The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings. Received: 19 May 2000 / Accepted: 3 August 2000     

Two Types of FtsZ Proteins in Mitochondria and Red-Lineage Chloroplasts: The Duplication of FtsZ Is Implicated in Endosymbiosis

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.     

Evolution of the chloroplast division machinery

Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution.Dramatic changes occurred during the process of the formation and evolution of chloroplasts,including the large-scale gene transfer from chloroplast to nucleus.However,there are still many essential characters remaining.For the chloroplast division machinery,FtsZ proteins,Ftn2,SulA and part of the division site positioning system- MinD and MinE are still conserved.New or at least partially new proteins,such as FtsZ family proteins FtsZl and ARC3,ARC6H,ARC5,PDV1,PDV2 and MCD1,were introduced for the division of chloroplasts during evolution.Some bacterial cell division proteins,such as FtsA,MreB,Ftn6,FtsW and Ftsl,probably lost their function or were gradually lost.Thus,the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

Oligomerization of plant FtsZ1 and FtsZ2 plastid division proteins

FtsZ was identified in bacteria as the first protein to localize mid-cell prior to division and homologs have been found in many plant species. Bacterial studies demonstrated that FtsZ forms a ring structure that is dynamically exchanged with a soluble pool of FtsZ. Our previous work established that Arabidopsis FtsZ1 and FtsZ2-1 are capable of in vitro self-assembly into two distinct filament types, termed type-I and type-II and noted the presence of filament precursor molecules which prompted this investigation. Using a combination of electron microscopy, gel chromatography and native PAGE revealed that (i) prior to FtsZ assembly initiation the pool consists solely of dimers and (ii) during assembly of the Arabidopsis FtsZ type-II filaments the most common intermediate between the dimer and filament state is a tetramer. Three-dimensional reconstructions of the observed dimer and tetramer suggest these oligomeric forms may represent consecutive steps in type-II filament assembly and a mechanism is proposed, which is expanded to include FtsZ assembly into type-I filaments. Finally, the results permit a discussion of the oligomeric nature of the soluble pool in plants.     

PARC6, a novel chloroplast division factor, influences FtsZ assembly and is required for recruitment of PDV1 during chloroplast division in Arabidopsis

Chloroplast division in plant cells is accomplished through the coordinated action of the tubulin-like FtsZ ring inside the organelle and the dynamin-like ARC5 ring outside the organelle. This coordination is facilitated by ARC6, an inner envelope protein required for both assembly of FtsZ and recruitment of ARC5. Recently, we showed that ARC6 specifies the mid-plastid positioning of the outer envelope proteins PDV1 and PDV2, which have parallel functions in dynamin recruitment. PDV2 positioning involves direct ARC6–PDV2 interaction, but PDV1 and ARC6 do not interact indicating that an additional factor functions downstream of ARC6 to position PDV1. Here, we show that PARC6 (paralog of ARC6), an ARC6-like protein unique to vascular plants, fulfills this role. Like ARC6, PARC6 is an inner envelope protein with its N-terminus exposed to the stroma and Arabidopsis parc6 mutants exhibit defects of chloroplast and FtsZ filament morphology. However, whereas ARC6 promotes FtsZ assembly, PARC6 appears to inhibit FtsZ assembly, suggesting that ARC6 and PARC6 function as antagonistic regulators of FtsZ dynamics. The FtsZ inhibitory activity of PARC6 may involve its interaction with the FtsZ-positioning factor ARC3. A PARC6–GFP fusion protein localizes both to the mid-plastid and to a single spot at one pole, reminiscent of the localization of ARC3, PDV1 and ARC5. Although PARC6 localizes PDV1, it is not required for PDV2 localization or ARC5 recruitment. Our findings indicate that PARC6, like ARC6, plays a role in coordinating the internal and external components of the chloroplast division complex, but that PARC6 has evolved distinct functions in the division process.     

衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E．coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。     

Studies on chloroplast division in young leaf tissues of some higher plants

Summary The process of chloroplast division in young leaves of four species (bean, spinach, wheat, and maize) was investigated by light and electron microscopy. Two types of division, i.e., by fission, and by partition were observed.Chloroplast division by fission prevailed in the plant species examined, as shown by the relative abundance of dumbbell-shaped plastids, the characteristic stage in this type of division. Electron dense material, most commonly in the shape of a ring structure in the isthmus of the dividing plastid, was nearly always present in wheat and maize. Similar, but less distinct structures were usually observed in the neck region of constricted bean and spinach chloroplasts.Chloroplast division by partition was found in young leaf tissues of bean and spinach, but was not observed in wheat and maize. The main indication of this type of division is a centripetal invagination of the inner limiting membrane of the plastid envelope which progressively divides the chloroplast stroma into two, nearly equal, parts. Specific membraneous structures resembling myelin figures were usually found close to a dividing chloroplast and may participate in chloroplastokinesis.     

Evolution of the chloroplast division machinery

Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution. Dramatic changes occurred during the process of the formation and evolution of chloroplasts, including the large-scale gene transfer from chloroplast to nucleus. However, there are still many essential characters remaining. For the chloroplast division machinery, FtsZ proteins, Ftn2, SulA and part of the division site positioning system—MinD and MinE are still conserved. New or at least partially new proteins, such as FtsZ family proteins FtsZ1 and ARC3, ARC6H, ARC5, PDV1, PDV2 and MCD1, were introduced for the division of chloroplasts during evolution. Some bacterial cell division proteins, such as FtsA, MreB, Ftn6, FtsW and FtsI, probably lost their function or were gradually lost. Thus, the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.     

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

A uniquely high number of ftsZ genes in the moss Physcomitrella patens

Plant FtsZ proteins are encoded by two small nuclear gene families (FtsZ1 and FtsZ2) and are involved in chloroplast division. From the moss Physcomitrella patens , four FtsZ proteins, two in each nuclear gene family, have been characterised and described so far. In the recently sequenced P. patens genome, we have now found a fifth fts Z gene. This novel gene has a genomic structure similar to Pp fts Z1-1. According to phylogenetic analysis, the encoded protein is a member of the FtsZ1 family, while PpFtsZ1-2, together with an orthologue from Selaginella moellendorffii , forms a separate clade. Further, this new gene is expressed in different gametophytic tissues and the encoded protein forms filamentous networks in chloroplasts, is found in stromules, and acts in plastid division. Based on all these results, we have renamed the PpFtsZ proteins of family 1 and suggest the existence of a third FtsZ family. No species is known to encode more FtsZ proteins per haploid genome than P. patens .     

Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction

The time courses of chloroplast and mitochondrial division and the morphological changes in the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) during chloroplast and mitochondrial division were studied in Cyanidioschyzon merolae De Luca, Taddei and Varano. To accomplish this, chloroplast and cell division of living cells were continuously video-recorded under light microscopy, and the morphological changes in the PD and MD rings were analyzed quantitatively and three-dimensionally by transmission electron microscopy (TEM). Under the light microscope, the diameters of the chloroplast and the cell decreased at uniform velocities, the speed depending on the temperature. To study in detail the sequential morphological change of the mitochondrion in M phase and the contractile mechanism in the divisional planes of the chloroplast and the mitochondrion, we observed the PD and MD rings, which are believed to promote contraction, under TEM, using the diameter of the chloroplast as an index of the time. Three PD rings (an outer PD ring on the cytoplasmic face of the outer envelope, a middle PD ring in the intermembrane space, and an inner PD ring on the stromal face of the inner envelope) were clearly observed, but only the outer MD ring could be observed. The PD ring started to contract soon after it formed, while the contraction of the MD ring did not occur immediately after formation, but was delayed until the contraction of the PD ring was almost complete. Once the MD ring began to contract, the rate of decrease of its circumference was 4 times as high as that of the PD ring. As the outer PD and MD rings contracted, they grew thicker and maintained a constant volume, while the thickness of the inner PD ring did not change and its volume decreased at a constant rate with contraction. In the early stage of contraction, the widths of the three PD rings increased in order, from the outer to the inner ring. With contraction, their widths changed at different rates until they came to have much the same width. In cross-section, the MD ring was wider where it was next to the chloroplast than at the opposite side, adjacent to the nucleus in the early stage of contraction. By the late stage, the widths of the two sides became equal. In our observations, the microbody elongated along the outer MD ring and touched the outer PD ring during contraction of the PD and MD rings. These results clearly revealed differences between the mode of contraction of the outer, middle, and inner PD rings, and between the PD and the MD rings. They also revealed the coordinated widening of the three PD rings, and suggested that the microbody plays a role in the contraction of the PD and MD rings. Received: 1 July 1998 / Accepted: 1 September 1998     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Making the connections--the crucial role of metabolite transporters at the interface between chloroplast and cytosol

