Protoplasma (2007) 232: 1–9 DOI 10.1007/s00709-007-0284-5
PROTOPLASMA Printed in Austria
An integrated physiological and genetic approach to the dynamics of FtsZ targeting and organisation in a moss, Physcomitrella patens I. Suppanz*, E. Sarnighausen, R. Reski Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg Received 1 June 2007; Accepted 15 July 2007; Published online 19 December 2007 © Springer-Verlag 2007
Summary. Plant FtsZ (filamentous temperature-sensitive Z) proteins are regarded as descendants of prokaryotic cell division proteins. We could show previously that four FtsZ isoforms of the moss Physcomitrella patens assemble into, and interact in, distinct structures inside the chloroplasts and in the cytosol. Their organisation and localisation patterns indicate an involvement in chloroplast and cell division and in the maintenance of chloroplast shape and integrity. The cellular processes of chloroplast division and maintenance of chloroplast shape were disturbed either by application of the -lactam antibiotic ampicillin or by a mutation that presumably affects signal transduction of the plant hormone cytokinin. When cells of these plants were analysed microscopically, there was no indication that cytosolic functions of FtsZ proteins were affected. Furthermore, FtsZ proteins continued to build three-dimensional plastoskeleton networks, even in considerably enlarged or malformed chloroplasts. On the other hand, macrochloroplast formation promoted the localisation of FtsZ proteins in filaments that emanate from the plastids and, therefore, most likely represent stromules. Annular FtsZ structures that are regarded as essential components of the division apparatus were absent from macrochloroplasts of ampicillin-treated cells. Thus, the distribution of FtsZ proteins after inhibition of chloroplast division further strengthens our hypothesis on the functions of distinct isoforms. In addition, the results provide further insight into the regulation of protein targeting and dynamics of plastoskeletal elements. Keywords: Ampicillin; Chloroplast division; Cytokinin; Plastoskeleton; Stromule; Systems biology. Abbreviations: FtsZ filamentous temperature-sensitive Z protein; GFP green-fluorescent protein.
Correspondence: Ralf Reski, Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Federal Republic of Germany. E-mail:
[email protected] * Present address: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Federal Republic of Germany
Introduction Chloroplasts developed from free-living cyanobacteria that were engulfed by a host cell (McFadden 1999). In the course of the endosymbiotic relationship, genomes of the host and endosymbiont were extensively reshuffled (Martin et al. 2002). During this process, the endosymbiont genome was drastically reduced: while the cyanobacterium Synechocystis sp. encodes about 3168 proteins, chloroplast genomes contain between 60 and 200 protein-coding genes (Martin and Herrmann 1998). A large number of genes, the products of which are considered important for the survival of the endosymbiont, were translocated to the host nucleus, which in turn required the establishment of a chloroplast protein import machinery (Dyall et al. 2004). Control of the endosymbiont functions was thus mainly transferred to the host gene expression system. Those genes related exclusively to the former free-living lifestyle of the endosymbiont became obsolete and were depleted from the genome. Concomitantly, a large number of the initial features of the endosymbiont that either hindered cooperation between host and endosymbiont or did not provide any advantage were lost during fine tuning of the endosymbiotic alliance. It was the detection of a plant homologue of the bacterial ftsZ gene that provided the first hints of an incorporation of the prokaryotic division apparatus into the division machinery of chloroplasts (Osteryoung and Vierling 1995). In most eubacteria and archea, cell division is initiated by the assembly of the filamentous temperature-sensitive Z (FtsZ) protein, which self-polymerises to form a contractile cytoplasmic ring at the future site of division. This Zring spans the whole circumference of the prokaryote and
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acts as a scaffold for the recruitment of the other proteins that together build the division apparatus (divisome) (Margolin 2000, 2005; Goehring and Beckwith 2005). While ftsZ is present as a single copy in almost all prokaryotes (Lutkenhaus and Addinall 1997), plant nuclear genomes contain multiple homologues of ftsZ genes in two small subfamilies (Osteryoung et al. 1998, Rensing et al. 2004). The gene products are targeted to the chloroplasts and participation in chloroplast division has been demonstrated or suggested for a number of these proteins (Osteryoung et al. 1998, Strepp et al. 1998, Wang et al. 2002). Homologues of most bacterial divisomal proteins are absent from the chloroplast division machinery (Miyagishima 2005, Maple and Møller 2007) and the question of why the evolution of chloroplasts required duplication and diversification of ftsZ genes is still a matter