Planta (2005) 223: 14 DOI 10.

1007/s00425-005-0078-y

P RO G RE SS R E P OR T

Jodi Maple Simon Geir Mller

An emerging picture of plastid division in higher plants

Received: 18 May 2005 / Accepted: 30 June 2005 / Published online: 1 September 2005 Springer-Verlag 2005

Keywords Plastid division FtsZ MinE MinD ARC GIANT CHLORPLAST 1 ARTEMIS Arabidopsis Plastids arose from an endosymbiotic event between a photosynthetic prokaryote and a proto-eukaryote and life on earth depends on this vital organelle for photosynthetic oxygen production (McFadden 2001). Plastids are derived from undierentiated meristematic proplastids (Pyke 1999) and arise by division from preexisting plastids in the cytosol (Aldridge et al. 2005). Plastid division therefore, is an essential process for plastid population accumulation and maintenance in plant cells. Research eorts during the last decade have revealed that plastid division represents a highly coordinated, multifaceted process involving both prokaryote-derived and host eukaryote-derived proteins however, how the dierent proteins act together to control the division process has only recently started to become understood. Plastid division is initiated by polymerization of FtsZ, an ancient tubulin-like protein with presumed GTPase activity (Osteryoung and Vierling, 1995). In contrast to bacteria, which contain a single FtsZ, higher plants harbor two FtsZ families, FtsZ1 and FtsZ2, thought to have arisen through a gene duplication event from a single cyanobacterial ftsZ gene (Stokes and Osteryoung, 2003). FtsZ1 and FtsZ2 are essential, nonredundant plastid division components, as both reduced or elevated levels of either transcript in transgenic plants result in severe plastid division inhibition (Osteryoung

J. Maple S. G. Mller (&) Department of Biology, University of Leicester, Leicester, LE1 7RH UK E-mail: sgm5@le.ac.uk Tel.: +44-116-252-5302 Fax: +44-116-252-3330 S. G. Mller Department of Science and Technology, University of Stavanger, 4036 Stavanger, Norway

et al. 1998). Both FtsZ1 and FtsZ2 are stromal proteins (McAndrew et al. 2001) and similar to bacterial FtsZ form ring structures (Z-ring) at the plastid midpoint (Fujiwara and Yoshida 2001; Vitha et al. 2001). The exact composition of this ring structure is unclear; however, recent studies have shed light on the complexity of its assembly and organization. Studies using yeast two-hybrid, uorescence resonance energy transfer (FRET) and bimolecular uorescence complementation (BiFC) assays have demonstrated that FtsZ1 and FtsZ2 can form both homopolymeric and heteropolymeric Zring structures in living plastids (Fig. 1a; Maple et al. 2005). Interestingly, a proportion of FtsZ2 appears to be associated with the envelope and it is speculated that, during division the FtsZ1 ring interacts with inner membrane-bound FtsZ2, allowing further protein recruitment to the site of division. In vitro cross-linking studies support this model, showing that only FtsZ1 can form GTP-dependent rod-shaped polymers but that FtsZ2 can promote GTP-independent FtsZ1 polymerization (El-Kafa et al. 2005). The organization of the FtsZ ring in plants is indeed intriguing, but why do plants harbor two forms of the FtsZ protein? One logical possibility is that the two FtsZ families have acquired dierent functions during evolution and the rst evidence comes from a recent study demonstrating that the stromal plastid division protein Accumulation and Replication of Chloroplasts 6(ARC6; Vitha et al. 2003) interacts with FtsZ2 but not with FtsZ1 (Fig. 1b; Maple et al. 2005). The Arabidopsis arc mutants (at least 12 loci with altered plastid division phenotypes) represent a rich source of plastid division components (Pyke and Leech 1991). The disrupted gene in arc6 encodes a protein with similarity to the cyanobacterial cell division gene Ftn2 (Koksharova and Wolk 2002; Vitha et al. 2003). Like the FtsZ proteins ARC6 can interact with itself and localize to a discontinuous ring structure at the chloroplast division site prior to and during constriction (Fig. 1b; McAndrew et al. 2001; Vitha et al. 2001; 2003; Maple et al. 2005). The interaction of ARC6 with FtsZ2

