Correspondence to: Ralf Reski, Freiburg University, Plant Biotechnology, Sonnenstrasse 5, D-79104 Freiburg, Germany. Tel:49-761-203-6969 Fax:49-761-203-6967
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Abstract |
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It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.
Key Words: FtsZ, plastid division, plastoskeleton, cytoskeleton, Physcomitrella
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Introduction |
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The functional complexity of eukaryotic cells is guaranteed at the structural level by different cell compartments; e.g., most eukaryotes possess mitochondria, and all plant cells possess plastids. These organelles are remnants of free-living prokaryotes which lost their autonomy during evolution by establishing an endosymbiosis with their host cells. In the course of this coevolution, the majority of prokaryotic genes were lost or transferred to the eukaryotic nucleus. Thus, recent eukaryotic cells are mosaics of at least two (animals and fungi) or three (plants) different genetic systems and their encoded structures (
It has been a long-standing canon in life sciences that a major difference between prokaryotes and eukaryotes is the cytoskeleton, a complex network of actin, tubulin, and intermediate filaments spanning the eukaryotic cytoplasm. This view was questioned by analysis of FtsZ, a protein which initiates bacterial cell division by polymerizing to a ring at the division site (
In plants, FtsZ has been retained, encoded by a small nuclear gene family whose proteins are re-imported to their evolutionary origins, plastids and mitochondria (
Here, we promote a novel, more complex view. As bacteria are kept in shape by their cell walls, they may only transiently need cytoskeletal elements in the course of their division. To facilitate endosymbiotic metabolite exchange, however, bacterial cell walls had to be reduced, concomitantly enhancing the need for internal structures that maintain the integrity and shape of the organelle. Thus, the eukaryotic cell may have been forced to generate structures based on preexisting bacterial compounds that keep their organelles in shape. Such an as yet undescribed structure has now been identified inside chloroplasts in this work and is termed the plastoskeleton.
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Materials and Methods |
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Plant Material
Physcomitrella patens (Hedw.) B.S.G. was cultivated as described previously (
Isolation of PpftsZ2
A cDNA of PpftsZ1 has been described previously (ZAP library (
Phylogenetic Tree Reconstruction
40 full-length FtsZ amino acid sequences were aligned (CLUSTAL W v1.8.; Proteobacteria, Gram+ Eubacteria, and Cyanobacteria, the number of sequences was reduced to 3 per cluster, leading to 27 sequences (accession numbers upon request) for final tree construction. Trees were reconstructed using TREECON (
PpftsZ1 and PpftsZ2 Gene Analysis
To analyze the genomic structure of the two ftsZ genes, different sets of primers were synthesized on the basis of the respective cDNA sequences. Genomic Physcomitrella DNA was extracted as described previously (
Cloning of PpftsZ Green Fluorescent Protein Fusions and Transfection Assays
The coding region of PpftsZ1 was PCR-amplified with primers F1 (5'-GCGGGGATCCGTATGGCTAGCGGTACCGCGTTGTTTAGTGG-GTGCTCGGG-3') and R1 (5'-GCGGTCGACCCCGGGATGACGTGTCTGGCCTCGCTTCCTTAAG-3') introducing BamHI and SmaI restriction sites 5' and 3' of the cDNA, respectively. The PCR product was cleaved and ligated into the BamHI-SmaIdigested green fluorescent protein (GFP)1 reporter plasmid pMAV4 (
Confocal Laser Scanning Microscopy
Localization of FtsZ-GFP was analyzed in transfected protoplasts by confocal laser scanning microscopy (CLSM) (TCS 4D; Leica) using 488-nm excitation and two-channel measurement of emission from 510580 nm (green/GFP) as well as >590 nm (red/chlorophyll).
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Results and Discussion |
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Isolation of PpftsZ2
Recently, a role of eukaryotic PpFtsZ1 in plastid division had been demonstrated in the moss Physcomitrella patens (
Genomic Sequences of the Two PpftsZ Genes
To investigate the evolutionary relationship of the two different plant ftsZ genes, genomic fragments of PpftsZ were PCR-amplified, cloned, and subsequently sequenced. Sequence assembly yielded two loci 3,088 and 3,459 bp in length (sequence data available from EMBL/GenBank/DDBJ under accession nos. AJ249138 and AJ249139). Each locus contains six introns ranging in size from 78 to 461 bp.