of debate. The location of four FtsZ isoforms has been investigated in Physcomitrella patens. FtsZ2-1 and FtsZ2-2 assemble into node-based networks in the chloroplasts of P. patens. It has been suggested that these structures build the plastoskeleton and play a role in the maintenance of chloroplast shape and integrity (Kiessling et al. 2000, Reski 2002). The importance of FtsZ2-1 in chloroplast division in P. patens was shown by targeted knockout of the respective nuclear gene (Strepp et al. 1998). FtsZ1-1 assembles into networks that are distinctively different from those built by FtsZ2-1 and FtsZ2-2. In addition, the protein is also detected in filaments that connect individual plastids (Gremillon et al. 2007). FtsZ1-2 is dually targeted to the chloroplasts and to the cytosol and forms annular structures in both compartments (Kiessling et al. 2004). -Lactam antibiotics, such as penicillin, ampicillin, and cefotaxime, act on bacterial enzymes that catalyse the final steps of peptidoglycan synthesis (Yocum et al. 1979, Waxman and Strominger 1983). As the presence of a peptidoglycan cell wall distinguishes eukaryotes from prokaryotes, -lactam antibiotics have been used extensively for the treatment of bacterial infections in humans, animals, and eukaryotic cell cultures (Rolinson and Geddes 2007). Only the chloroplasts (cyanelles) of the glaucocystophytes have retained the murein cell wall of their cyanobacterial ancestors (Loffelhardt et al. 1997). As a consequence, cyanelle division is sensitive to -lactam antibiotics (Kies 1988). It is, however, not understood why the same is true for chloroplasts of the liverwort Marchantia polymorpha (Tounou et al. 2002), the mosses Funaria hygrometrica and P. patens (Kasten and Reski 1997, Katayama et al. 2003), and the pteridophyte Selaginella nipponica (Izumi et al. 2003). Physcomitrella patens expresses nine homologues of bacterial cell wall synthesis genes. A penicillin-binding protein (PpPBP) is as-
sumed to be the target of -lactam antibiotics. Disruption of the pbp gene results in an inhibition of chloroplast division in P. patens (Machida et al. 2006). The requirement of the putative activity (transglycosylase, transpeptidase) of this protein for chloroplast division is not understood, as the chloroplasts of P. patens do not contain any peptidoglycans. It seems plausible that plastid division in P. patens represents an intermediate state, probably linking cyanobacteria and the plastids of seed plants, which are nonresponsive to -lactams (Kasten and Reski 1997, Glynn et al. 2007). Analysis of the chloroplast division machinery of moss, therefore, holds out the prospect of gaining insights not only into this division process itself but also into the overall process of endosymbiosis evolution. The mutant PC22 of P. patens was generated via untargeted X-ray mutagenesis. It is impaired in plastid division (pdi) and in gametophore development (gad) (Abel et al. 1989). The defects in chloroplast division can be partly compensated for by elevation of cytokinin levels or irradiation with blue light (Reski et al. 1991, Kasten et al. 1997, Reutter et al. 1998), indicating that functions unrelated to pbp and ftsZ genes are affected in the mutant. Endogenous cytokinin levels are not reduced in PC22. It is, therefore, probable that the mutation interferes with signal transduction mechanisms rather than with cytokinin metabolism (Reutter et al. 1998). It appears that FtsZ assembly and disassembly in plants is as dynamic as that of the classical cytoskeleton. As a prerequisite for the study of the function of FtsZ in this highly dynamic context, we here analyse the effects of an ampicillin-induced inhibition of chloroplast division and the consequences of a block in chloroplast division in the pdi mutant PC22 on the formation of cellular FtsZ structures. This integrated physiological and genetic approach will increase understanding of the particular functions of distinct FtsZ isoforms and aid modelling of their interactions and dynamics in a systems biology approach. Material and methods Plant growth Physcomitrella patens Hedw. (B.S.G.) protonemata of wild type and the mutant PC22 (Abel et al. 1989) were propagated in liquid culture in modified Knop medium as described previously (Reski et al. 1994). The moss protonemata were transferred to fresh medium and chopped once a week to retain the plants in the developmental state of young protonema (chloronema). In order to improve transformation efficiency, protoplasts were prepared from plant material that had been grown in medium with a calcium concentration reduced by a factor of ten for 23 days (Rother et al. 1994). Chloroplast division of wild-type moss was blocked by supplementing the Knop medium with 100 M ampicillin as described by Kasten and Reski (1997). Moss protonemata were grown in the presence of the antibi-
I. Suppanz et al.: FtsZ polymerisation in division-blocked chloroplasts of Physcomitrella patens otic for up to 72 days. Cytokinin (N6-,-(dimethylallyl)aminopurine) was applied to the growth medium at a final concentration of 5 M from a 10,000-fold stock solution.
