et al., 1997; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and leaf flattening (Sakai et al., 2001; Sakamoto and Briggs, 2002). Rapid inhibition of hypocotyl elongation is specifically mediated by phot1 (Folta and Spalding, 2001), whereas the chloroplast avoidance response (Jarillo et al., 2001; Kagawa et al., 2001) and palisade cell development (Kozuka et al., 2011) are mediated primarily by phot2.It is thought that the phototropin-regulated photoresponses are mediated by mechanisms in which gene expression is not involved primarily. For example, chloroplast photorelocation movement can be observed even in enucleated fern cells (Wada, 1988), and phototropins show only a minor contribution to blue-light-induced gene expression in Arabidopsis (Jiao et al., 2003; Ohgishi et al., 2004; Lehmann et al., 2011). Furthermore, both phot1 and phot2 are localized on the plasma membrane despite the absence of a transmembrane domain (Sakamoto and Briggs, 2002; Kong et al., 2006). During chloroplast movement, phototropins, in particular phot2, associate not only with the plasma membrane but also with the chloroplast outer membrane (Kong et al., 2013b). In addition, phot1 shows blue-light-dependent internalization into the cytoplasm (Sakamoto and Briggs, 2002; Knieb et al., 2004; Wan et al., 2008; Kaiserli et al., 2009), whereas phot2 exhibits a blue-light-dependent association with the Golgi apparatus (Kong et al., 2006).PHOT genes have been identified from various green plants and are indicated to be duplicated in respective lineages such as seed plants, ferns, lycophytes, and mosses (Li et al., 2014). In the fern A. capillus-veneris, chloroplast accumulation and avoidance responses are induced by both blue and red light (Yatsuhashi et al., 1985). This fern has three phototropin family proteins, two phototropins (Acphot1 and Acphot2; Kagawa et al., 2004), and one neochrome that possesses the chromophore-binding domain of phytochrome and complete phototropin domains (Nozue et al., 1998). Neochrome is the red-light receptor that mediates chloroplast movement (Kawai et al., 2003) and possibly blue-light-induced chloroplast movement through its LOV domains (Kanegae et al., 2006). Because the Acphot2 mutant is defective in the chloroplast avoidance response and dark positioning (Kagawa et al., 2004; Tsuboi et al., 2007), similar to the phot2 mutant in Arabidopsis (Jarillo et al., 2001; Kagawa et al., 2001; Suetsugu et al., 2005a), the function of phot2 in the regulation of chloroplast movement is highly conserved in these vascular plants. In the moss P. patens, in which chloroplast accumulation and avoidance responses are induced by both blue and red light (Kadota et al., 2000), seven phototropin genes are present in the draft genome sequences (Rensing et al., 2008). The phototropins encoded by four of these genes (PpphotA1, PpphotA2, PpphotB1, and PpphotB2) function in the blue-light-induced chloroplast movement (Kasahara et al., 2004). Moreover, red-light-induced chloroplast movements are mediated by both conventional phytochromes (Mittmann et al., 2004; Uenaka and Kadota, 2007) and phototropins (Kasahara et al., 2004). Because the direct association between phytochromes and phototropins is suggested to be involved in red-light-induced chloroplast movement (Jaedicke et al., 2012), phototropins should be essential components in the chloroplast movement signaling pathway (Kasahara et al., 2004).A single PHOT gene was isolated in a unicellular green alga, Chlamydomonas reinhardtii (Huang et al., 2002; Kasahara et al., 2002). When expressed in Arabidopsis phot1 phot2 double-mutant plants, C. reinhardtii phototropin rescued the defects in chloroplast photorelocation movement in phot1 phot2 plants (Onodera et al., 2005), indicating that the initial step of the phototropin-mediated signal transduction mechanism for chloroplast movements is conserved in the green plant lineage. Although the existence of only one PHOT gene is ideal for elucidation of phototropin-mediated responses, C. reinhardtii cells contain a single chloroplast and show no chloroplast photorelocation movement.Liverworts represent the most basal lineage of extant land plants and offer a valuable experimental system for elucidation of various physiological responses commonly seen in land plants (Bowman et al., 2007). Marchantia polymorpha has emerged as a model liverwort because molecular biological techniques, such as genetic transformation and gene-targeting technologies, have been established for the species (Ishizaki et al., 2008, 2013a; Kubota et al., 2013; Sugano et al., 2014). Furthermore, an ongoing M. polymorpha genome sequencing project under the Community Sequencing Program at the Joint Genome Institute has indicated that many biological mechanisms found in other groups of land plants are conserved in a much less complex form. Blue-light-induced chloroplast movement was briefly reported in M. polymorpha (Senn, 1908; Nakazato et al., 1999). However, information on chloroplast photorelocation movement in liverworts, including M. polymorpha, is very limited.In this study, we investigated chloroplast photorelocation movement in detail in M. polymorpha and analyzed the molecular mechanism underlying the photoreceptor system through molecular genetic analysis of M. polymorpha phototropin.     