2 b Fig. 1 Interactions of the Arabidopsis stromal plastid division proteins. a FtsZ1 and FtsZ2 fusion proteins were co-expressed in tobacco leaves by particle bombardment. Fluorescence of CFP and YFP in leaf chloroplasts was detected by epiuorescence microscopy and the cells subject to FRET analysis. The FRET signal was false colored red. Scale bar = 5 lm. b BiFC assays were preformed by co-expressing ARC6-NY or FtsZ2-NY (NY = N-terminal half of YFP) with ARC6-CY or FtsZ2-CY (CY = C-terminal half of YFP) in tobacco leaf cells and the reconstituted YFP uorophore was detected by epiuorescence microscopy. Scale bar = 2.5 lm. c AtMinE1 and AtMinD1 fusion proteins were co-expressed in tobacco leaves by particle bombardment and subject to FRET analysis as in (a). Scale bar = 5 lm. d Working model for plastid division showing the identied protein components to date, their localization patterns and proteinprotein interaction properties. AtMinE1 and AtMinD1 localize to one or two spots in proximity to the inner envelope membrane. AtMinE1 and AtMinD1 can both form homodimers and AtMinE1 and AtMinD1 can also interact. GC1 localizes to the stromal side of the inner envelope membrane and forms dimers but is unable to interact with other plastid division components. AtFtsZ1-1 and AtFtsZ2-1 (F) form a Z-ring at the center of the chloroplast and can form homodimers and heterodimers. ARC6 (6) also localizes to a ring like structure at the site of chloroplast division and interacts specically with AtFtsZ21. ARC5 and ARC3 localize to ring-like structures on the cytosolic surface of the outer envelope membrane. ARTEMIS localizes to the chloroplast inner membrane but its distribution is unknown so is not included in this model

is dependent on a conserved peptide sequence at the extreme C-terminus of FtsZ2 known as the core domain (Ma and Margolin 1999; Maple et al. 2005). This highly conserved stretch of six to eight amino acids is present in

most bacterial FtsZ proteins and in all plant FtsZ2 proteins studied to date, but absent in FtsZ1. In Escherichia coli, two cell division proteins, FtsA and ZipA, have been linked to the regulation or control of FtsZring formation and both of these proteins interact with the bacterial FtsZ protein through the core domain (Ma and Margolin 1999). Although ARC6 shows no homology to FtsA and ZipA, evidence points towards ARC6 playing an analogous role: In the absence of both FtsA and ZipA, bacterial cell division is inhibited and FtsZ rings fail to assemble (Picho and Lutkenhaus 2002) and similarly, in arc6 chloroplast division is blocked (mesophyll cells harbor one or two large chloroplasts) and both FtsZ proteins fail to form Z-rings (Pyke et al. 1994; Vitha et al. 2003). Additionally, ZipA and ARC6 are both membrane proteins and could stabilize the Z-ring by anchoring FtsZ to the membrane and supporting the Z-ring structure (Hale and de Boer 1997). It is interesting that both FtsZ proteins fail to localize in arc6 as this indicates that ring formation by either FtsZ protein is dependent on a functional ARC6 protein, adding another level of complexity to the control and execution of plastid division. A further noteworthy feature of the ARC6 protein is a N-terminal J-domain motif characteristic of DnaJ chaperones which projects into the chloroplast stroma. J-Domain proteins are found in all organisms and one of these, DnaJ (Hsp40), interacts with Hsp70 stimulating the Hsp70 ATPase activity necessary for stable binding to its protein substrate (Walsh et al. 2004). HscA (an Hsp70 family protein) is involved in FtsZ-ring formation in E. coli, through a chaperon-like interaction with FtsZ (Uehara et al. 2001) and ARC6 may play a similar role in Arabidopsis.