Transient transfection of protoplasts Cellular localisations of four P. patens FtsZ proteins, FtsZ1-1, FtsZ1-2, FtsZ2-1, and FtsZ2-2, were investigated by transient transfection of protoplasts with ftsZ-gfp fusion constructs, the synthesis of which has been described elsewhere (Kiessling et al. 2000, 2004; Gremillon et al. 2007). Preparation and transfection of moss protoplasts were performed according to standard procedures (Frank et al. 2005). Briefly, the protonemata were harvested by filtration and incubated with 1% driselase (Sigma, St. Louis, Mo., U.S.A.) in 0.5 M mannitol, pH 5.8, for 45 min at room temperature with gentle agitation. Protoplasts were obtained after filtration through 100 m and subsequently 45 m pore size gauze and sedimentation at 45 g for 10 min. Protoplasts were washed twice with 0.5 M mannitol and subsequently resuspended in 3 M medium (15 mM MgCl2, 0.1% 2(N-morpholino)ethanesulfonic acid, 0.48 M mannitol, pH 5.6) at a density of 1.2 106 protoplasts per ml. Finally, protoplasts were transfected with 25 g of circular DNA in the presence of 20% polyethylene glycol 4000 in a total volume of 700 l and kept at 25 °C in the growth chamber under 16 h long day conditions prior to microscopic analysis.
Confocal laser scanning microscopy Confocal laser scanning microscopy was performed using a Leica TCS 4D CLS microscope (Leica, Wetzlar, Federal Republic of Germany). Samples were excited at 488 nm, green-fluorescent protein (GFP) fluorescence was detected through a 520–550 nm bandpass emission filter, while a 590 nm longpass filter was used to monitor chlorophyll autofluorescence. Optical sections were taken at 1 m intervals. An oil immersion objective PLAN APO 100 (numerical aperture, 1.4) (Nikon, Düsseldorf, Federal Republic of Germany) was used.
Results FtsZ structures in ampicillin-treated cells of P. patens Growing the moss in the presence of ampicillin led to a severe inhibition of chloroplast division. In order to investigate the effect of this inhibition on the formation of the different FtsZ structures, transfection experiments were performed with moss protoplasts prepared from protonemata that had grown in the presence of 100 M ampicillin for ten weeks. At this time, each protonema cell contained one to only a few macrochloroplasts. The polymerisation characteristics of the FtsZ1-1–GFP protein were obviously not severely affected by the dramatic increase in chloroplast size. The protein assembled into network structures inside the regular chloroplasts of untreated plants (Fig. 1A) and behaved similarly in the giant chloroplasts of ampicillin-treated moss cells (Fig. 1B). However, apart from the network structures inside the chloroplasts, the fluorescent label was detectable in thin filaments that formed connections between different regions of the same plastid (Fig. 1C).
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FtsZ1-2 is dually targeted and builds annular structures in both the chloroplasts and the cytosol (Fig. 1D). The distribution of the FtsZ1-2–GFP protein was obviously connected to the size of the chloroplasts. While the fusion protein was sometimes found to build spooled filaments inside enlarged plastids (Fig. 1E), the label was completely absent from many macrochloroplasts (Fig. 1F). In untreated protoplasts, there was never a connection between the chloroplasts and cytosolic FtsZ1-2, but some of the macrochloroplasts now appeared to be pierced by cytosolic FtsZ filaments (Fig. 1E). Plastidic FtsZ2-1–GFP networks in protoplasts prepared from untreated moss protonemata (Fig. 1G) closely resembled the structures detectable in ampicillin-induced macrochloroplasts (Fig. 1H). The only difference was the extrusion of fluorescent filaments from the plastids into the cytoplasm, which was only detectable after prolonged ampicillin exposure (Fig. 1H). The typical structure of FtsZ2-2–GFP networks consisting of large nodes connected by thin filaments in the chloroplasts (Fig. 1I) was also found in ampicillin-induced macrochloroplasts (Fig. 1J). Cytosolic localisation of FtsZ2-2 was never observed. FtsZ structures in P. patens plastid division mutant PC22 The P. patens mutant PC22 is characterised by a repression of plastid division, which is observed when plants are grown under standard conditions. Three days after transfection of PC22 protoplasts with the ftsZ1-1–gfp fusion construct, networks of fluorescent filaments were present, mainly in the cytoplasm (Fig. 2 A, B). A delocalisation of the protein took place with time and later stages were characterised by a clear predominance of dense plastidic networks (Fig. 2C, D). FtsZ1-2–GFP fusions could always be detected forming cytoplasmic rings. If plastids of almost regular size were present in the protoplasts, these showed annular FtsZ12–GFP structures comparable to the wild-type control. Filaments pervaded macrochloroplasts, on the other hand, in a rather unordered manner (Fig. 2E, F). In the protoplasts of the P. patens PC22 mutant, the structures built by FtsZ2-1–GFP (Fig. 2G) and FtsZ2-2–GFP (Fig. 2H) were barely distinguishable. Both proteins assembled into the characteristic networks known from the wild type, also within the macrochloroplasts. The FtsZ2-2 network was again characterised by distinct nodes that were connected by rather thin filaments, whereas in the case of FtsZ2-1 the network structure was more symmetric. Cytoplasmic fluorescent filaments were sometimes detectable in the case of FtsZ2-2–GFP (Fig. 2H).