The two ftsZ loci are highly conserved: (a) all six introns are in exactly the same position within the coding sequence of the two genes, and (b) five out of the six pairs of introns show only small differences in length (116 bp). The remaining pair of introns (intron 6) differs in length by 180 bp. The precise conservation of intron positions and their nearly identical lengths indicate that a single copy of the prokaryotic ftsZ was transferred to the nucleus during establishment of endosymbiosis and acquired eukaryotic features like introns before duplication and subsequent divergence occurred.
Phylogeny of FtsZ
27 deduced FtsZ protein sequences were compared with each other, 14 of them from photosynthetic eukaryotes and 13 from photosynthetically inactive bacteria, mitochondria, and Archaea (Fig 1). Archaea, Gram+ Eubacteria, Cyanobacteria, nongreen algae, as well as and
Proteobacteria form monophyletic clusters. As recently described, the mitochondrial Mallomonas sequence clusters within the
Proteobacteria (
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In addition, one Cyanidioschyzon sequence is placed in that clade as well, identifying a second eukaryote with an Proteobacteriumrelated FtsZ that, therefore, most likely is located in mitochondria. Interestingly, Mallomonas and Cyanidioschyzon are nongreen algae. Despite huge sequencing projects, a mitochondrial FtsZ has never been identified from any organism other than the nongreen algae. Thus, only their mitochondria might divide by an ancient FtsZ-dependent mechanism whereas in yeast, chlorobionts, and higher eukaryotes, mitochondria may be divided by dynamin (
Surprisingly, the land plant sequences formed two monophyletic clusters designated types a and b. Both Physcomitrella sequences cluster in clade a, whereas the two divergent Arabidopsis ftsZ genes, whose products are putatively located in different cell compartments (
Both FtsZ Proteins Are Imported into Plastids
The existence of two distinct monophyletic FtsZ clusters in land plants indicates differences in function or localization, respectively, between these two protein classes. Arabidopsis possesses at least two different ftsZ genes, the products of which were suggested to be localized in different cell compartments: in vitro studies demonstrated that AtFtsZ1 was imported into plastids (
Because in vitro as well as in silico studies are indirect and may easily generate contradictory and inconclusive data, we analyzed subcellular localization of both Physcomitrella FtsZ proteins in vivo as COOH-terminal GFP fusion proteins. Physcomitrella protoplasts were transiently transfected with expression plasmids PpFtsZ1-GFP or PpFtsZ2-GFP, respectively, and GFP fluorescence was visualized by CLSM. 2 d after transfection, GFP signals could be solely detected within plastids (Fig 2, ag). The absence of GFP fluorescence in all other cell compartments, including the plastid outer membrane, was confirmed by series of optical sections through protoplasts and single plastids (Fig 2, dg), respectively.
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As controls, a COOH-terminal fusion of GFP to a short NH2-terminal fragment of PpFtsZ1, designated pFtsZ1(135)-GFP, accumulated in the cytoplasm (Fig 2 h), whereas pFtsZ1(193)-GFP accumulated exclusively in the plastids (Fig 2 i), revealing that the NH2-terminal 93 amino acid residues, but not the first 35, contain the functional transit peptide of PpFtsZ1.
Thus, in vivo both Physcomitrella FtsZ proteins are exclusively imported into plastids.
FtsZ-GFP Fusion Proteins Are Functional in Plastid Division
In bacteria, FtsZ acts together with a variety of associated proteins in forming the cell division apparatus. The molar ratios of some of its constituents, such as FtsZ, FtsA, and ZipA, were shown to be critical for its functionality: a slight overexpression of FtsZ enhanced division frequency, whereas high levels of FtsZ inhibited bacterial cell division (
Like their bacterial progenitors, both Physcomitrella proteins acted in a dose-dependent manner on the division process despite the presence of the COOH-terminal GFP fusion domain. Low FtsZ-GFP levels (indicated by weak GFP fluorescence) led to an enhancement of the division process and resulted in plastids that were significantly reduced in size (Fig 3). In contrast, high FtsZ levels (indicated by strong GFP fluorescence) blocked plastid division (Fig 4, ad), leading to undivided, abnormally large plastids closely resembling the PpftsZ1-knockout phenotype (
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The correlation between FtsZ-GFP dosage and plastid division was most strikingly evident in regenerating Physcomitrella plants: the initial cell, the protoplast, regenerates into a protonema. As only apical cells divide, the protonema filament represents a cell lineage from the initial protoplast to the dividing tip cell. In our transfection experiments, cells immediately after the initial protoplast showed strong GFP fluorescence in one giant plastid (Fig 4, ad). In subsequent cells, GFP fluorescence as well as plastid sizes decreased, finally resulting in cells with wild-typelike plastid number and size (Fig 4, a, b, and d). As a control, pFtsZ(193)-GFP had no influence on plastid division but exhibited the same gradient in fluorescence intensities as FtsZ-GFPs (Fig 4e and Fig f).
