The Plant Cell, Vol. 30: 1807–1823, August 2018, www.plantcell.org © 2018 ASPB.

INTRODUCTION

Chloroplasts are specialized photosynthetic organelles in plants that evolved from an ancient photosynthesizing cyanobacterium through endosymbiosis (Gray, 1992). Like their ancestors, chlo­roplasts replicate by binary fission, which is important for allowing plant cells to maintain an appropriate population of chloroplasts during cell division and expansion (Pyke, 1999). Bacterial cell division is driven by a Z­ring in which the cytoskel­etal protein FtsZ localizes at mid­cell and recruits other proteins, forming a divisome (Bi and Lutkenhaus, 1991; de Boer, 2010). Some chloroplast division proteins have been retained from bacteria, and similar to bacterial cell division, chloroplast di­vision is driven by a ring­like dynamic division machinery across the two envelope membranes at mid­chloroplast (Miyagishima et al., 1999; Yoshida et al., 2012; Osteryoung and Pyke, 2014). In Arabidopsis thaliana, the first molecular assembly of this ma­chinery, the chloroplast Z­ring, is formed by the association of FtsZ1 and FtsZ2 heteropolymers to the inner envelope mem­brane via the membrane­tethering protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6) (Pyke et al., 1994; McAndrew et al., 2001; Vitha et al., 2003; Olson et al., 2010). Spatial control over Z­ring assembly is crucial for ensuring correct placement of the division machinery and symmetric

bacterium/chloroplast division (Yu and Margolin, 1999; Mazouni et al., 2004; Glynn et al., 2007; de Boer, 2010). In bacteria, Z­ring assembly is directed to the mid­cell region mainly by a negative regulatory system known as the Min (mini­cell) system, which consists of three Min proteins, MinC, MinD, and MinE (de Boer et al., 1989). The cytosolic protein MinC is a direct inhibitor of FtsZ assembly (Hu et al., 1999; Dajkovic et al., 2008) and is recruited to the membrane by the membrane­ associated protein MinD through direct interaction (Hu and Lutkenhaus, 2000; Lutkenhaus and Sundaramoorthy, 2003). In Escherichia coli, the oscillation of the MinCD complex, which is driven by the topological factor MinE, causes its time­averaged concentration at the membrane to be highest at the cell poles and lowest at the cell center, guiding Z­ring formation at the mid­cell position (Raskin and de Boer, 1999; Bisicchia et al., 2013). In plants, the mechanism for chloroplast Z­ring positioning is more complicated. Homologs of bacterial MinD and MinE, termed MinD1 and MinE1 in algae and plants, function in Z­ring placement in the chloroplast (Colletti et al., 2000; Kanamaru et al., 2000; Itoh et al., 2001; Adams et al., 2008). However, bacterial MinC is functionally replaced by the stromal protein ARC3, which directly inhibits chloroplast FtsZ assembly through in­teractions with both FtsZ1 and FtsZ2 (Shimada et al., 2004; Maple et al., 2007; Zhang et al., 2013). In Arabidopsis, ARC3 can interact with both MinD1 and MinE1, and similar to bac­terial MinD, plant MinD1 is an ATPase whose activity is stim­ulated by MinE1 (Hu et al., 2002; Aldridge and Møller, 2005; Maple et al., 2007). Nevertheless, bacterium­like oscillation of the chloroplast Min system has not been reported. Strikingly, the plant­specific protein MULTIPLE CHLOROPLAST DIVISION SITE1 (MCD1) plays a novel role in chloroplast division site

MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division

Li Chen,1 Bing Sun,1 Wei Gao,1 Qi-yang Zhang, Huan Yuan, and Min Zhang2

College of Life Sciences, Capital Normal University, Beijing 100048, China

ORCID IDs: 0000­0001­7406­7820 (L.C.); 0000­0003­2221­3448 (B.S.); 0000­0001­5873­0442 (W.G.); 0000­0002­9046­3273 (Q.­y.Z.); 0000­0001­5806­8233 (H.Y.); 0000­0002­3716­4565 (M.Z.)

Chloroplasts replicate by binary fission, a process driven by ring-like dynamic division machinery at mid-chloroplast. In Ara-bidopsis thaliana, the first molecular assembly of this machinery, the Z-ring, forms via the association of FtsZ1 and FtsZ2 heteropolymers with the inner envelope membrane through the membrane-tethering protein ACCUMULATION AND REP-LICATION OF CHLOROPLASTS6 (ARC6). Spatial control of Z-ring assembly ensures the correct placement of the division machinery and, therefore, symmetric chloroplast division. The plant-specific protein MULTIPLE CHLOROPLAST DIVISION SITE1 (MCD1) plays a role in Z-ring positioning and chloroplast division site placement, but its mechanism of action is un-known. Here, we provide evidence that MCD1 is a bitopic inner membrane protein whose C terminus faces the chloroplast stroma. Interaction analysis showed that MCD1 and ARC6 directly interact in the stroma and that MCD1 binds to FtsZ2 in an ARC6-dependent manner. These results are consistent with the in vivo observation that ARC6 influences the localization of MCD1 to membrane-tethered FtsZ filaments. Additionally, we found that MCD1 is required for the regulation of Z-ring positioning by ARC3 and MinE1, two components of the chloroplast Min (minicell) system, which negatively regulates Z-ring placement. Together, our findings indicate that MCD1 is part of the chloroplast Min system that recognizes membrane-tethered FtsZ filaments during chloroplast division-ring positioning.

1These authors contributed equally to this work.2Address correspondence to minzhang@cnu.edu.cn.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Min Zhang (minzhang@cnu.edu.cn).www.plantcell.org/cgi/doi/10.1105/tpc.18.00189

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placement, as evidenced by the observation that mcd1 mutant chloroplasts have multiple constrictions and Z­rings similar to those of the arc11 (an allele of MinD1) and arc3 mutants (Marrison et al., 1999; Glynn et al., 2007; Nakanishi et al., 2009). MCD1 localizes to the mid­chloroplast ring and to puncta dispersed on the envelope membrane and influences MinD1 localization to the FtsZ filaments on the membrane (Nakanishi et al., 2009). Therefore, it appears that MCD1 is able to recog­nize membrane­tethered FtsZ filaments ahead of MinD1, but the underlying mechanism is unclear. The functional relation­ships of MCD1 with ARC3 and MinE1 have not been investi­gated, so it is currently unclear if MCD1 functions as a part of the chloroplast Min system. Here, we performed a detailed topological analysis of Arabi­dopsis MCD1, investigated its interactions with other division factors, and assessed the role of MCD1 in chloroplast division site positioning. Additionally, we developed an improved system for expressing soluble recombinant FtsZ2 in E. coli, which is fundamental for interaction analysis in vitro. Our findings pro­vide evidence that MCD1 is a bitopic inner membrane protein whose large C terminus resides in the stroma. We show that MCD1 recognizes membrane­tethered FtsZ filaments through a direct interaction with ARC6 rather than interacting with FtsZ1 and FtsZ2 per se. In vivo functional analysis revealed that MCD1 closely cooperates with ARC3 and MinE1 in addition to MinD1 to spatially prevent excess Z­ring assembly. These findings provide important insights into the molecular regulatory mechanism un­derlying the positioning of the chloroplast­division site in plants, showing that the emergence of MCD1 in plants likely facilitated the precise localization of the ARC3 and MinD1 complex to the membrane­tethered FtsZ filaments and therefore improved the efficiency of this complex in inhibiting FtsZ assembly on the membrane.