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I. Suppanz et al.: FtsZ polymerisation in division-blocked chloroplasts of Physcomitrella patens
Fig. 1 A–J. Localisation of FtsZ isoforms in protoplasts of P. patens. A In control experiments, FtsZ1-1–GFP assembled into network structures inside the chloroplasts. B and C The network structure was retained in ampicillin-induced macrochloroplasts (B), and FtsZ1-1–GFP was also found in filaments connecting distinct regions of the macrochloroplast (C). D FtsZ1-2–GFP is dually targeted and built annular structures in the chloroplasts but also in the cytosol of untreated protoplasts. E Following ampicillin treatment, FtsZ1-2–GFP still built annular structures in the cytosol and in macrochloroplasts, and cytosolic FtsZ appeared to pierce the macrochloroplasts (arrow). F In protoplasts harbouring large macrochloroplasts, FtsZ1-2 was mostly detected in the cytosol. G FtsZ2-1–GFP built node-based networks in the chloroplasts in control experiments. H These network structures were also found in ampicillin-induced macrochloroplasts, but short filaments sometimes extended from the macrochloroplast (arrow). I In untreated protoplasts, FtsZ2-2 structures closely resembled FtsZ2-1 networks, although the filaments connecting the nodes were usually thinner in the FtsZ2-2 plastoskeleton. J The plastoskeleton was also found in ampicillin-induced macrochloroplasts. Confocal laser scanning micrographs show the merge of GFP fluorescence (green) and chlorophyll autofluorescence (red). Except for panel C, which shows a single optical layer, all images are three-dimensional reconstructions derived from 7 to 16 optical sections. Bars: 5 m
The structure and distribution of the FtsZ2-1–GFP fusion protein, however, were profoundly affected when protoplasts were allowed to regenerate protonemata. While still being present inside chloroplasts in the form of characteristic networks, the protein was also detectable in cytoplasmic filaments that appeared to be released into the
surrounding cytosol from the chloroplasts (Fig. 3A). This effect was markedly increased when regeneration took place in the presence of the phytohormone cytokinin (Fig. 3B). Appearance of cytosolic filaments was also observable in the case of FtsZ2-2, albeit to a much lower extent (Fig. 3C, D).