As both PpFtsZ proteins are independently acting on plastid division, they arealthough highly conservednot redundant in function.
FtsZ Proteins Contribute to a Filamentous Plastoskeleton
It has been long believed that cytoskeletal filaments occur solely in the cytoplasm of eukaryotes. Nonetheless, in the late 1960s, some authors by means of electron microscopy detected microtubule-like structures within plastids of several plant species (
In vivo localization by CLSM showed that both PpFtsZ-GFP fusions form organized, branched filamentous structures within Physcomitrella plastids (Fig 5). This polymerization is due to the FtsZ part of the fusion protein, since the truncated pFtsZ(193)-GFP is distributed more diffusely within the organelle (see Fig 2 i) compared with the complete FtsZ-GFP fusions (see Fig 2, a and b). Both FtsZ scaffolds span the entire plastids and thus resemble the cytoskeleton in eukaryotic cytoplasm. We therefore suggest the term "plastoskeleton" to describe these structures. Additionally, these networks represent the first report to date on eukaryotic FtsZ filamentation in vivo. As FtsZ1-GFP and FtsZ2-GFP independently influence plastid division and even in low concentration are functional in division, we postulate that the scaffolds built by these fusion proteins mimic a naturally occurring state.
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Although PpFtsZ1 and PpFtsZ2 share 84% identity at the peptide sequence level, they could be discriminated according to their spatial organization in plastids. FtsZ1-GFP formed highly regular networks (Fig 5, a and b), whereas FtsZ2-GFP networks were not as well organized (Fig 5c and Fig d). In both cases, the filaments were connected by nodes that seemed to anchor the skeleton to the inner chloroplast membrane.
Although bacterial and eukaryotic FtsZ proteins are highly conserved in molecular terms, our results indicate that there are striking differences between them: in E. coli, structures formed by FtsZ are only transiently detected as Z rings during cell division (
This striking difference between bacteria and plastids may be due to differences in the FtsZ molecule itself. Bacterial and eukaryotic FtsZ proteins are highly conserved in the NH2-terminal region, which is responsible for FtsZ polymerization to protofilaments (
In addition to these networks, we occasionally identified an S-shaped structure at the constriction site of dividing chloroplasts in regenerating protoplasts transfected with FtsZ2-GFP (Fig 5, cf; arrows). This S-shaped structure may represent a PpFtsZ2-based plastid division ring, indicating that the eukaryotic PpFtsZ2 has maintained a conserved function besides polymerizing into filamentous networks.
In this study, we identified two types of plant FtsZ proteins. Our phylogenetic analysis revealed that both Physcomitrella proteins group in land plant clade a. In contrast, the two Arabidopsis proteins group in different clades: AtFtsZ1, which was shown to be imported into plastids (
The existence of two clearly distinct clades of land plant FtsZ, both involved in plastid division, strongly suggests differences in localization or function, respectively, between both protein groups. The existing data indicate that type b may represent a basal FtsZ type that is involved in the ancient function of plastid division, whereas type a may be a more evolved type that is additionally involved in the maintenance of plastid shape and flexibility via the newly described plastoskeleton.
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Footnotes |
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1 Abbreviations used in this paper: CLSM, confocal laser scanning microscope; GFP, green fluorescent protein.
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Acknowledgements |
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We thank William Martin, Randall Cassada, Gunther Neuhaus, and Eberhard Schäfer for helpful discussions.
Financial support by the Deutsche Forschungsgemeinschaft (Re 837/4) is gratefully acknowledged.
Submitted: 11 July 2000
Revised: 2 October 2000
Accepted: 3 October 2000
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References |
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