The presence of two FtsZ protein forms in Arabidopsis may also have implications for the placement of the FtsZ-ring in higher plants. In E. coli, correct Z-ring placement depends on the proteins MinD, MinE and MinC (de Boer et al. 1990). MinC is an antagonist of FtsZ polymerization preventing Z-ring formation at sites other than midcell by the coordinated oscillation of MinD and MinE (Hu and Lutkenhaus 1999). Higher plants contain both MinD and MinE homologs but lack MinC. In Arabidopsis, reduced levels of the MinD homolog AtMinD1 leads to asymmetric plastid division (Colletti et al. 2000), a phenotype reminiscent of the asymmetric cell division phenotype of MinD-decient E. coli (de Boer et al. 1989). In E. coli, MinE confers topological specicity on MinD and it was predicted that the plant MinE homolog had a similar role. Indeed Arabidopsis plants overexpressing the MinE homolog AtMinE1 show Z-ring misplacement and chloroplast size heterogeneity within cells (Maple et al. 2002). Despite the low amino acid sequence similarity ($20%) between E. coli MinE and AtMinE1 the evolutionary conservation at the functional level is remarkable in that AtMinE1 overexpression in E. coli can induce minicelling (Maple et al. 2002) and moreover AtMinE1 can counteract the MinCD-induced division inhibition in E. coli null for MinE (Maple and Mller, unpublished). In E. coli the Min proteins form a dynamic complex to coordinate correct Z-ring positioning. Given the high level of functional conservation between the E. coli Min proteins and their plant homologs, it was speculated that such a complex would exist in Arabidopsis. AtMinD1 and AtMinE1 have been shown to localize to one or two discrete spots often at opposite ends of chloroplasts (Maple et al. 2002; Fujiwara et al. 2004) and through a combination of yeast two hybrid, FRET and BiFC studies, AtMinD1 and AtMinE1 have not only been shown to homodimerize but also to interact with each other in the chloroplast stroma (Fujiwara et al. 2004; Maple et al. 2005; Fig. 1c). The importance of correct Min protein localization and interaction is also starting to become unraveled. arc11 chloroplasts frequently divide asymmetrically due to misplacement of the Z-ring and cloning of the ARC11 loci revealed a C-terminal A296G mutation in AtMinD1 resulting in loss of dimerisation and appropriate localization (Fujiwara et al. 2004). Recent studies have also demonstrated that AtMinD1 is a Ca2+ -activated ATPase, stimulated by its interaction with AtMinE1, and that ATPase inactivation leads to loss of AtMinD1AtMinE1 interactions and correct localization (Aldridge and Mller 2005). Two further plastid division components, ARTEMIS (Ar abidopsis t haliana envelope membrane integra se; Fulgosi et al. 2002) and GIANT CHLOROPLAST 1 (GC1; also called AtSulA, Raynaud et al. 2004) can be found in cyanobacteria but not in other prokaryotes suggesting they do not form part of the classical Min protein-mediated division pathway. ARTEMIS is an integral inner membrane protein with an Alb3/ Oxa1/YidC family C-terminal domain, suggesting an

evolutionary conserved protein sorting pathway which inserts plastid division components into the chloroplast envelope, and an N-terminal region similar to receptor protein kinases which may enable the eukaryotic host to gain nuclear control over organelle division. GC1 is a stromal protein (Maple et al. 2004) and although GC1 does not interact with Arabidopsis FtsZ, GC1 overexpression can rescue plastid division inhibition caused by AtFtsZ1-1 or AtFtsZ2-1 accumulation (Raynaud et al. 2004). Both ARTEMIS and GC1 play important roles in plastid division and it will now be important to integrate these proteins into the stromal plastid division machinery. Recently, two cytosolic plastid division proteins were identied. One of these, ARC5, shows similarity to a dynamin-like protein distantly related to mitochondrial division proteins from plants, yeast, mammals and red alga (Gao et al. 2003). Several of these proteins (ADL2b, Arimura and Tsutsumi 2002; Dnm1p, Bleazard et al. 1999; and CmDnm1, Nishida et al. 2003) localize to mitochondrial constriction sites and analogous to this ARC5 forms a ring at the site of chloroplast constriction on the chloroplast outer surface. ARC5 is of eukaryotic origin as no obvious prokaryotic counterparts exist. Like ARC5, ARC3 localizes to a cytosolic ring-like structure at the site of chloroplast division. ARC3 appears to have arisen by a fusion between a prokaryotic FtsZ gene and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) gene (Shimada et al. 2004). The evolutionary signicance of this fusion event is, however, unclear since the FtsZ domain lacks key conserved motifs and the PIP5K-homologous region has no detectable kinase activity (Shimada et al. 2004). However, due to its cytosolic localization ARC3 may act in concert with ARC5. The identication of cytosolic plastid division proteins has certainly highlighted the complexity of plastid division in higher plants.

Future prospects
During the last decade a number of plastid division proteins have been identied and this has provided valuable insight into the intricate nature of plastid division in higher plants. Although it has recently become evident that plastid division proteins act together in protein complexes and that, these protein interactions aect protein localization and activities little is still known how these interactions inuence the actual division process. There is now a need to use the available information not only to analyse the physiological importance of the various proteinprotein interactions but to dissect the stoichiometric relationship between the various plastid division proteins.

References
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