I. Suppanz et al.: FtsZ polymerisation in division-blocked chloroplasts of Physcomitrella patens
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Fig. 2 A–H. FtsZ structures in macrochloroplasts of the P. patens mutant PC22. Three days after transient transfection, FtsZ1-1–GFP was mainly found as cytosolic filaments (A and B), and 11 days after transfection, the fluorescent label was mostly detected in the macrochloroplasts (C and D). FtsZ1-2–GFP was dually targeted to the cytosol and the chloroplasts (E and F). FtsZ2-1–GFP (G) and FtsZ2-2–GFP (H) assembled into network structures in the macrochloroplasts. FtsZ22–GFP was also found in filaments emanating from the macrochloroplast (arrow in H). Confocal laser scanning micrographs A, C, and D show GFP fluorescence (green), all other images show the merge of GFP fluorescence (green) and chlorophyll autofluorescence (red). All images are three-dimensional reconstructions derived from 15 to 20 optical sections. Bars: 5 m
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Fig. 3 A–D. Distribution of FtsZ2 isoforms in regenerating protoplasts of the P. patens mutant PC22. A In regenerating protoplasts, FtsZ2-1–GFP was found in network structures inside the macrochloroplasts and also in short cytosolic filaments emanating from the chloroplasts (arrow). B After application of cytokinin, large amounts of FtsZ2-1–GFP appeared to be extruded from the macrochloroplasts. C and D Cytosolic localisation of FtsZ2-2–GFP was also observable in regenerating chloroplasts of PC22 (C, arrow). Here the effect of cytokinin application was far less pronounced (D, arrow). Bars: 5 m
Discussion The moss P. patens is a model organism in the field of plant biology, mainly due to its small size, the ease of highly standardised cultivation, and its amenability to genetic engineering via direct gene targeting. The growth cycle of mosses is dominated by the haploid gametophytic generation, where induced alterations of gene functions cannot be masked by dominant alleles. As a consequence, genetic manipulations can directly affect the moss phenotype (Reski and Cove 2004). The functions of FtsZ proteins and their diversification have long been investigated in P. patens (Strepp et al. 1998; Kiessling et al. 2000, 2004; Reski 2002; Rensing et al. 2004; Gremillon et al. 2007). The results suggest a coordinated involvement of these proteins in the static and dynamic behaviour of the chloroplasts and even of the whole cell. To further test these hypotheses, we deliberately put the FtsZ system out of equilibrium. To this end, interference with the chloroplast division process was accomplished via physiological mechanisms, by application of ampicillin (Kasten and Reski 1997, Katayama et al. 2003) or cytokinin (Reski et al. 1991, Kasten et al. 1997). In a genetic approach, formation of FtsZ structures in the
chloroplast division-deficient mutant PC22 was analysed. The young moss gametophyte, the protonema, consists of uniseriate branched filaments that grow by division of the tip cells; therefore, each protonema thread represents a cell lineage (Reski 1998). When protoplasts generated from protonemata grown in the presence of ampicillin were transiently transfected with ftsZ1-1–gfp, the protein was detected in network structures and in filaments that connected different regions of the single macrochloroplast. It has been shown before that FtsZ1-1–GFP fusion proteins are found in strands interconnecting chloroplasts in P. patens (Gremillon et al. 2007). The outer surface of chloroplasts has the ability to form thin stroma-filled tubules, named stromules (Kohler and Hanson 2000). Whether FtsZ1-1 merely prefers to enter stromules or even induces their formation is not known. The function of stromules is still not resolved. It has been suggested that these highly dynamic structures are built to increase the total surface area of the chloroplasts in order to facilitate or promote exchange of compounds with the cytosol (Natesan et al. 2005). This hypothesis is consistent with our finding that inhibition of chloroplast division probably promotes stromule formation. As the size of chloroplasts increases
I. Suppanz et al.: FtsZ polymerisation in division-blocked chloroplasts of Physcomitrella patens
after application of ampicillin, the surface-to-volume ratio of the organelle might well decrease towards critical values. The exchange of compounds across the chloroplast surface might hence become a bottleneck for chloroplast and/or cellular function. However, an increased formation of stromules would counteract this relative loss of surface area. On the other hand, plastid interconnections built by stromules may have a pipeline function. In fact, GFP fusions of aspartate aminotransferase and of the large subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase have been shown to move between chloroplasts via the stromules (Kwok and Hanson 2004b). In this functional context, the stromules in a giant chloroplast might act as shortcuts facilitating transport and equilibrium processes inside the chloroplast itself. Formation of cytosolic FtsZ1-1–GFP filaments was even more pronounced in protoplasts of the mutant PC22. However, the distribution of the fusion protein shifted with time from a predominant cytosolic location to the formation of network structures inside the chloroplasts. It is assumed that shortly after transfection, FtsZ1-1–GFP is imported into the chloroplasts and subsequently efficiently translocated to the stromules, which are exceedingly numerous at this time point. Apparently, preparation of protoplasts promotes the formation of stromules in PC22 cells. In fact, the involvement of stromules in the adaptation of plant cells to various stresses has been suggested (Shiina et al. 2001) but is still a matter of debate (Kwok and Hanson 2004a). The formation of annular structures by FtsZ1-2 in ampicillin-treated plants was obviously connected to the size of the chloroplasts, as large macrochloroplasts were found to not