RESULTS

MCD1 Is a Bitopic Inner Membrane Protein Whose N Terminus Resides within the Intermembrane Space and Whose Long C Terminus Faces the Stroma

MCD1 is an inner envelope membrane (IEM) protein, but its precise topology on the membrane has been unclear, since trypsin­resistant fragments of this protein were not observed in previous protease protection assays (Nakanishi et al., 2009). We utilized a transient expression system in protoplasts to in­vestigate the topology of Arabidopsis MCD1. First, we tested our expression system using the IEM protein ARC6, whose N terminus resides in the stroma (Vitha et al., 2003; Froehlich and Keegstra, 2011; Zhang et al., 2016). We fused the ARC6 cDNA fragment without its transit peptide sequence to the C terminus of eYFP, yielding the fusion protein eYFP­ARC6. To enable chloroplast targeting, we fused the chloroplast transit peptide sequence from the nucleus­encoded protein RecA to the 5′ end of eYFP-ARC6 (Köhler et al., 1997) (Figure 1A). After protoplast transformation and incubation, eYFP­ARC6 fusion protein was guided into the chloroplast and localized to the chloroplast envelope membrane (Figure 1B). By contrast, in pro­toplasts transfected with RecATP-eCFP or eYFP, RecATP­eCFP fusion protein was detected in the stroma, whereas empty eYFP was specifically observed in the cytosol (Figure 1B). Consis­tent with these fluorescence observations, immunoblot analy­sis showed that bands from RecATP­eYFP­ARC6, RecATP­eCFP, and eYFP were specifically detected by anti­GFP monoclonal antibody in total protein extracts from the corresponding trans­fected protoplasts (Figure 1C). Next, we treated chloroplasts isolated from protoplasts expressing RecATP-eYFP-ARC6 with

The Role of MCD1 in Regulating Z­Ring Positioning 1809

Figure 1. Membrane Topology Analysis of Arabidopsis MCD1.

(A) Diagram of the MCD1 and ARC6 constructs used for transient expression analysis. RecATP, chloroplast transit peptide of RecA; TP, MCD1 transit peptide; TM, transmembrane domain.(B) Fluorescent micrographs showing the localization patterns of various fluorescent fusion proteins transiently expressed in Arabidopsis protoplasts isolated from 4­week­old Col­0 plants. eYFP and eCFP signals are falsely colored green; chlorophyll signals are falsely colored magenta. Bar =10 µm.(C) Total proteins from the transfected protoplasts shown in (B) were extracted for further immunoblotting analysis. Bands on the blots were detected with GFP monoclonal antibody.(D) Membrane­association analysis of the region of MCD1 at amino acids 115 to 139. Chloroplasts isolated from protoplasts expressing MCD11-141-eYFP, MCD11-114-eYFP, or RecATP-eCFP were subfractionated into stroma (S) and membrane (P) fractions and analyzed by immunoblotting.

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(+) or without (–) trypsin, followed by fractionation. Without trypsin treatment, a band from RecATP­eYFP­ARC6 of ∼120 kD was detected in the pellet (Figure 1E; Supplemental Figure 1A). By contrast, the GFP antibody detected an ∼100­kD fragment below RecATP­eYFP­ARC6 in the pellet fractions after trypsin treatment (Figure 1E; Supplemental Figure 1A), suggesting that the ∼18­kD C­terminal region of ARC6 was located in the intermembrane space and digested by trypsin. To further con­firm that these smaller bands correspond to specific trypsin­ resistant fragments, chloroplasts isolated from protoplasts expressing ARC6­eCFP (eCFP fused to the C terminus of ARC6) were also treated with (+) or without (–) trypsin. After trypsin treatment, no smaller fragments were detected below the bands of ARC6­eCFP (Supplemental Figure 2). These data are in agreement with previous results of chloroplast import as­says using in vitro­translated, radiolabeled ARC6 (Froehlich and Keegstra, 2011; Zhang et al., 2016) and suggest that our newly developed transient expression system, in combination with localization analysis in protoplasts, is suitable for topological analysis of chloroplast membrane proteins. Next, we separately transformed the fusion genes MCD1- eYFP, RecATP-eYFP-MCD1, MCD11-141-eYFP, and MCD11-114-eYFP into protoplasts (Figure 1A; Supplemental Figure 3A). MCD1­eYFP, RecATP­eYFP­MCD1, and MCD11­141­eYFP exhibited an identical localization pattern. In transfected protoplasts, these proteins were primarily observed on the chloroplast enve­lope membrane, whereas MCD11­114­eYFP was mainly found in the stroma (Figure 1B; Supplemental Figure 3B). We confirmed the localizations of these fusion proteins in the corresponding samples via immunoblot analysis (Figure 1C; Supplemental Figure 3C). We then directly fractionated chloroplasts isolated from protoplasts expressing MCD11-141-eYFP or MCD11-114-eYFP. Immunoblot analysis of these chloroplast subfractions re­vealed that MCD11­141­eYFP and MCD11­114­eYFP were localized in the membrane and stromal fractions, respectively (Figure 1D). These results, which are consistent with bioinformatics predictions, suggest that the 115­ to 139­amino acid region of MCD1 is indeed a membrane­spanning domain. To investigate whether MCD1 has the same topology as ARC6, we treated chloroplast samples isolated from protoplasts expressing RecATP-eYFP-MCD1 with trypsin at various con­centrations, followed by immunodetection. The GFP antibody detected RecATP­eYFP­MCD1 but failed to detect the predicted trypsin­resistant fragment equivalent to MCD11­141­eYFP no mat­ter what concentration of trypsin was used (Supplemental Fig­ure 3C), suggesting that unlike ARC6, MCD1 might span the inner membrane in the opposite orientation. Subsequently, we performed trypsin protection assays of MCD1­eYFP fusion pro­tein using chloroplasts isolated from protoplasts expressing

MCD1-eYFP. After fractionation and immunoblotting of trypsin­ treated proteins, a trypsin­resistant fragment below the MCD1­ eYFP band was detected by the GFP antibody (Figure 1F; Supple­mental Figure 1B). The size of this protected fragment was ap­proximately equal to the calculated molecular mass of MCD114­349 plus eYFP (57 kD), suggesting that the N terminus of MCD1 not including the transit peptide sequence was digested in the

(E) and (F) Topology analysis of ARC6 (E) and MCD1 (F) at the inner membrane. Chloroplasts (equivalent to 10 µg of chlorophyll) isolated from proto­plasts separately expressing RecATP­eYFP-ARC6 (E) and MCD1-eYFP (F) were treated with (+) or without (−) trypsin, lysed, and fractionated into total membrane (P) and soluble (S) fractions. All fractions were analyzed by SDS­PAGE and immunoblotting. GFP monoclonal antibody was used to detect RecATP­eYFP­ARC6, MCD1­eYFP and the trypsin­resistant fragments. Red asterisks indicate trypsin­protected fragments of RecATP­eYFP­ARC6 in (E) and MCD1­eYFP in (F). Toc75, outer membrane marker; Tic40, inner membrane marker; RbsS, Rubisco small subunit as a stromal marker. All assays were performed at least three times.

