Jodi Maple

ARC3 is a stromal Z‐ring accessory protein essential for plastid division

In plants, chloroplast division is an integral part of development, and these vital organelles arise by binary fission from pre‐existing cytosolic plastids. Chloroplasts arose by endosymbiosis and although they have retained elements of the bacterial cell division machinery to execute plastid division, they have evolved to require two functionally distinct forms of the FtsZ protein and have lost elements of the Min machinery required for Z‐ring placement. Here, we analyse the plastid division component accumulation and replication of chloroplasts 3 (ARC3) and show that ARC3 forms part of the stromal plastid division machinery. ARC3 interacts specifically with AtFtsZ1, acting as a Z‐ring accessory protein and defining a unique function for this family of FtsZ proteins. ARC3 is involved in division site placement, suggesting that it might functionally replace MinC, representing an important advance in our understanding of the mechanism of chloroplast division and the evolution of the chloroplast division machinery.

The Arabidopsis DJ-1a protein confers stress protection through cytosolic SOD activation

Mutations in the DJ-1 gene (also known as PARK7) cause inherited Parkinsons disease, which is characterized by neuronal death. Although DJ-1 is thought to be an antioxidant protein, the underlying mechanism by which loss of DJ-1 function contributes to cell death is unclear. Human DJ-1 and its Arabidopsis thaliana homologue, AtDJ-1a, are evolutionarily conserved proteins, indicating a universal function. To gain further knowledge of the molecular features associated with DJ-1 dysfunction, we have characterized AtDJ-1a. We show that AtDJ-1a levels are responsive to stress treatment and that AtDJ-1a loss of function results in accelerated cell death in aging plants. By contrast, transgenic plants with elevated AtDJ-1a levels have increased protection against environmental stress conditions, such as strong light, H2O2, methyl viologen and copper sulfate. We further identify superoxide dismutase 1 (SOD1) and glutathione peroxidase 2 (GPX2) as interaction partners of both AtDJ-1a and human DJ-1, and show that this interaction results in AtDJ-1a- and DJ-1-mediated cytosolic SOD1 activation in a copper-dependent fashion. Our data have highlighted a conserved molecular mechanism for DJ-1 and revealed a new protein player in the oxidative stress response of plants.

GIANT CHLOROPLAST 1 Is Essential for Correct Plastid Division in Arabidopsis

Plastids are vital plant organelles involved in many essential biological processes. Plastids are not created de novo but divide by binary fission mediated by nuclear-encoded proteins of both prokaryotic and eukaryotic origin. Although several plastid division proteins have been identified in plants, limited information exists regarding possible division control mechanisms. Here, we describe the identification of GIANT CHLOROPLAST 1 (GC1), a new nuclear-encoded protein essential for correct plastid division in Arabidopsis. GC1 is plastid-localized and is anchored to the stromal surface of the chloroplast inner envelope by a C-terminal amphipathic helix. In Arabidopsis, GC1 deficiency results in mesophyll cells harbouring one to two giant chloroplasts, whilst GC1 overexpression has no effect on division. GC1 can form homodimers but does not show any interaction with the Arabidopsis plastid division proteins AtFtsZ1-1, AtFtsZ2-1, AtMinD1, or AtMinE1. Analysis reveals that GC1-deficient giant chloroplasts contain densely packed wild-type-like thylakoid membranes and that GC1-deficient leaves exhibit lower rates of CO(2) assimilation compared to wild-type. Although GC1 shows similarity to a putative cyanobacterial SulA cell division inhibitor, our findings suggest that GC1 does not act as a plastid division inhibitor but, rather, as a positive factor at an early stage of the division process.

Functional conservation of the MIN plastid division homologues of Chlamydomonas reinhardtii

Chloroplasts arise by binary fission from pre-existing plastids, thus division plays a key role in the development of these essential photosynthetic organelles. To ensure that actively dividing tissues accumulate large numbers of chloroplasts prior to cell division, chloroplast division and the cell cycle must be intimately linked. However, little is known about the regulation of the plastid division machinery during cell division and these questions are difficult to address in higher plants. For this purpose we have studied the unicellular green alga Chlamydomonas reinhardtii for its potential as a new system for chloroplast division research. Here we show the functional conservation of key components of the higher plant chloroplast machinery in Chlamydomonas. The highly conserved Chlamydomonas MinD homologue, CrMinD1, retains crucial protein–protein interactions, sub-cellular localisation and the ability to affect both higher plant plastid division and bacterial cell division. Furthermore, using the coupling of chloroplast and cell division in Chlamydomonas we have established that transcript levels of chloroplast division homologues significantly increase during cell division, with levels falling as division reaches completion.