contain FtsZ1-2 filaments. Cytosolic FtsZ1-2 filaments displayed the rather surprising feature of piercing the macrochloroplasts. A comparable phenomenon has never been observed with regularly sized chloroplasts and its physiological relevance therefore remains to be elucidated. Dual targeting of FtsZ1-2 to the cytosol and to the chloroplasts is due to the use of different translation initiation sites (Kiessling et al. 2004). It is, however, not known how targeting is controlled. In our experiments, the distribution of FtsZ1-2 between cytosol and macrochloroplasts differed depending on the causes of macrochloroplast formation. When chloroplast division was inhibited by addition of ampicillin, large macrochloroplasts did not contain FtsZ1-2–GFP. However, ampicillin does not seem to exert direct control on FtsZ1-2 targeting as formation of FtsZ1-2 filaments in the chloroplasts was not immediately repressed but still occurred after chloroplast division had obviously ceased. Absence of FtsZ1-2
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from the chloroplasts is a consequence and not the cause of ampicillin-induced inhibition of chloroplast division. It is likely that chloroplast-to-cytosol signals influence targeting specificity, i.e., selection of the translation start codon on cytosolic ribosomes might be controlled by the chloroplast. In the mutant PC22, annular structures are formed by FtsZ1-2–GFP even in large chloroplasts. Probably the arrest in chloroplast division in PC22 and after ampicillin treatment occurs at different time points during the chloroplast division cycle. The results show that the inability of FtsZ1-2 to assemble into ring structures is not (merely) related to chloroplast size. In the case of ampicillin-treated plants, conditions permitting FtsZ1-2 assembly inside the chloroplast would need to be restored before chloroplast division could be resumed. In the mutant PC22, the ability of the macrochloroplasts to still form the annular structure will allow them to immediately proceed with chloroplast division. The consequences are obvious: in contrast to ampicillin-treated moss, inhibition of chloroplast division in PC22 is rather “leaky”; therefore, the cells contain variable numbers of chloroplasts of different sizes. When targeted to the cytosol by the depletion of the transit peptide, proteins of the FtsZ2 family do not build network structures but form aggregates (Kiessling et al. 2004). It is assumed that these proteins require factors specific to the chloroplast for correct assembly. Our results demonstrate that these factors are obviously still present when chloroplast division is inhibited and that network formation does not require the limited volume of regular chloroplasts. The presence of FtsZ2-1 or FtsZ2-2 in stromules or “cytosolic filaments” has never been observed before. Our results again suggest that an increase in chloroplast volume promotes the formation of stromules. Cytokinins have long been known as stimulators of development and replication of chloroplasts (Tsuji et al. 1979, Naito et al. 1981). In mosses, cytokinins induce the formation of a bud cell from a single initial protonema cell, which represents the transition to a three-dimensional growth habit (Brandes and Kende 1968, Reski and Abel 1985). The massive incorporation of FtsZ2-1–GFP into stromules from chloroplasts in regenerating protoplasts of the PC22 mutants, as observed after cytokinin treatment, might be indicative of an enhanced metabolic activity induced in the macrochloroplast by the hormone treatment. In line with this is the observation that cytokinin treatment promotes the expression of key enzymes of the chloroplast energy conversion system in this mutant (Kasten et al.
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1997). It cannot be ruled out completely, however, that these observations are artefacts due to overexpression of FtsZ-GFP fusion proteins. As P. patens is highly amenable to reverse genetics (Hohe and Reski 2003, Hohe et al. 2004), this hypothesis will be challenged by targeted knockouts of the respective nuclear-encoded genes. Our findings further support the proposed function of the distinct FtsZ isoforms in P. patens (Kiessling et al. 2000, 2004; Gremillon et al. 2007). Those structures that are presumably relevant for the maintenance of the integrity of the chloroplasts, built by the plastoskeleton-forming isoforms FtsZ1-1, FtsZ2-1, and FtsZ2-2, are retained in macrochloroplasts. In large macrochloroplasts induced by ampicillin treatment, assembly of FtsZ1-2 is repressed, while annular structures are still built in the cytosol of the same protoplasts. FtsZ1-2 rings are assumed to participate in chloroplast and cell division in P. patens (Kiessling et al. 2004). Our observations made after ampicillin treatment are in accordance with the repression of chloroplast division but not of cell division by the antibiotic. The FtsZ ring in Escherichia coli has been shown to be a highly dynamic structure which continually remodels itself (Stricker et al. 2002). While dynamic behaviour is to be expected from a contractile structure like the Z-ring, the plastoskeleton should preferentially be stable. However, evidence suggests that the FtsZ1-1 network in P. patens disassembles or undergoes reconstruction during plastokinesis (Gremillon et al. 2007). The massive relocation of FtsZ1-1 from cytosolic filaments to plastoskeletal networks observed in protoplasts of the PC22 mutant supports the view that FtsZ proteins build highly flexible structures in P. patens. On the basis of the data presented here, we will determine precise functions for specific FtsZ proteins by the production of loss-of-function mutants via targeted gene knockout and start to model the dynamics of the plastoskeleton in a systems biology approach. Acknowledgments Funding by Deutsche Forschungsgemeinschaft (SFB388) to R.R. is gratefully acknowledged. We are grateful to Anne Katrin Prowse for help with the draft of the manuscript.