Figure 1. (continued).

Figure 2. Investigation of the Interactions of MCD1 with FtsZ1, FtsZ2, ARC6, and PARC6 Using a Yeast Two­Hybrid System.

Prey, constructs in the pGAD­T7 vector backbone; Bait, constructs in the pGBK­T7 vector backbone; AbA, 60 ng/mL of Aureobasidin A; MCD1C, amino acids 141 to 349 of MCD1; MCDN, MCD1 amino acids 53 to 114; PARC6N, PARC6 amino acids 77 to 573; ARC6N, ARC6 amino acids 154 to 509; ARC6C, ARC6 amino acids 637 to 801; PARC6C, PARC6 amino acids 595 to 819. Two biological replicates were performed.

The Role of MCD1 in Regulating Z­Ring Positioning 1811

intermembrane space by trypsin. These results suggest that MCD1 is a bitopic IEM protein whose C terminus faces the stroma.

The Stromal Regions of MCD1 and ARC6 Directly Interact

Based on the topology of MCD1, we performed a yeast two­ hybrid assay to investigate interactions of the stromal region of MCD1 (MCD1C) with FtsZ1 and FtsZ2. Yeast cells cotransfected with MCD1C and FtsZ1 or MCD1C and FtsZ2 did not survive on synthetic dropout (SD) medium lacking histidine (Figure 2, first panel), indicating that the association of MCD1 to the FtsZ filaments does not occur through an interaction with FtsZ1 or FtsZ2. The IEM protein ARC6 and its paralog, PARC6, have the same topologies, and both of their N termini reside in the stroma and interact with FtsZ2 rather than FtsZ1 in yeast two­hybrid assays (Vitha et al., 2003; Zhang et al., 2016) (Figure 2, second panel). Thus, we next examined the interactions of MCD1 with ARC6 and PARC6. Yeast cells containing MCD1C and ARC6N grew well on SD medium lacking histidine, whereas cells har­boring MCD1C and PARC6N did not survive on this medium (Figure 2, second and third panels), suggesting that MCD1C and ARC6N interact with each other. Additionally, neither ARC6C nor PARC6C interacted with MCD1N in yeast cells (Figure 2, forth panel). Interestingly, the region of MCD1 at amino acids 277 to 314, which is predicted to form a coiled­coil helix, did not in­terfere with the interaction between MCD1C and ARC6N, since cells harboring MCD1C(∆277­314) and ARC6N still survived on SD medium lacking histidine (Figure 2, second panel). The interaction between the stromal regions of MCD1 and ARC6 was further confirmed by in vitro pull­down assays. Recom­binant His­ARC6N was precipitated from crude E. coli extracts by Glutathione­Sepharose beads coated with GST­tagged MCD1C and MCD1C(∆277­314), but not by GST­MCD1N­coated, GST­coated, or empty beads (Figures 3A and 3B). However, recombinant malt­ose binding protein (MBP)­PARC6N­His was not precipitated by the beads coated with GST­MCD1C (Figure 3B, lane 8), consistent with the negative results from the yeast two­hybrid assays (Fig­ure 2, third panel). As a negative control, recombinant His­ARC6C was not precipitated by the beads coated with GST­MCD1C either (Figure 3B, lane 7). These results indicate that MCD1 and ARC6 directly interact with each other through their stromal regions.

The Stromal Region of ARC6 Binds Directly to FtsZ2 Independently of Other Proteins

ARC6 tethers FtsZ­heteropolymer filaments to the membrane via an interaction with FtsZ2, promoting Z­ring formation (Vitha et al., 2003; Maple et al., 2005; Irieda and Shiomi, 2017). To determine whether FtsZ2 and the stromal region of ARC6 (ARC6N) interact directly and independently of other factors, we tested their interaction using an in vitro pull­down assay. We used a small ubiquitin­like modifier (SUMO) as a tag to avoid the problem of the insolubility of recombinant FtsZ2 expressed in E. coli. With the SUMO tag, we successfully obtained solu­ble FtsZ2 protein, although the recombinant His­SUMO­FtsZ2 protein always copurified with some degradation products (Fig­ure 4A). His­SUMO­FtsZ2 was precipitated by amylose resin beads coated with MBP­tagged ARC6N but not by MBP­coated

Figure 3. In Vitro Pull­Down Analysis of the Stromal Regions of MCD1 and ARC6.

(A) Recombinant His­ARC6N binds to GST­MCD1C or GST­MCD1C(∆277­314). Glutathione­Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST­tagged MCD1C (lane 4), MCD1C(∆277­314) (lane 5), or MCD1N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His­ARC6N. Proteins were eluted and analyzed by immunoblotting with anti­His and anti­GST antibodies.(B) Recombinant MBP­PARC6N­His or His­ARC6C was not precipitated from crude E. coli extracts by Glutathione­Sepharose 4B beads coated with GST­MCD1C. All assays were performed more than three times.

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or empty beads (Figure 4B). By contrast, His­SUMO protein failed to bind to MBP­tagged ARC6N or MBP (Figure 4C). These results demonstrate that in vitro, FtsZ2 binds directly to ARC6 independently of other proteins.

ARC6 Bridges the Binding of FtsZ2 to MCD1 in Vitro

Consistent with the results of yeast two­hybrid assays (Figure 2), GST pull­down assays showed that His­SUMO­FtsZ2 did not bind to GST­MCD1C (Figure 5, lane 10). Like His­ARC6N, recombinant MBP­ARC6N was also precipitated by Glutathione­ Sepharose beads coated with GST­MCD1C, but MBP­ARC6C or MBP was not (Figure 5, lanes 5 to 8). Since the stromal region of ARC6 binds directly to both FtsZ2 and the C terminus of MCD1, we asked if ARC6N might bridge the binding of FtsZ2 to MCD1C. To this end, we incubated Glutathione­Sepharose beads coated with GST­MCD1C with purified recombinant His­SUMO­FtsZ2 in the presence of recombinant MBP­ARC6N, MBP­ARC6C, or MBP. As shown in Figure 5, His­SUMO­FtsZ2 was only retained by beads coated with GST­MCD1C in the presence of ARC6N. These results suggest that ARC6 might form a bridge that facilitates the asso­ciation of MCD1 with membrane­tethered FtsZ filaments in vivo.