Autophagy Plays a Role in Chloroplast Degradation during Senescence in Individually Darkened Leaves

Chloroplasts contain approximately 80% of total leaf nitrogen and represent a major source of recycled nitrogen during leaf senescence. While bulk degradation of the cytosol and organelles in plants is mediated by autophagy, its role in chloroplast catabolism is largely unknown. We investigated the effects of autophagy disruption on the number and size of chloroplasts during senescence. When leaves were individually darkened, senescence was promoted similarly in both wild-type Arabidopsis (Arabidopsis thaliana) and in an autophagy-defective mutant, atg4a4b-1. The number and size of chloroplasts decreased in darkened leaves of wild type, while the number remained constant and the size decrease was suppressed in atg4a4b-1. When leaves of transgenic plants expressing stroma-targeted DsRed were individually darkened, a large accumulation of fluorescence in the vacuolar lumen was observed. Chloroplasts exhibiting chlorophyll fluorescence, as well as Rubisco-containing bodies, were also observed in the vacuole. No accumulation of stroma-targeted DsRed, chloroplasts, or Rubisco-containing bodies was observed in the vacuoles of the autophagy-defective mutant. We have succeeded in demonstrating chloroplast autophagy in living cells and provide direct evidence of chloroplast transportation into the vacuole.Chloroplasts contain 75% to 80% of total leaf nitrogen mainly as proteins (Makino and Osmond, 1991). During leaf senescence, chloroplast proteins are gradually degraded as a major source of nitrogen for new growth (Wittenbach, 1978; Friedrich and Huffaker, 1980; Mae et al., 1984), correlating with a decline in photosynthetic activity, while chloroplasts gradually shrink and transform into gerontoplasts, characterized by the disintegration of the thylakoid membranes and accumulation of plastoglobuli (for a recent review, see Krupinska, 2006). Concomitantly, a decline in the cellular population of chloroplasts is also evident in many cases, for example, during natural (Kura-Hotta et al., 1990; Inada et al., 1998), dark-induced (Wittenbach et al., 1982), and nutrient-limited senescence (Mae et al., 1984; Ono et al., 1995), suggesting the existence of a whole chloroplast degradation system. Some electron microscopic studies have shown whole chloroplasts in the central vacuole, which is rich in lytic hydrolases (Wittenbach et al., 1982; Minamikawa et al., 2001). However, there is no direct evidence of chloroplasts moving into the vacuole in living cells and the mechanism of transport is not yet understood (Hörtensteiner and Feller, 2002; Krupinska, 2006).The most abundant chloroplast protein is Rubisco (EC 4.1.1.39), comprising approximately 50% of the soluble protein (Wittenbach, 1978). The amount of Rubisco decreases rapidly in the early phase of leaf senescence, although more slowly in the later phase (Friedrich and Huffaker, 1980; Mae et al., 1984). In contrast, the chloroplast number remains relatively constant, making it impossible to explain Rubisco loss solely by whole chloroplast degradation. However, the mechanism of intrachloroplastic Rubisco degradation is still unknown (for review, see Feller et al., 2008). Using immunoelectron microscopy, we previously demonstrated in naturally senescing wheat (Triticum aestivum) leaves that Rubisco is released from chloroplasts into the cytoplasm and transported to the vacuole for subsequent degradation in small spherical bodies, named Rubisco-containing bodies (RCBs; Chiba et al., 2003). Similar chloroplast-derived structures were also subsequently confirmed in senescent leaves of soybean (Glycine max) and/or Arabidopsis (Arabidopsis thaliana) by electron microscopy (Otegui et al., 2005), and recently in tobacco (Nicotiana tabacum) leaves by immunoelectron microscopy, although the authors gave them a different name, Rubisco vesicular bodies (Prins et al., 2008). RCBs have double membranes, which seem to be derived from the chloroplast envelope; thus, the RCB-mediated degradation of stromal proteins represents a potential mechanism for chloroplast shrinkage during senescence. We recently demonstrated that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole by ATG-dependent autophagy via RCBs, using leaves treated with concanamycin A, a vacuolar H⁺-ATPase inhibitor (Ishida et al., 2008). To investigate further, we wished to observe chloroplast autophagy and degradation directly in living cells to determine whether autophagy is responsible for chloroplast shrinkage and whether it