References Abel WO, Knebel W, Koop HU, Marienfeld JR, Quader H, Reski R, Schnepf E, Sporlein B (1989) A cytokinin-sensitive mutant of the moss, Physcomitrella patens, defective in chloroplast division. Protoplasma 152: 1–13 Brandes H, Kende H (1968) Studies on cytokinin-controlled bud formation in moss protonemata. Plant Physiol 43: 827–837 Dyall SD, Brown MT, Johnson PJ (2004) Ancient invasions: from endosymbionts to organelles. Science 304: 253–257
Frank W, Decker EL, Reski R (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7: 220–227 Glynn JM, Miyagishima S, Yoder DW, Osteryoung KW, Vitha S (2007) Chloroplast division. Traffic 8: 451–461 Goehring NW, Beckwith J (2005) Diverse paths to midcell: assembly of the bacterial cell division machinery. Curr Biol 15: R514–R526 Gremillon L, Kiessling J, Hause B, Decker EL, Reski R, Sarnighausen E (2007) FtsZ isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens. New Phytol 176: 299–310 Hohe A, Reski R (2003) A tool for understanding homologous recombination in plants. Plant Cell Rep 21: 1135–1142 Hohe A, Egener T, Lucht JM, Holtorf H, Reinhard C, Schween G, Reski R (2004) An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted geneknockouts in a moss, Physcomitrella patens. Curr Genet 44: 339–347 Izumi Y, Ono K, Takano H (2003) Inhibition of plastid division by ampicillin in the pteridophyte Selaginella nipponica Fr. et Sav. Plant Cell Physiol 44: 183–189 Kasten B, Reski R (1997) -lactam antibiotics inhibit chloroplast division in a moss (Physcomitrella patens) but not in tomato (Lycopersicon esculentum). J Plant Physiol 150: 137–140 Kasten B, Buck F, Nuske J, Reski R (1997) Cytokinin affects nuclearand plastome-encoded energy-converting plastid enzymes. Planta 201: 261–272 Katayama N, Takano H, Sugiyama M, Takio S, Sakai A, Tanaka K, Kuroiwa H, Ono K (2003) Effects of antibiotics that inhibit the bacterial peptidoglycan synthesis pathway on moss chloroplast division. Plant Cell Physiol 44: 776–781 Kies L (1988) The effect of penicillin on the morphology and ultrastructure of Cyanophora, Gleochaete and Glaucocystis (Glaucocystophycea) and their cyanelles. Endocyt Cell Res 5: 361–372 Kiessling J, Kruse S, Rensing SA, Harter K, Decker EL, Reski R (2000) Visualization of a cytoskeleton-like FtsZ network in chloroplasts. J Cell Biol 151: 945–950 Kiessling J, Martin A, Gremillon L, Rensing SA, Nick P, Sarnighausen E, Decker EL, Reski R (2004) Dual targeting of plastid division protein FtsZ to chloroplasts and the cytoplasm. EMBO Rep 5: 889–894 Kohler RH, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and developmentally regulated. J Cell Sci 113: 81–89 Kwok EY, Hanson MR (2004a) Stromules and the dynamic nature of plastid morphology. J Microsc 214: 124–137 Kwok EY, Hanson MR (2004b) GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids. J Exp Bot 55: 595–604 Loffelhardt W, Bohnert HJ, Bryant DA (1997) The cyanelles of Cyanophora paradoxa. Crit Rev Plant Sci 16: 393–413 Lutkenhaus J, Addinall SG (1997) Bacterial cell division and the Z ring. Annu Rev Biochem 66: 93–116 Machida M, Takechi K, Sato H, Chung SJ, Kuroiwa H, Takio S, Seki M, Shinozaki K, Fujita T, Hasebe M, Takano H (2006) Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss. Proc Natl Acad Sci USA 103: 6753–6758 Maple J, Møller SG (2007) Plastid division: evolution, mechanism and complexity. Ann Bot 99: 565–579 Margolin W (2000) Themes and variations in prokaryotic cell division. FEMS Microbiol Rev 24: 531–548 Margolin W (2005) FtsZ and the division of prokaryotic cells and organelles. Nat Rev Mol Cell Biol 6: 862–871 Martin W, Herrmann RG (1998) Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol 118: 9–17 Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M, Penny D (2002) Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci USA 99: 12246–12251