ARC6 Affects the Interaction between MCD1 and FtsZ2 in Vivo

To further determine whether ARC6 mediates the interaction between MCD1 and FtsZ2 in vivo, we performed bimolecular fluorescence complementation (BiFC) assays in Arabidopsis protoplasts isolated from wild­type, arc6, and parc6 plants (Glynn et al., 2008, 2009). Based on the topologies of MCD1 and ARC6, we used the MCD1­YFPN and RecATP­YFPC­ARC6 con­structs to ensure that the YFPN and YFPC fusion proteins were physically located in the same compartment (stroma). Since FtsZ2 is a stromal protein (McAndrew et al., 2001), YFPN or YFPC was fused to the C terminus of FtsZ2 for the BiFC assays. In wild­type protoplasts, reconstituted YFP signals were detected on the chloroplast envelope membrane when MCD1­YFPN and FtsZ2­YFPC were coexpressed, which also occurred when RecATP­YFPC­ARC6 was coexpressed with either MCD1­YFPN or FtsZ2­YFPN, suggesting that MCD1 interacts with FtsZ2 in vivo (Figure 6A). However, since MCD1 and FtsZ2 did not directly interact, and ARC6 is required for the binding of MCD1 to FtsZ2 in vitro (Figures 2 and 5), we performed BiFC assays using arc6 protoplasts. YFP signals were detected in arc6 protoplasts when RecATP­YFPC­ARC6 was coexpressed with MCD1­YFPN or FtsZ2­YFPN, but not when MCD1­YFPN and FtsZ2­YFPC were coexpressed (Figure 6B), indicating that ARC6 affects the inter­action between MCD1 and FtsZ2 in vivo. When we transformed parc6 protoplasts with the same combinations of plasmids, YFP signals were detected in protoplasts cotransformed with either

Figure 4. In Vitro Pull­Down Assays Showing That FtsZ2 Directly Binds to the Stromal Region of ARC6.

(A) Coomassie­stained SDS­PAGE gel showing that recombinant His­SUMO­FtsZ2 is a soluble protein in E. coli, but protein degradation could not be avoided; the degraded product (indicated by a white arrowhead) accumulated in the lysate and was copurified with His­SUMO­FtsZ2.(B) Pull­down assays showing that His­SUMO­FtsZ2 binds to the stro­mal region of ARC6 (ARC6N).

(C) Negative controls. His­SUMO cannot be precipitated with MBP only or MBP­tagged ARC6N.Proteins retained on amylose resin beads were visualized using anti­6xHis antibody to detect His­SUMO­FtsZ2 and His­SUMO or anti­MBP antibody to detect MBP or MBP­tagged ARC6N. Black asterisks indicate the degra­dation bands of MBP­ARC6N. Assays were performed at least two times.

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MCD1­YFPN and FtsZ2­YFPC, MCD1­YFPN and RecATP­YFPC­ ARC6, or FtsZ2­YFPN and RecATP­YFPC­ARC6 (Figure 6C). These results indicate that the association of MCD1 with FtsZ2 is specifically mediated by ARC6 in vivo.

The Effect of MCD1 on Chloroplast Z-Ring Positioning Is Dependent on ARC6 in Vivo

To assess whether the role of MCD1 in chloroplast division­ring positioning is dependent on ARC6 in vivo, we generated the mcd1 arc6 double mutant. Cells from the green tissues of arc6 mutants contain one to two giant chloroplasts, whereas mcd1 cells contain chloroplasts with multiple membrane constrictions, resulting in a heterogeneous population of chloroplasts in terms of number, size, and shape (Vitha et al., 2003; Nakanishi et al., 2009) (Figure 7A). The phenotype of mcd1 arc6 chloroplasts was similar to that of the single mutant arc6 rather than mcd1 (Figure 7A), suggesting that MCD1 acts downstream of ARC6 in Arabidopsis. Furthermore, we introduced FtsZ1-CFP driven by the FtsZ1 native promoter into the arc6, mcd1, and mcd1 arc6 mutants and evaluated the morphology of FtsZ in these transgenic plants, which expressed FtsZ1­CFP at relatively low levels (Figures 7B and 7C). Consistent with previous findings (Vitha et al., 2001), FtsZ1­CFP localized to the mid­chloroplast

ring in wild­type plants and did not affect normal chloroplast division (Figure 7B; Supplemental Figure 4). FtsZ1­CFP signals were detected as short filaments in arc6 transgenic plants but as multiple rings in mcd1 transgenic plants (Figure 7B). In the mcd1 arc6 double mutant, FtsZ1­CFP was localized to short filaments and dots, a pattern similar to that in arc6 (Figure 7B), suggesting that the presence of ARC6 is a prerequisite for the regulation of Z­ring positioning by MCD1 in vivo. In agreement with a previous observation (Nakanishi et al., 2009), MCD1 localized to a ring structure at mid­chloroplast and to puncta on the envelope membrane in wild­type plants (Figure 8A). We analyzed the immunolocalization of MCD1 in arc6 and found only a few punctate structures on the envelope membranes of giant chloroplasts (Figure 8A). In the control, sim­ilar bright spots were not detected in mcd1 by the anti­MCD1 antibody (Figure 8A). Immunoblot analysis showed that MCD1 protein levels in wild­type and arc6 plants were nearly identical (Figure 8C). We also produced transgenic wild­type and mcd1 plants expressing ARC6-eGFP and analyzed the localization pat­tern of ARC6. In wild­type transgenic plants, ARC6­eGFP was localized to the mid­chloroplast ring (Figure 8B), which is consis­tent with previous observations (Vitha et al., 2003). By contrast, ARC6­eGFP formed multiple rings in elongated chloroplasts in mcd1 transgenic plants (Figure 8B), which is reminiscent of the multiple Z­rings observed in the mcd1 mutant (Nakanishi et al., 2009) (Figure 7B). Collectively, these results suggest that ARC6 influences the localization of MCD1 to membrane­tethered FtsZ filaments in vivo.

MCD1, MinD1, and ARC3 Regulate Z-Ring Positioning in the Same Pathway

Besides MCD1, three other proteins, ARC3, MinD1, and MinE1, play roles in chloroplast Z­ring positioning (Glynn et al., 2007; Fujiwara et al., 2008). Previous studies have shown that MCD1 interacts with MinD1 and affects its localization to the FtsZ fil­aments (Nakanishi et al., 2009). In addition, MinD1 requires a direct interaction with ARC3, a direct inhibitor of FtsZ assembly, to inhibit FtsZ assembly (Maple et al., 2007; Zhang et al., 2013). Finally, chloroplasts with multiple membrane constrictions and Z­rings have been observed in mcd1, arc3, and arc11 (a mu­tant allele of MinD1) or minD1-1 (Pyke and Leech, 1992; Glynn et al., 2007; Nakanishi et al., 2009; Zhang et al., 2013). Hence, these findings suggest that these three proteins function as a complex. To investigate this possibility, we first analyzed chloroplast phenotype and FtsZ morphology in the mcd1 arc11 double mutant and transgenic mcd1 arc11 plants expressing FtsZ1pro-FtsZ1-CFP. An additive genetic effect was not detected in the mcd1 arc11 double mutant, as these plants exhibited the same chloroplast phenotype as the single mutants mcd1 and arc11 (Figure 9A). FtsZ1­CFP was expressed at relatively low levels and was localized to multiple rings in transgenic plants in the mcd1, arc11, and mcd1 arc11 backgrounds (Figures 9B and 9C). We also analyzed the chloroplast phenotypes and FtsZ morphol­ogies in the mcd1 arc3 double mutant and mcd1 arc3 plants expressing FtsZ1­CFP. However, no obvious differences were de­tected between mcd1 arc3 and arc3, mcd1, arc11, mcd1 arc11,

Figure 5. ARC6 Bridges the Binding of FtsZ2 to MCD1 in Vitro.