Mutagenesis in Arabidopsis

Ethyl methane sulfonate (EMS) mutagenesis in Arabidopsis is the most widely used mutagenesis technique. EMS has high mutagenicity and low mortality and can be used in any laboratory with a fume hood. The chemical principle of EMS mutagenesis is simple; it is based on the ability of EMS to alkylate guanine bases, which results in base mispairing. An alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions, which ultimately results in an amino acid change or deletion. There are several advantages to EMS mutagenesis compared with other mutagenesis techniques available for Arabidopsis. First, EMS generates a high density of nonbias irreversible mutations in the genome, which permits saturation mutagenesis without having to screen a large number of individual mutants. Second, EMS mutagenesis not only generates loss-of-function mutants, but can also generate novel mutant phenotypes, which include dominant or gain-of-function versions of proteins owing to alterations of specific amino acids. This chapter describes the use of EMS mutagenesis in Arabidopsis and how mutagenized plant populations should be handled after the mutagenesis event.

Plastid division coordination across a double-membraned structure.

Chloroplasts still retain components of the bacterial cell division machinery and research over the past decade has led to an understanding of how these stromal division proteins assemble and function as a complex chloroplast division machinery. However, during evolution plant chloroplasts have acquired a number of cytosolic division proteins, indicating that unlike the cyanobacterial ancestors of plastids, chloroplast division in higher plants require a second division machinery located on the chloroplast outer envelope membrane. Here we review the current understanding of the stromal and cytosolic plastid division machineries and speculate how two protein machineries coordinate their activities across a double‐membraned structure.

The complexity and evolution of the plastid-division machinery

Plastids are vital organelles, fulfilling important metabolic functions that greatly influence plant growth and productivity. In order to both regulate and harness the metabolic output of plastids, it is vital that the process of plastid division is carefully controlled. This is essential, not only to ensure persistence in dividing plant cells and that optimal numbers of plastids are obtained in specialized cell types, but also to allow the cell to act in response to developmental signals and environmental changes. How this control is exerted by the host nucleus has remained elusive. Plastids evolved by endosymbiosis and during the establishment of a permanent endosymbiosis they retained elements of the bacterial cell-division machinery. Through evolution the photosynthetic eukaryotes have increased dramatically in complexity, from single-cell green algae to multicellular non-vascular and vascular plants. Reflected with this is an increasing complexity of the division machinery and recent findings also suggest increasing complexity in the molecular mechanisms used by the host cell to control the process of plastid division. In the present paper, we explore the current understanding of the process of plastid division at the molecular and cellular level, with particular respect to the evolution of the division machinery and levels of control exerted on the process.

Interdependency of formation and localisation of the Min complex controls symmetric plastid division

Plastid division represents a fundamental biological process essential for plant development; however, the molecular basis of symmetric plastid division is unclear. AtMinE1 plays a pivotal role in selection of the plastid division site in concert with AtMinD1. AtMinE1 localises to discrete foci in chloroplasts and interacts with AtMinD1, which shows a similar localisation pattern. Here, we investigate the importance of Min protein complex formation during the chloroplast division process. Dissection of the assembly of the Min protein complex and determination of the interdependency of complex assembly and localisation in planta allow us to present a model of the molecular basis of selection of the division site in plastids. Moreover, functional analysis of AtMinE1 in bacteria demonstrates the level of functional conservation and divergence of the plastidic MinE proteins.

Yeast Two-Hybrid Screening

Yeast two-hybrid screening represents a sensitive in vivo method for the identification and analysis of protein-protein interactions. The principle is based on the ability of a separate DNA-binding domain (DNA-BD) and activation domain (AD) to reconstitute a functional transactivator when brought into proximity. In the MATCHMAKER yeast two-hybrid system, a bait protein is expressed as a fusion to the GAL4 DNA-BD, whereas the prey protein is expressed as a fusion to the GAL4 AD. When a bait and a prey protein interact, the DNA-BD and AD form a functional transactivator, resulting in activation of reporter gene expression in yeast reporter strains. The method described in this chapter can be used to identify novel protein interactions, analyze protein-protein interactions between two known proteins, as well as dissect interacting protein domains.

An emerging picture of plastid division in higher plants

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 undifferentiated 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 efforts 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 different 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 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, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence 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-Kafafi 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 different functions during evolution and the first 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 J. Maple Æ S. G. Møller (&) 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