is involved in the vacuolar degradation of whole chloroplasts during leaf senescence.Autophagy is known to be a major system for the bulk degradation of intracellular proteins and organelles in the vacuole in yeast and plants, or the lysosome in animals (for detailed mechanisms, see reviews by Ohsumi, 2001; Levine and Klionsky, 2004; Thompson and Vierstra, 2005; Bassham et al., 2006). In those systems, a portion of the cytoplasm, including entire organelles, is engulfed in membrane-bound vesicles and delivered to the vacuole/lysosome. A recent genome-wide search confirmed that Arabidopsis has many genes homologous to the yeast autophagy genes (ATGs; Doelling et al., 2002; Hanaoka et al., 2002; for detailed functions of ATGs, see the reviews noted above). Using knockout mutants of ATGs and a monitoring system with an autophagy marker, GFP-ATG8, numerous studies have demonstrated the presence of the autophagy system in plants and its importance in several biological processes (Yoshimoto et al., 2004; Liu et al., 2005; Suzuki et al., 2005; Thompson et al., 2005; Xiong et al., 2005, 2007; Fujiki et al., 2007; Phillips et al., 2008). These articles suggest that autophagy plays an important role in nutrient recycling during senescence, especially in nutrient-starved plants. The atg mutants exhibited an accelerated loss of some chloroplast proteins, but not all, under nutrient-starved conditions and during senescence, suggesting that autophagy is not the sole mechanism for the degradation of chloroplast proteins; other, as yet unidentified systems must be responsible for the degradation of chloroplast contents when the ATG system is compromised (Levine and Klionsky, 2004; Bassham et al., 2006). However, it still remains likely that autophagy is responsible for the vacuolar degradation of chloroplasts in wild-type plants.Prolonged observation is generally required to follow leaf senescence events in naturally aging leaves and senescence-associated processes tend to become chaotic over time. To observe chloroplast degradation over a short period, and to draw clear conclusions, a suitable experimental model of leaf senescence is required. Weaver and Amasino (2001) reported that senescence is rapidly induced in individually darkened leaves (IDLs) of Arabidopsis, but retarded in plants subjected to full darkness. In addition, Keech et al. (2007) observed a significant decrease of both the number and size of chloroplasts in IDLs within 6 d.In this study, using IDLs as a senescence model, we aimed to investigate the involvement of autophagy in chloroplast degradation. We show direct evidence for the transport of whole chloroplasts and RCBs to the vacuole by autophagy.     

The Stromal Chloroplast Deg7 Protease Participates in the Repair of Photosystem II after Photoinhibition in Arabidopsis

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).Chloroplasts of higher plants carry out one of the most important biochemical reactions: the capture of light energy and its conversion into chemical energy. Although light is the ultimate source of energy for photosynthesis, it can also be harmful to plants. Light-induced loss of photosynthetic efficiency, which is generally termed as photoinhibition, limits plant growth and lowers productivity, especially when combined with other abiotic stresses.The main target of photoinhibition is PSII, which catalyzes the light-dependent water oxidation concomitantly with oxygen production (for review, see Prasil et al., 1992; Aro et al., 1993; Adir et al., 2003). In higher plants, PSII consists of more than 20 subunits, including the reaction center D1 and D2 proteins, cytochrome (Cyt) b₅₅₉, the light-harvesting chlorophyll a-binding proteins CP47 and CP43, the oxygen-evolving 33-kD protein (PsbO), and several low molecular mass proteins (Nelson and Yocum, 2006). The PSII reaction center D1 protein has been identified among PSII proteins as the primary target of light-induced damage (Kyle et al., 1984; Mattoo et al., 1984; Ohad et al., 1984; Adir et al., 1990), but several studies have shown that the D2, CP47, and CP43 proteins are degraded under photoinhibitory conditions (Schuster et al., 1988; Yamamoto and Akasaka, 1995; Jansen et al., 1999; Adir et al., 2003). Moreover, several small PSII subunits, such as PsbH, PsbW, and Cyt b₅₅₉, were also found to be frequently replaced within PSII (Hagman et al., 1997; Ortega et al., 1999; Bergantino et al., 2003). Evidence for the involvement of two families of proteases, FtsH and Deg, in the degradation of the D1 protein in thylakoids of higher plants has