Recombinant MBP­ARC6N or His­SUMO­FtsZ2 was incubated with Glutathione­Sepharose beads coated with GST or GST­MCD1N (lanes 5, 6, 9, and 10). MBP or MBP­ARC6C was incubated with beads coated with GST­MCD1N (lanes 7 and 8). His­SUMO­FtsZ2 was incubated with beads coated with GST and recombinant GST­MCD1N in the presence of MBP­ARC6N, MBP­ARC6C, or MBP (lanes 11 to 13). For the input, 2% MBP­ARC6N and MBP­ARC6C or 1% His­SUMO­FtsZ2 and MBP were utilized. Proteins retained on Glutathione­Sepharose beads were visu­alized using anti­6xHis antibody to detect His­SUMO­FtsZ2, anti­GST antibody to detect GST, or GST­MCD1C or anti­MBP antibody to detect MBP or MBP­ARC6N. Asterisks indicate the degradation bands of His­SUMO­FtsZ2 and MBP­ARC6N. More than three replicates were performed.

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or arc3 arc11 (Figures 9A, 9B, 10A, and 10B) (Marrison et al., 1999; Glynn et al., 2007; Zhang et al., 2013). Together, these results indicate that MCD1, MinD1, and ARC3 regulate Z­ring positioning and placement of the chloroplast division site via the same pathway.

In wild­type chloroplasts, MinD1, like MCD1, was localized to a ring structure at the constriction site and to puncta on the envelope membrane (Fujiwara et al., 2009; Nakanishi et al., 2009) (Figure 8A; Supplemental Figure 5A). However, in the absence of ARC3, MCD1 and MinD1 protein levels were not altered, but

Figure 6. In Vivo Interaction of MCD1 and FtsZ2 Examined by BiFC.

BiFC assays were performed by coexpressing the indicated combinations of plasmids in protoplasts isolated from wild­type (A), arc6 (B), and parc6 (C) Arabidopsis plants. Fluorescence of the reconstituted YFP fluorophore was detected by epifluorescence microscopy. In all types of protoplasts cotransformed with MCD1­YFPN and RecATP­YFPC­ARC6 or FtsZ2­YFPN and RecATP­YFPC­ARC6, the reconstitution of YFP signals serves as an indicator of interaction. Protoplasts cotransformed with MCD1­YFPN and FtsZ2­YFPC show reconstituted YFP signals in the wild type (A) and parc6 (C), but not in arc6 (B). The chloroplast­specific localization of empty YFPN and YFPC was detected by expressing the RecATP­YFPN and RecATP­YFPC plasmids. RecATP­YFPN and FtsZ2­YFPC, MCD1­YFPN and RecATP­YFPC, or RecATP­YFPN and RecATP­YFPC­ARC6 were coexpressed in protoplasts as the negative controls, showing no YFP signals. YFP and chlorophyll signals were falsely colored green and magenta, respectively. Bars = 10 µm.

The Role of MCD1 in Regulating Z­Ring Positioning 1815

both formed more than one ring­like structure (Figures 10D and 10E; Supplemental Figure 5), which is reminiscent of the multiple Z­rings detected in arc3 mutants (Glynn et al., 2007; Zhang et al., 2013) (Figure 10B). These results suggest that ARC3 does not affect the localization of MCD1 or MinD1 to membrane­tethered FtsZ filaments.

MCD1 Is Required for the Role of MinE1 in Z-Ring Positioning

Next, we analyzed the functional relationship of MCD1 with MinE1, a topological specificity factor in the chloroplast Min

system. The arc12 mutant, bearing a mutation in MinE1, con­tained one to two drastically enlarged chloroplasts (Glynn et al., 2007) (Figure 10A). FtsZ1­CFP, which was expressed at a relatively low level, formed numerous short filaments and dots in the giant chloroplasts of transgenic arc12 plants (Figures 10B and 10C). Previous genetic studies of the minD1-1 arc12 and arc3 arc12 double mutants suggested that MinE1 acts downstream of MinD1 and ARC3 (Zhang et al., 2013). Here, we created the mcd1 arc12 double mutant and found that it contained a het­erogeneous population of chloroplasts, including some with multiple membrane constrictions similar to those in the mcd1 single mutant (Figure 10A). FtsZ1­CFP signals were observed as

Figure 7. The mcd1 arc6 Double Mutant Exhibits a Chloroplast Phenotype and FtsZ Morphology Similar to Those of the Single Mutant arc6 Rather Than mcd1.

(A) Differential interference contrast images showing chloroplast morphology in living cells of the wild type, the arc6 and mcd1 single mutants, and the mcd1 arc6 double mutant. Blue arrowheads indicate multiple chloroplast membrane constrictions. Bars = 10 µm.(B) Fluorescence micrographs showing the localization patterns of FtsZ1­CFP in chloroplasts of the indicated transgenic plants expressing FtsZ1pro-FtsZ1-CFP. More than 20 cells were observed in each indicated transgenic plants, and representative photographs are shown. Fluorescent FtsZ1­CFP and chlorophyll signals were falsely colored green and magenta, respectively. White arrowheads indicate multiple Z­rings. Bar = 10 µm.(C) Immunoblot analysis showing relative FtsZ1­CFP levels in the indicated transgenic plants in (B) compared with wild­type transgenic plants with low levels of FtsZ1-CFP expression. FtsZ1­CFP was detected with GFP monoclonal antibody. Actin served as a loading control.

1816 The Plant Cell

multiple rings in mcd1 arc12 transgenic plants, which were iden­tical to those in mcd1 plants expressing FtsZ1-CFP (Figure 10B). These results demonstrate that MinE1 also acts downstream of MCD1. Finally, we analyzed the localization of MCD1 and MinD1 in arc12 plants and detected numerous puncta on the envelope membranes of giant chloroplasts (Figure 10E; Supple­mental Figure 5A). The immunolocalization patterns of MCD1 and MinD1 differed between the wild type and arc12 (Figures 8A and 10E; Supplemental Figure 5A), but their protein levels were nearly equal in these lines (Figure 10D; Supplemental Fig­ure 5B). Together, these results suggest that MinE1 regulates Z­ring positioning indirectly through conferring spatial specificity to MCD1 as well as MinD1.

DISCUSSION

Correct positioning of the division machinery at the mid­ chloroplast is vital for maintaining a relatively constant popula­tion of chloroplasts in algae and plant cells during cell division and expansion. In Arabidopsis, symmetric chloroplast division begins with the assembly of tubulin­like proteins FtsZ1 and FtsZ2 into a ring structure just underneath the inner mem­brane (Osteryoung et al., 1998; Vitha et al., 2001). Spatial control over Z­ring assembly is achieved by the chloroplast Min system, a system derived from a negative regulatory system in bacteria that restricts Z­ring formation everywhere except at the division site (Colletti et al., 2000; Itoh et al., 2001; Shimada et al., 2004; Maple et al., 2007; Zhang et al., 2013). However, in addition to ARC3, MinD1, and MinE1, plants have acquired a new chloroplast Z­ring regulator, MCD1, which is important for MinD1 localization (Nakanishi et al., 2009). In this study, we dissected the precise topology of MCD1 on the membrane, revealed the mechanism for the re­cruitment of MCD1 to membrane­tethered FtsZ filaments, and provided evidence that MCD1 is a pivotal component of the chloroplast Min system. A working model depicting the roles of MCD1 at the early stage of Z­ring positioning is shown in Figure 11. The nucleus­encoded MCD1 protein is predicted to contain a chloroplast transit peptide and a hydrophobic region (Nakanishi et al., 2009). We performed a series of experiments showing that the MCD1 region at amino acids 115 to 139 is involved in the insertion of this protein into the inner membrane (Figures 1B and 1D). Trypsin protection assays combined with localization analysis of MCD1­eYFP indicated that MCD1 spans the chlo­roplast inner membrane, with a large portion of its C terminus residing in the stroma (Figures 1B and 1F; Supplemental Figure 1B). Thus, our results demonstrate that MCD1 is a bitopic inner membrane protein that resembles the inner membrane protein ARC6; however, these two proteins span the inner membrane in the opposite orientation (Figure 11). Most IEM proteins with a