Eukaryotic cells are most fascinating because of their high degree of compartmentation. This is particularly true for plant cells, due to the presence of chloroplasts, photosynthetic organelles of endosymbiotic origin that can be traced back to a single cyanobacterial ancestor. Plastids are major hubs in the metabolic network of plant cells, their metabolism being heavily intertwined with that of the cytosol and of other organelles. Solute transport across the plastid envelope by metabolite transporters is key to integrating plastid metabolism with that of other cellular compartments. Here, we review the advances in understanding metabolite transport across the plastid envelope membrane.     

The 5S ribosomal RNA sequences of a red algal rhodoplast and a gymnosperm chloroplast. Implications for the evolution of plastids and cyanobacteria

Summary The 5S ribosomal RNA sequences have been determined for the rhodoplast of the red algaPorphyra umbilicalis and the chloroplast of the coniferJuniperus media. The 5S RNA sequence of theVicia faba chloroplast is corrected with respect to a previous report. A survey of the known sequences and secondary structures of 5S RNAs from plastids and cyanobacteria shows a close structural similarity between all 5S RNAs from land plant chloroplasts. The algal plastid 5S RNAs on the other hand show much more structural diversity and have certain structural features in common with bacterial 5S RNAs. A dendrogram constructed from the aligned sequences by a clustering algorithm points to a common ancestor for the present-living cyanobacteria and the land plant plastids. However, the algal plastids branch off at an early stage within the plastid-cyanobacteria cluster, before the divergence between cyanobacteria and land plant chloroplasts. This evolutionary picture points to the occurrence of multiple endosymbiotic events, with the ancestors of the present algal plastids already established as photosynthetic endosymbionts at a time when the ancestors of the present land plant chloroplasts were still free-living cells.     

A cytokinin-sensitive mutant of the moss,Physcomitrella patens,defective in chloroplast division

Summary An X-ray induced mutant (PC22) of the moss,Physcomitrella patens was analysed with respect to its morphology, physiology and suitability for microculture techniques. The mutant protonemata are defective in bud formation and in chloroplast division. As a consequence of the latter, giant chloroplasts are formed which disturb the development of the phragmoplast, the formation of regular cross walls, and cell division. Abnormal cross walls are rich in callose. The actin cytoskeleton was found to be less regularly developed in the mutant than in the wild type. Three-dimensional analysis of the microtubular arrangement with confocal laser scan microscopy demonstrates a close association between spindle- or phragmoplast- and interphase-microtubules. The deficiencies in chloroplast division and in bud formation can partly be compensated for by exogeneously applied cytokinin. The suitability of this particular developmental mutant for further studies was shown by regeneration of protoplasts in microculture and microinjection of the fluorochrome Lucifer yellow into the chloroplast.Abbreviations CLSM confocal laser scan microscope - DAPI diamidinophenyl indole - DiOC 3,3-dihexyloxacarbocyanine iodide - EGTA ethylene glycol-bis-(-amino-ethylether-N,N,N,N-tetraacetic acid - i⁶Ade N⁶-(²-isopentenyladenine) - PIPES piperazine-N, N-bis-2-ethanesulfonic acid - ptDNA chloroplast DNA Devoted to the memory of Prof. Dr. O. Kiermayer, our colleague and friend.