I. Suppanz et al.: FtsZ polymerisation in division-blocked chloroplasts of Physcomitrella patens McFadden GI (1999) Endosymbiosis and evolution of the plant cell. Curr Opin Plant Biol 2: 513–519 Miyagishima S (2005) Origin and evolution of the chloroplast division machinery. J Plant Res 118: 295–306 Naito K, Ueda K, Tsuji H (1981) Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age. Protoplasma 105: 293–306 Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56: 787–797 Osteryoung KW, Vierling E (1995) Conserved cell and organelle division. Nature 376: 473–474 Osteryoung KW, Stokes KD, Rutherford SM, Percival AL, Lee WY (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10: 1991–2004 Rensing SA, Kiessling J, Reski R, Decker EL (2004) Diversification of ftsZ during early land plant evolution. J Mol Evol 58: 154–162 Reski R (1998) Development, genetics and molecular biology of mosses. Bot Acta 111: 1–15 Reski R (2002) Rings and networks: the amazing complexity of FtsZ in chloroplasts. Trends Plant Sci 7: 103–105 Reski R, Abel WO (1985) Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine. Planta 165: 354–358 Reski R, Cove DJ (2004) Physcomitrella patens. Curr Biol 14: R261–R262 Reski R, Wehe M, Hadeler B, Marienfeld JR, Abel WO (1991) Cytokinin and light quality interact at the molecular level in the chloroplast mutant PC22 of the moss Physcomitrella. J Plant Physiol 138: 236–243 Reski R, Faust M, Wang XH, Wehe M, Abel WO (1994) Genome analysis of the moss Physcomitrella patens (Hedw.) B.S.G. Mol Gen Genet 244: 352–359 Reutter K, Atzorn R, Hadeler B, Schmulling T, Reski R (1998) Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division. Planta 206: 196–203
9
Rolinson GN, Geddes AM (2007) The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA). Int J Antimicrob Agents 29: 3–8 Rother S, Hadeler B, Orsini JM, Abel WO, Reski R (1994) Fate of a mutant macrochloroplast in somatic hybrids. J Plant Physiol 143: 72–77 Shiina T, Hayashi K, Ishii N, Morikawa K, Iwai K, Yasuda H, Yamashita H, Toyoshima Y (2001) Characterization of stromules visualized in transplastomic plants expressing green fluorescent protein (GFP). In: PS2001 Proceedings: 12th International Congress on Photosynthesis. CSIRO Publishing, Melbourne, S23-004 Strepp R, Scholz S, Kruse S, Speth V, Reski R (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci USA 95: 4368–4373 Stricker J, Maddox P, Salmon ED, Erickson HP (2002) Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching. Proc Natl Acad Sci USA 99: 3171–3175 Tounou E, Takio S, Sakai A, Ono K, Takano H (2002) Ampicillin inhibits chloroplast division in cultured cells of the liverwort Marchantia polymorpha. Cytologia 67: 429–434 Tsuji H, Naito K, Hatakeyama I, Ueda K (1979) Benzyladenine-induced increase in DNA content per cell, chloroplast size, and chloroplast number per cell in intact bean leaves. J Exp Bot 30: 1145–1151 Wang D, Kong D-D, Ju C-L, Hu Y, He Y-K, Sun J-S (2002) Effects of tobacco plastid division genes NtFtsZ1 and NtFtsZ2 on the division and morphology of chloroplasts. Acta Bot Sin 44: 838–844 Waxman DJ, Strominger JL (1983) Penicillin-binding proteins and the mechanism of action of -lactam antibiotics. Annu Rev Biochem 52: 825–869 Yocum RR, Waxman DJ, Rasmussen JR, Strominger JL (1979) Mechanism of penicillin action: penicillin and substrate bind covalently to the same active site serine in two bacterial D-alanine carboxypeptidases. Proc Natl Acad Sci USA 76: 2730–2734