been recently described (Lindahl et al., 1996, 2000; Bailey et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; Kapri-Pardes et al., 2007; Sun et al., 2007a, 2007b). However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases.DegP (or HtrA) proteases were initially identified based on the fact that they are required for the survival of Escherichia coli at high temperatures and for the degradation of abnormal periplasmic proteins (Lipinska et al., 1988; Strauch and Beckwith, 1988). DegP is an ATP-independent Ser endopeptidase, and it contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (Gottesman, 1996; Pallen and Wren, 1997; Clausen et al., 2002). PDZ domains appear to be important for complex assembly and substrate binding through three or four residues in the C terminus of their target proteins (Doyle et al., 1996; Harris and Lim, 2001). DegP switches between chaperone and protease functions in a temperature-dependent manner. The chaperone function dominates at low temperatures, and DegP becomes proteolytically active at elevated temperatures (Spiess et al., 1999). Crystal structures of different members of the DegP protein family (Krojer et al., 2002; Li et al., 2002; Kim et al., 2003; Wilken et al., 2004) have revealed the structure-function relationship of these PDZ-containing proteases. Trimeric DegP is the functional unit, and the hexameric DegP is formed via the staggered association of trimers (Clausen et al., 2002; Kim and Kim, 2005). At normal growth temperatures, the active site of the protease is located within the chamber of hexameric DegP, which is not accessible to the substrates. However, at high temperatures, conformational changes induce the activation of the protease function (Krojer et al., 2002). Recent studies have shed light on the substrate binding-induced formation of larger oligomeric complexes of DegP (Jiang et al., 2008; Krojer et al., 2008).In Arabidopsis (Arabidopsis thaliana), 16 genes coding for DegP-like proteases have been identified, and at least seven gene products are predicted to be located in chloroplasts (Kieselbach and Funk, 2003; Huesgen et al., 2005; Adam et al., 2006; Sakamoto, 2006; Kato and Sakamoto, 2009). Based on proteomic data, four Deg proteases have been shown to be localized to the chloroplast (Peltier et al., 2002; Schubert et al., 2002) and functionally characterized. Deg1, Deg5, and Deg8 are located in thylakoid lumen, and Deg2 is peripherally associated with the stromal side of thylakoid membranes (Itzhaki et al., 1998; Haußühl et al., 2001; Sun et al., 2007a). Recombinant DegP1, now renamed Deg1, has been shown to be proteolytically active toward thylakoid lumen proteins such as plastocyanin and PsbO of PSII in vitro (Chassin et al., 2002). A 5.2-kD C-terminal fragment of the D1 protein was detected in vitro after incubation of recombinant Deg1 with inside-out thylakoid membranes. In transgenic plants with reduced levels of Deg1, fewer of its 16- and 5.2-kD degradation products were observed (Kapri-Pardes et al., 2007). Deg5 and Deg8 form a dodecameric complex in the thylakoid lumen, and recombinant Deg8 is able to degrade the photodamaged D1 protein of PSII in an in vitro assay (Sun et al., 2007a). The 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in a deg5 deg8 double mutant after high-light treatment. The deg5 deg8 double mutant showed increased sensitivity to high light and high temperature in terms of growth and PSII activity compared with the single mutants deg5 and deg8, suggesting that Deg5 and Deg8 have overlapping functions in the primary cleavage of the CD loop of the D1 protein (Sun et al., 2007a, 2007b). In vitro analysis has demonstrated that recombinant stroma-localized Deg2 was also shown to be involved in the primary cleavage of the DE loop of the D1 protein (Haußühl et al., 2001). However, analysis of a mutant lacking Deg2 suggested that Deg2 may not be involved in D1 degradation in vivo (Huesgen et al., 2006).Here, we have expressed and purified a recombinant DegP protease, His-Deg7. In vitro experiments showed that His-Deg7 is proteolytically active toward the PSII proteins D1, D2, CP43, and CP47. In vivo analyses of a deg7 mutant revealed that the mutant is more sensitive to high light stress than the wild-type plants. We demonstrated that Deg7 is a chloroplast stroma protein associated with the thylakoid membranes and that it interacts with PSII, which suggests that it can cleave the stroma-exposed region of substrate proteins. Our results also provide evidence that Deg7 is important for maintaining PSII function.     