Anti­MCD1 and ARC6­eGFP signals were falsely colored green; chlo­rophyll signals were falsely colored magenta. Arrows, asterisks, and ar­rowheads indicate the mid­chloroplast ring, puncta, and multiple ring structures, respectively. Bars = 10 µm.

Figure 8. Localization Patterns of ARC6 and MCD1 in Chloroplasts.

(A) Immunofluorescence micrographs showing the localization of MCD1 in chloroplasts of the indicated plants.(B) Fluorescence micrographs showing the localization patterns of ARC6­eGFP in transgenic plants in the wild­type and mcd1 backgrounds expressing ARC6pro-ARC6-eGFP. More than three individual lines were observed in the indicated transgenic plants, and representative photo­graphs are shown.(C) and (D) Immunoblot analysis showing MCD1 and ARC6­eGFP pro­tein levels in the indicated plants. As a loading control, the samples were also probed with antibody against Arabidopsis Actin.

The Role of MCD1 in Regulating Z­Ring Positioning 1817

transit peptide cross the envelope membranes using the TOC/TIC (translocons at the outer and inner envelope membrane, respectively) machinery (Lee et al., 2017). Two different path­ways are utilized for IEM protein targeting: the stop­transfer pathway and the post­import pathway (Viana et al., 2010). The IEM protein ARC6 uses the stop­transfer pathway for targeting (Froehlich and Keegstra, 2011), whereas the topology of MCD1 suggests that it likely exploits the post­import pathway for tar­geting to the chloroplast inner membrane. Hence, MCD1 is likely another good candidate for studying the mechanism underlying inner membrane­protein sorting in the future. In the mcd1 mutant, ARC6, FtsZ1, and FtsZ2 localize to mul­tiple ring structures (Nakanishi et al., 2009) (Figures 7B and 8B), and our in vivo genetic analysis demonstrated that ARC6 acts upstream of MCD1 (Figure 7), indicating that MCD1 mediates Z­ring assembly in an ARC6­dependent manner. Cytoskeletal proteins FtsZ1 and FtsZ2 colocalize to short filaments in the arc6 mutant (McAndrew et al., 2001), implying that in vivo, these short filaments are likely heteropolymers of FtsZ1 and FtsZ2, although their properties are currently unclear. The stromal region of ARC6 interacts with FtsZ2 rather than FtsZ1 (Maple et al., 2005) (Figure 2), and this region can directly interact with FtsZ2 in the absence of other proteins (Figure 4), strongly favoring the hypothesis that ARC6 tethers FtsZ heteropolymers to the membrane, mainly through a direct interaction with FtsZ2 (Figure 11). Based on the topology of MCD1 (Figure 1), we found that neither FtsZ1 nor FtsZ2 interacts with MCD1 but that ARC6 and MCD1 can directly interact through their stromal regions (Figures 2 and 3). A comparison of the localization patterns of MCD1 between the wild type and arc6 (Figure 8) suggested that ARC6 may affect the association of MCD1 to FtsZ filaments on the membrane. This was evidenced by our in vitro and in vivo analyses. The C terminus of MCD1 was able to bind to FtsZ2 in vitro in the presence of the N terminus of ARC6 (Figure 5). Furthermore, BiFC assays showed that in vivo, MCD1 interacted with FtsZ2 in wild­type and parc6 protoplasts but not in protoplasts from the arc6 mutant (Figure 6). These findings suggest that the associa­tion of MCD1 to FtsZ filaments on the membrane is specifically mediated by ARC6 in vivo (Figure 11). In fact, the single mcd1, arc3, and arc11 mutants exhibit similar heterogeneity in terms of chloroplast size and number and share the same FtsZ morphology, i.e., multiple Z­rings. Moreover, the mcd1 arc11, mcd1 arc3, and arc3 arc11 double mutants did not exhibit additive genetic effects (Figures 9, 10A, and 10B) (Marrison et al., 1999; Glynn et al., 2007; Zhang et al., 2013). These findings suggest that MCD1, MinD1, and ARC3 act as a complex to inhibit FtsZ assembly. Recent in vitro studies have suggested that ARC3, a replacement for bacterial MinC in plants, directly inhibits FtsZ assembly, likely through the

Figure 9. The mcd1 arc11 Double Mutant Exhibits a Similar Chloroplast Phenotype and FtsZ Morphology to the Single mcd1 and arc11 Mutants.

(A) Differential interference contrast images showing that chloroplast mor­phology was observed in living mesophyll cells of the wild type, the mcd1 and arc11(minD1) single mutants, and the mcd1 arc11 double mutant. The mcd1, arc11, and mcd1 arc11 mutants exhibit similar heterogeneity in chloroplast size and number compared with the wild type. Bar = 10 µm.(B) Fluorescence micrographs showing the localization patterns of FtsZ1­CFP in chloroplasts of the indicated transgenic plants expressing

FtsZ1pro-FtsZ1-CFP. Multiple Z­rings were observed in the mcd1, arc11, and mcd1 arc11 mutants. More than 30 cells were observed in each indi­cated transgenic lines, and representative photographs are shown. Flu­orescent FtsZ1­CFP and chlorophyll signals were falsely colored green and magenta, respectively. Bar = 10 µm.(C) Immunoblot analysis showing relative FtsZ1­CFP levels in the indicated transgenic plants in (B) compared with wild­type transgenic plants with low levels of FtsZ1-CFP expression. Actin served as a loading control.

1818 The Plant Cell

debundling and disassembly of existing FtsZ filaments, in a concentration­dependent manner (Johnson et al., 2015; Shaik et al., 2018). To inhibit FtsZ assembly on the membrane in vivo, the stromal protein ARC3 must be recruited to the membrane or directly to membrane­tethered FtsZ filaments. Since ARC3 interacts with MinD1 (Maple et al., 2007), and we did not detect a positive interaction between MCD1 and ARC3 (Supplemental Figure 6), it appears that ARC3 is first recruited to the mem­brane by MinD1, which is a membrane­associated protein pos­sibly due to an amphipathic helix at its C terminus (Colletti et al., 2000; Fujiwara et al., 2004). The similar multiple ring structures

of MCD1, FtsZ1, and FtsZ2 observed in the arc3 and arc11 (an allele of MinD1) mutants (Figures 9B and 10B) (Nakanishi et al., 2009) imply that MCD1 associates with the membrane­ tethered FtsZ filaments ahead of both MinD1 and ARC3 (Figure 11). MCD1 is important for MinD1 localization to the FtsZ filaments on the membrane (Nakanishi et al., 2009) (Supplemental Figure 5), and like MCD1, MinD1 also localized to the multiple ring structures in arc3 chloroplasts (Figure 10E; Supplemental Figure 5A). Thus, these data suggest that MCD1 might recruit a membrane­ associated complex comprising MinD1 and ARC3 to FtsZ fil­aments through an interaction with MinD1, precisely delivering

Figure 10. MCD1 Regulates Chloroplast Division Site Positioning in Close Cooperation with ARC3 and MinE1.