Chloroplast Phosphoglycerate Kinase Is Involved in the Targeting of Bamboo mosaic virus to Chloroplasts in Nicotiana benthamiana Plants

The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.Bamboo mosaic virus (BaMV) is a single-stranded, positive-sense RNA virus. The genomic RNA of BaMV contains five open reading frames (ORFs) and is 6,366 nucleotides in length with a 5′ cap and a 3′ poly(A) tail (Lin et al., 1994; Yang et al., 1997). ORF1 encodes a 155-kD replicase comprised of a capping enzyme domain that exhibits S-adenosylmethionine-dependent guanylyltransferase activity (Li et al., 2001a; Huang et al., 2004), a helicase-like domain with RNA 5′-triphosphatase activity (Li et al., 2001b), and an RNA-dependent RNA polymerase domain (Li et al., 1998; Cheng et al., 2001). The three overlapping ORFs (i.e. ORF2, ORF3, and ORF4) are known as the triple gene block. They encode for proteins involved in viral movement (Lin et al., 2004, 2006; Vijaya Palani et al., 2006). ORF5 encodes the viral capsid protein (CP), required for virion assembly and viral movement (Cruz et al., 1998).The genomes of positive-strand RNA viruses are templates for both translation and replication. Viral replication complexes are likely to be assembled using host factors to synthesize the minus-strand RNA and then the plus-strand progeny RNA. Recent studies have shown that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral replication templates, activating the complex for RNA synthesis, and other steps (Ahlquist et al., 2003; Patarroyo et al., 2012). The translation and the minus-strand RNA synthesis of poliovirus are regulated by host poly(C) and poly(A) binding proteins and viral polymerase precursor 3CD (Waggoner and Sarnow, 1998; Herold and Andino, 2001; Walter et al., 2002). A number of host genes required for Brome mosaic virus replication have been identified systemically by the yeast (Saccharomyces cerevisiae) genetic approach (Ishikawa et al., 1997; Kushner et al., 2003; Mas et al., 2006; Gancarz et al., 2011). The same approach was used to identify the host factors involved in the replication of Tomato bushy stunt virus (TBSV; Panavas et al., 2005; Li et al., 2009b). A heat shock protein90 homolog (Huang et al., 2012) and the Nicotiana benthamiana glutathione transferase U4 (NbGSTU4; Chen et al., 2013), were identified to interact with the 3′ untranslated region (UTR) of BaMV RNA and enhanced the minus-strand RNA synthesis at the early replication step. The Ser/Thr kinase-like protein localized on cell membrane facilitates the BaMV intercellular movement (Cheng et al., 2013).Previously, we have identified two host proteins (i.e. p51 and p43) interacting specifically with the 3′ UTRs of BaMV by using electrophoretic mobility shift assay (EMSA) and the UV cross-linking competition technique. The results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and BLAST indicate that the protein sequences of p43 and p51 match the chloroplast phosphoglycerate kinase (chl-PGK) and elongation factor1a (EF1a) of Nicotiana benthamiana, respectively (Lin et al., 2007). Phosphoglycerate kinase is an ATP-generating enzyme that acts in the glycolytic, gluconeogenic, and photosynthetic pathways (Banks et al., 1979; McHarg et al., 1999). chl-PGK is encoded in the nucleus and translated to produce a 50-kD precursor protein and is then processed into mature 43 kD in the chloroplast. In a knockdown experiment through virus-induced gene silencing, the reduction of PGK decreased the accumulation of BaMV coat protein (Lin et al., 2007).Eukaryotic EF1a has been shown to play a role in binding to the tRNA-like structure and upstream pseudoknot in the 3′ UTR of Tobacco mosaic virus to regulate the gene expression and viral replication (Pathak et al., 2008). EF1a has also been involved in the recruitment of viral RNA and has facilitated the replicase complex assembly of TBSV (Pogany et al., 2008). The 3′ UTR of BaMV cannot only bind its replicase but also the EF1a and has been proposed to regulate viral RNA replication (Lin et al., 2007).In this study, we transiently expressed two mislocalized PGK mutants to study the possible functions of chl-PGK that is involved in viral RNA replication. In addition, we used confocal microscopy to investigate the localization of BaMV RNA. Finally, we provided evidence that the down-regulation of BaMV accumulation in PGK-knockdown plants can be restored by the expression of the BaMV RNA binding protein EF1a that is fused to a chloroplast transit peptide.     

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.In plants, organelles move within the cell and become appropriately positioned to accomplish their functions and adapt to the environment (for review, see Wada and Suetsugu, 2004). Light-induced chloroplast movement (chloroplast photorelocation movement) is one of the best characterized organelle movements in plants (Suetsugu and Wada, 2012). Under weak light conditions, chloroplasts move toward light to capture light efficiently (the accumulation response; Zurzycki, 1955). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response; Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012; Cazzaniga et al., 2013). In most green plant species, these responses are induced primarily by the blue light receptor phototropin (phot) in response to a range of wavelengths from UVA to blue light (approximately 320–500 nm; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). Phot-mediated chloroplast movement has been shown in land plants, such as Arabidopsis (Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001), the fern Adiantum capillus-veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), and the liverwort Marchantia polymorpha (Komatsu et al., 2014). Two phots in Arabidopsis, phot1 and phot2, redundantly mediate the accumulation response (Sakai et al., 2001), whereas phot2 primarily regulates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Luesse et al., 2010). M. polymorpha has only one phot that mediates both the accumulation and avoidance responses (Komatsu et al., 2014), although two or more phots mediate chloroplast photorelocation movement in A. capillus-veneris (Kagawa et al., 2004) and P. patens (Kasahara et al., 2004). Thus, duplication and functional diversification of PHOT genes have occurred during land plant evolution, and plants have gained a sophisticated light sensing system for chloroplast photorelocation movement.In general, movements of plant organelles, including chloroplasts, are dependent on actin filaments (for review, see Wada and Suetsugu, 2004). Most organelles common in eukaryotes, such as mitochondria, peroxisomes, and Golgi bodies, use the myosin motor for their movements, but there is no clear evidence that chloroplast movement is myosin dependent (for review, see Suetsugu et al., 2010a). Land plants have innovated a novel actin-based motility system that is specialized for chloroplast movement as well as a photoreceptor system (for review, see Suetsugu et al., 2010a; Wada and Suetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found in Arabidopsis, are short actin filaments specifically localized around the chloroplast periphery at the interface between the chloroplast and the plasma membrane (Kadota et al., 2009). Strong blue light induces the rapid disappearance of cp-actin filaments and then, their subsequent reappearance preferentially at the front region of the moving chloroplasts. This asymmetric distribution of cp-actin filaments is essential for directional chloroplast movement (Kadota et al., 2009; Kong et al., 2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts becomes, the faster the chloroplasts move, in which the magnitude of the difference is determined by fluence rate (Kagawa and Wada, 2004; Kadota et al., 2009; Kong et al., 2013a). Strong blue light-induced disappearance of cp-actin filaments is regulated in a phot2-dependent manner before the intensive polymerization of cp-actin filaments at the front region occurs (Kadota et al., 2009; Ichikawa et al., 2011; Kong et al., 2013a). This phot2-dependent response contributes to the greater difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts. Similar behavior of cp-actin filaments has also been observed in A. capillus-veneris (Tsuboi and Wada, 2012) and P. patens (Yamashita et al., 2011).Like chloroplasts, nuclei also show light-mediated movement and positioning (nuclear photorelocation movement) in land plants (for review, see Higa et al., 2014b). In gametophytic cells of A. capillus-veneris, weak light induced the accumulation responses of both chloroplasts and nuclei, whereas strong light induced avoidance responses (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). However, in mesophyll cells of Arabidopsis, strong blue light induced both chloroplast and nuclear avoidance responses, but weak blue light induced only the chloroplast accumulation response (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In Arabidopsis pavement cells, small numbers of tiny plastids were found and showed autofluorescence under the confocal laser-scanning microscopy (Iwabuchi et