(A) Chloroplast morphology and number in living cells of the indicated mutant plants.(B) Fluorescence micrographs showing the localization of FtsZ1­CFP in chloroplasts of the indicated transgenic plants expressing FtsZ1pro-FtsZ1-CFP. More than 20 cells were observed in each indicated transgenic lines, and representative photographs are shown.(C) and (D) Immunoblot analysis showing relative FtsZ1­CFP protein levels (C) in the indicated transgenic plants in (B) compared with wild­type transgenic plants with low levels of FtsZ1-CFP expression, and MCD1 protein levels (D) in wild­type, arc3, and arc12 plants. Actin probed on the blot served as a loading control.(E) Immunofluorescence micrographs showing different MCD1 localization patterns in the chloroplasts of arc3 and arc12 mutant plants.Blue, white, and yellow arrowheads indicate multiple chloroplast membrane constriction sites, multiple FtsZ rings, and multiple MCD1 ring structures, respectively. FtsZ1­CFP and anti­MCD1 signals were falsely colored green; chlorophyll signals were falsely colored magenta. Bars = 10 µm.

The Role of MCD1 in Regulating Z­Ring Positioning 1819

ARC3 to the existing FtsZ filaments on the membrane (Figure 11). The emergence of MCD1 in land plants might have served to enhance the accuracy and efficiency of the inhibitory complex in finding and inhibiting FtsZ filaments on the membrane. However, this division­inhibiting complex lacks site specificity, since both MCD1 and MinD1 are localized throughout the mem­brane, and Z­ring formation is prevented at all potential division sites unless MinE1 is also present (Figure 10; Supplemental Figure 5), strongly suggesting that MinE1 regulates Z­ring po­sitioning through conferring spatial specificity to the MCD1, MinD1, and ARC3 complex. The mcd1 arc12 (an allele of MinE1) double mutant, which resembles the arc3 arc12 and minD1-1 arc12 double mutants, exhibited multiple Z­rings, which is similar to mcd1, minD1-1, and arc3 rather than arc12 (Figure 9) (Glynn et al., 2007; Fujiwara et al., 2008; Zhang et al., 2013), indicating that the role of MinE1 in Z­ring positioning requires MCD1 in addition to MinD1 and ARC3. Collectively, these data suggest that MinE1, a factor that helps determine the chloroplast divi­sion site, promotes Z­ring formation, perhaps through spatially inhibiting the activity of the ARC3, MinD1, and MCD1 complex on the membrane. However, we did not detect an interaction between MCD1 and MinE1 (Supplemental Figure 6), but ARC3 and MinD1 do interact with MinE1 (Maple et al., 2005, 2007). Therefore, MinE1 likely acts on the inhibitory complex mainly through MinD1 and ARC3, although the biochemical activity of plant MinE1 must still be investigated. A recent study has re­ported pole­to­pole oscillation of the MinCD complex driven by MinE in Synechococcus elongatus, which has internal photosyn­thetic membranes (MacCready et al., 2017). However, MCD1 is a plant­specific protein and is not found in cyanobacteria (Nakanishi et al., 2009), indicating that green plants might have a distinct mechanism for site selection during chloroplast division. Thus, more studies of MinE1 are needed in the future to understand how MinE1 spatially restricts the inhibitory effect of the MCD1, ARC3, and MinD1 complex on FtsZ assembly underneath the membrane.

In summary, our in vivo analyses showed that MCD1 cooper­ates with ARC3, MinD1, and MinE1 to regulate FtsZ assembly underneath the inner membrane. These results, combined with the known effect of MCD1 on MinD1 localization (Nakanishi et al., 2009), suggest that the chloroplast division site­selection system (the chloroplast Min system) in land plants consists of ARC3, MinD1, MinE1, and MCD1.

METHODS

Plant Materials

Arabidopsis thaliana (Columbia­0) was used as the wild type. The Arabidopsis T­DNA insertion mutants mcd1 (SALK_015389), arc6 (SAIL_693_G04), parc6 (SALK_100009), and arc3 (SALK_057144) and the EMS­induced mutant arc12, all in the Col­0 background, were de­scribed previously (Shimada et al., 2004; Glynn et al., 2008; Nakanishi et al., 2009; Zhang et al., 2009) and were obtained from the ABRC Stock Center. The above single mutants were used to generate the mcd1 arc6, mcd1 arc3, and mcd1 arc12 double mutants. Homozygous mu­tants among F2 progeny were identified by PCR as previously described (Nakanishi et al., 2009; Wilson et al., 2011; Zhang et al., 2013). After vernalization for 3 d in the dark, seeds were sown in soil and grown at 23°C, 70% humidity, and in broad­spectrum light (110 µmol m–2 s–1) with a 16 h­light/8­h­dark photoperiod.

To obtain transgenic plants expressing ARC6pro­ARC6-eGFP and FtsZ1pro­FtsZ1-CFP, the eGFP and CFP fragments were amplified with primer set A032/A033 and separately cloned into the SalI/SacI site of pCAMBIA1300­20 (Zhang et al., 2013), resulting in binary vectors pCAMBIA1300­21 and pCAMBIA1300­22. The ∼3.3­kb FtsZ1 and ∼4.1­kb ARC6 DNA fragments were amplified from genomic DNA using primer pairs A043/A046 and C169/C122, digested with SbfI/BamHI and SbfI/SalI, and cloned into pCAMBIA1300­21 and pCAMBIA1300­22, respec­tively. Both clones were verified by sequencing. The constructs were introduced into plants by the floral dip method using Agrobacterium tumefaciens GV3101 (Clough and Bent, 1998). T1 and T2 seeds were sterilized and selected on 0.5× Linsmaier and Skoog medium (Caisson Laboratories) containing 30 µg/mL hygromycin B.

Figure 11. Proposed Working Model of the Role of MCD1 in the Onset of Z­Ring Positioning in Chloroplasts.

The model shows the topologies, interactions, and functional relationships of MCD1 with ARC6, FtsZ1, FtsZ2, MinD1, ARC3, and MinE1 based on our genetic, cytological, and interaction data, emphasizing the role of MCD1 in the Min system. In this model, ARC6 interacts with FtsZ2 to tether heteropolymers (Z­filaments) of FtsZ1 and FtsZ2 to the chloroplast IEM; MCD1 and ARC6 then interact with each other through their stromal regions, resulting in the recruitment of MCD1 to the FtsZ heteropolymers coupled to ARC6 in the membrane. Subsequently, MCD1 recruits MinD1 combined with ARC3 to the FtsZ heteropolymers, permitting ARC3 to function in the disassembly of FtsZ filaments. Finally, MinE1 spatially restricts the activity of this inhibitory complex of MCD1, MinD1, and ARC3 via an unknown mechanism, resulting in Z­ring assembly. Many details of this model remain to be elucidated. The proteins shown are not meant to represent stoichiometric ratios. OEM, outer envelope membrane; IMS, intermembrane space; N, N terminus; C, C terminus.