al., 2010; Higa et al., 2014a). Hereafter, the plastid in the pavement cells is called the pavement cell plastid. Strong blue light-induced avoidance responses of pavement cell plastids and nuclei were induced in a phot2-dependent manner, but the accumulation response was not detected for either organelle (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In both Arabidopsis and A. capillus-veneris, phots mediate nuclear photorelocation movement, and phot2 mediates the nuclear avoidance response (Iwabuchi et al., 2007, 2010; Tsuboi et al., 2007). The nuclear avoidance response is dependent on actin filaments in both mesophyll and pavement cells of Arabidopsis (Iwabuchi et al., 2010). Recently, it was shown that the nuclear avoidance response relies on cp-actin-dependent movement of pavement cell plastids, where nuclei are associated with pavement cell plastids of Arabidopsis (Higa et al., 2014a). In mesophyll cells, nuclear avoidance response is likely dependent on cp-actin filament-mediated chloroplast movement, because the mutants deficient in chloroplast movement were also defective in nuclear avoidance response (Higa et al., 2014a). Thus, phots mediate both chloroplast (and pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin filaments.Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast photorelocation movement have identified many molecular factors involved in signal transduction and/or motility systems as well as those involved in the photoreceptor system for chloroplast photorelocation movement (and thus, nuclear photorelocation movement; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1; Oikawa et al., 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC; Suetsugu et al., 2010b) are key factors for generating and/or maintaining cp-actin filaments. Both proteins are highly conserved in land plants and essential for the movement and attachment of chloroplasts to the plasma membrane in Arabidopsis (Oikawa et al., 2003, 2008; Suetsugu et al., 2010b), A. capillus-veneris (Suetsugu et al., 2012), and P. patens (Suetsugu et al., 2012; Usami et al., 2012). CHUP1 is localized on the chloroplast outer membrane and binds to globular and filamentous actins and profilin in vitro (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008). Although KAC is a kinesin-like protein, it lacks microtubule-dependent motor activity but has filamentous actin binding activity (Suetsugu et al., 2010b). An actin-bundling protein THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement (Whippo et al., 2011) and interacts with cp-actin filaments (Kong et al., 2013a). chup1 and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al., 2009; Suetsugu et al., 2010b; Ichikawa et al., 2011; Kong et al., 2013a). Similarly, cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al., 2013a), indicating that THRUM1 also plays an important role in maintaining cp-actin filaments.Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE1 (JAC1; Suetsugu et al., 2005), WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodama et al., 2010), and PLASTID MOVEMENT IMPAIRED2 (PMI2; Luesse et al., 2006; Kodama et al., 2010) are involved in the light regulation of cp-actin filaments and chloroplast photorelocation movement. JAC1 is an auxilin-like J-domain protein that mediates the chloroplast accumulation response through its J-domain function (Suetsugu et al., 2005; Takano et al., 2010). WEB1 and PMI2 are coiled-coil proteins that interact with each other (Kodama et al., 2010). Although web1 and pmi2 were partially defective in the avoidance response, the jac1 mutation completely suppressed the phenotype of web1 and pmi2, suggesting that the WEB1/PMI2 complex suppresses JAC1 function (i.e. the accumulation response) under strong light conditions (Kodama et al., 2010). Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response to strong blue light (Kodama et al., 2010). However, the exact molecular functions of these proteins are unknown.In this study, we characterized mutant plants deficient in the PMI1 gene and two homologous genes PLASTID MOVEMENT IMPAIRED1-RELATED1 (PMIR1) and PMIR2. PMI1 was identified through molecular genetic analyses of pmi1 mutants that showed severe defects in chloroplast accumulation and avoidance responses (DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang and Aravind, 2010), but its roles and those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation movements remained unclear. Thus, we analyzed chloroplast and nuclear photorelocation movements in the single, double, and triple mutants of pmi1, pmir1, and pmir2.     

Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly

Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.Chloroplasts evolved from an ancient cyanobacterium through endosymbiosis (Gould et al., 2008; Keeling, 2013). Like their prokaryotic relatives, chloroplasts replicate by binary fission, which is driven by a dynamic macromolecular complex located at the middle of the organelle (Falconet, 2011; Miyagishima et al., 2011; Osteryoung and Pyke, 2014). The major contractile components of the division complex include the FtsZ ring (Z ring), which assembles on the stromal surface of the inner envelope membrane (IEM; McAndrew et al., 2001; Vitha et al., 2001), and the DYNAMIN-RELATED PROTEIN 5B (DRP5B; also called ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 [ARC5]) ring, which assembles on the cytosolic surface of the outer envelope membrane (OEM; Gao et al., 2003; Miyagishima et al., 2003; Yoshida et al., 2006). In green algae and land plants, the Z ring is composed of the tubulin-like, heteropolymer-forming proteins FtsZ1 and FtsZ2, which are both required for normal Z-ring function (Schmitz et al., 2009; TerBush and Osteryoung, 2012). DRP5B is a member of the dynamin family of membrane fission proteins, which polymerize into collar-like structures to mediate a variety of membrane fission processes in eukaryotes (Morlot and Roux, 2013). The Z ring and DRP5B ring function together to drive the simultaneous constriction of the IEM and OEM during chloroplast division.The assembly and constriction of the inner Z ring and outer DRP5B ring are coordinated across the two membranes by the activities of midplastid-localized envelope membrane proteins whose functions have been studied in Arabidopsis (Arabidopsis thaliana). ARC6 (Pyke et al., 1994) is a bitopic IEM protein of cyanobacterial origin that is conserved throughout green-lineage chloroplasts (Koksharova and Wolk, 2002; Vitha et al., 2003; Osteryoung and Pyke, 2014). Its N-terminal region extends into the stroma, where it interacts directly and specifically with FtsZ2 (Maple et al., 2005). As FtsZ1 and FtsZ2 are soluble (McAndrew et al., 2001), this interaction probably serves both to tether the Z ring to the IEM and to promote FtsZ polymerization at the division site (Vitha et al., 2003). The C-terminal region of ARC6 protrudes into the intermembrane space (IMS) and interacts with the IMS region of the plant-specific bitopic OEM protein PLASTID DIVISION2 (PDV2). ARC6-PDV2 interaction is required for the localization of PDV2 to the midplastid (Glynn et al., 2008). PDV2 and its paralog PDV1, also in the OEM, in turn recruit DRP5B from a cytosolic pool to the OEM (Miyagishima et al., 2006), probably through direct interaction with their cytosolic regions (Holtsmark et al., 2013). Thus, interactions between FtsZ2 and ARC6 in the stroma, ARC6 and PDV2 in the IMS, and PDV2 (and PDV1) and DRP5B in the cytosol connect and coordinate the FtsZ and DRPB5B rings across the IEM and OEM.Previously, we showed that, despite the fact that an interaction between the IMS regions of ARC6 and PDV1 could not be detected, ARC6 was nevertheless required for the equatorial localization of PDV1 as well as PDV2, suggesting the existence of a factor that acted downstream of ARC6 to position PDV1 (Glynn et al., 2008). This downstream factor was subsequently shown to be the nucleus-encoded chloroplast division protein PARALOG OF ARC6 (PARC6; Glynn et al., 2009), also called CDP1 (Zhang et al., 2009) and ARC6H (Ottesen et al., 2010). parc6 mutants exhibited mislocalization of PDV1 but not PDV2, demonstrating a specific role for PARC6 in PDV1 positioning. PARC6 is restricted to vascular plants, suggesting that it arose by the duplication and divergence of ARC6 following separation of the nonvascular and vascular lineages. As suggested by its name, PARC6 shares significant sequence similarity with ARC6 and is similarly imported to the chloroplast by a cleavable N-terminal transit peptide and localized in the IEM. However, whereas ARC6 has a single transmembrane domain (TMD), PARC6 is predicted to bear two, and while a portion of its N terminus was clearly shown to reside in the stroma, its full topology has not been established (Glynn et al., 2009). Furthermore, genetic analysis suggested that, unlike ARC6, which positively regulates FtsZ assembly (Vitha et al., 2003), PARC6 functions partly as a negative regulator of FtsZ assembly. Interaction assays provided evidence that this negative regulation may be mediated by interaction of the N terminus of PARC6 with the stromal division protein ARC3 (Pyke et al., 1994; Shimada et al., 2004; Maple et al., 2007), a Z-ring positioning factor recently shown to inhibit Z-ring assembly and/or promote FtsZ filament and Z-ring destabilization (TerBush and Osteryoung, 2012; Zhang et al., 2013; Johnson et al., 2015). Although the interaction of PARC6 with FtsZ was not detected previously, the significance of this finding has remained uncertain in the absence of definitive data on PARC6 topology (Glynn et al., 2009).Here, we report a detailed topological analysis of Arabidopsis PARC6, investigate its interactions with other division factors, and assess the effect of PARC6 on chloroplast FtsZ assembly. Our findings provide evidence that the negative effect of PARC6 on Z-ring assembly results from its interaction with ARC3 and reveal a role for PARC6 in coordinating the inner Z ring and outer DRP5B ring partially analogous to the role of ARC6.