1820 The Plant Cell

Transient Expression Assays in Arabidopsis Protoplasts

A series of modified pUC19 constructs were used for protoplast transfor­mation. A 1925 bp HindIII/EcoRI-digested fragment containing the CaMV 35S promoter, multiple cloning sites, the eYFP coding region, and the nopaline synthase (Nos) gene terminator from pCAMBIA1300­20 (Zhang et al., 2013) was inserted into the HindIII/EcoRI site of pUC19, yield­ing the pUC19­35S­MCS­eYFP­Ter construct. The coding sequence of MCD1 without the stop codon was amplified from cDNA using the primer pair C064/C065. The PCR product was digested with XbaI/SalI and inserted between the unique XbaI and SalI sites of pUC19­35S­MCS­eYFP­Ter, resulting in the MCD1­eYFP construct. Two linear fragments were inversely amplified using primer pairs C214/C069 and C214/C215, using the MCD1­eYFP plasmid as template, and self­ligated individually, generating the MCD11­114­eYFP and MCD11­141­eYFP vectors. To construct the RecATP­eCFP vector, nucleotides at positions 1 to 195 bp of RecA and the full­length sequence of eCFP were amplified using primer sets A067/A068 and A069/A033, respectively. These two fragments were joined together by overlapping PCR using primer set A067/A033 and cloned into the XbaI/SalI site of pUC19­35S­MCS­eYFP­Ter after restric­tion enzyme digestion. A BamHI recognition site was introduced between RecA and eCFP. The RecATP­eYFP­ARC6 construct was generated via the following steps. First, eYFP without the stop codon and ARC6 lacking the first 201 bp were amplified using primer pairs C129/C124 and C125/C145, respectively. Overlapping PCR was then performed to fuse the two fragments using primer set C129/C145. The resulting PCR fragment was digested with BglII and KpnI and cloned into the BamHI/KpnI site of RecATP­eCFP to replace the original eCFP, yielding RecATP­eYFP­ARC6. To generate RecATP­eYFP­MCD1, the eYFP fragment without the stop codon and the MCD1 coding region for amino acids 53 to 349 were amplified with primer sets A069/A076 and A077/A078, digested with BamHI/XhoI and XhoI/SacI, respectively, and ligated with the BamHI/SacI­digested RecATP­eCFP construct. The full­length coding region of ARC6 was amplified with the primer set C127/C122, digested with SpeI/SalI, and cloned into the pUC19­35S­MCS­eCFP­Ter vector, yielding ARC6­eCFP construct.

To produce the RecATP­YFPN and RecATP­YFPC constructs, the YFPN and YFPC fragments were amplified using primer pairs C254/C252 and C255/C253, digested with XhoI/SacI, and cloned into RecATP­eCFP, re­placing its original eCFP sequence. The full­length eYFP sequence of MCD1­eYFP was replaced by YFPN using primers C254/C252, resulting in MCD1­YFPN. To obtain the RecATP­eYFPC­ARC6 construct, the YFPC- ARC6 fragment was amplified from the RecATP­eYFP­ARC6 construct using primer pair C269/C145 and cloned into the RecATP­eYFP­ARC6 vector after BamHI/KpnI digestion, replacing the original eYFP-ARC6 sequence. The fusion fragments of FtsZ2-YFPN and FtsZ2-YFPC were SOE­PCR amplified using primer sets C256/C258/C259/C252 and C256/C260/C261/C253, respectively, and separately cloned into the XbaI/SacI­digested pUC19­35S­MCS­eYFP­Ter vector, resulting in the pro­duction of the FtsZ2­YFPN and FtsZ2­YFPC constructs. All vectors were verified by sequencing.

Protoplast isolation was performed as previously described (Yoo et al., 2007). Rosette leaves from 4­week­old Arabidopsis plants of Col­0, arc6, or parc6 were used for protoplast preparation. The isolated protoplasts were counted with a hemocytometer and diluted to 2.5 × 105 to 5 × 105 protoplasts mL−1. For each transformation, 150 μL of protoplasts (corre­sponding to ∼20 µg of chlorophyll) was transformed with a total of 10 to 15 µg plasmid and incubated in the dark prior to examination.

Chloroplast Isolation, Protease Treatment, Fractionation, and Immunoblotting

To analyze the topology of each IEM protein, chloroplasts were isolated from the transformed protoplast samples. Following 16 h of incubation

in the dark and fluorescence observation, each protoplast pellet was collected, incubated in a 1.5­mL tube with 400 μL hypotonic buffer (0.3 M mannitol, 20 mM Tricine­KOH, pH 8.0, 5 mM EDTA, 5 mM EGTA, 10 mM NaHCO3, and 0.1% BSA), and rapidly ruptured by vigorous mixing for 40 s in a disruptor instrument (Digital Disruptor Genie; Scientific Indus­tries). The green chloroplast pellets were collected by centrifugation at 1500g for 2 min at 4°C. For trypsin treatment, chloroplast samples were incubated on ice for 30 min in 100 μL reaction buffer (50 mM HEPES, pH 7.9, 0.33 M sorbitol, and 0.8 mM CaCl2) including trypsin. The reactions were stopped by adding an equal volume of quenching buffer (50 mM HEPES­KOH, pH 7.9, 0.33 M sorbitol, and 1× cocktail inhibitor). After 15 min of incubation on ice, the chloroplasts were directly lysed by add­ing an additional 800 μL lysis buffer (20 mM HEPES, pH 7.9, and 1× cocktail inhibitor) to the reaction tube and incubated on ice for 10 min. Stroma and total membranes from the lysed chloroplasts were separated by centrifugation at 435,000g for 20 min at 4°C. Subsequently, the pel­lets were directly resuspended in 30 μL 2× SDS loading buffer, while the supernatants were precipitated in four volumes of cold acetone for 1 h at −20°C and pelleted by centrifugation at 15,000g for 20 min at 4°C. The precipitated pellets were resuspended in 30 μL 2× SDS loading buffer. All fractionated samples were subjected to SDS­PAGE and blotted onto PVDF membranes (Millipore). The blots were probed with a 1:2500 dilution of mouse anti­GFP (Clontech; JL­8, lot A0042539) or a 1:1000 dilution of rabbit anti­Toc75, anti­Tic40 (Agrisera; AS06 150 and AS10 709), or anti­ RbcS (PhytoAB; PHY0067), respectively, followed by a 1:10,000 dilution of goat anti­mouse or goat anti­rabbit IgG conjugated to horseradish peroxi­dase (Pierce; lot PA196831, PC1833773). The blots were imaged using an Amersham Imager 600 UV (GE Healthcare) after mixing with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). The images were processed using Adobe Photoshop (Adobe Systems).

Microscopy

To analyze chloroplast morphology, living, expanding rosette leaves with­out the lower epidermis layers were directly examined. Light micrographs depicting chloroplast morphology in living mesophyll and petiole cells of the wild type and each mutant were taken using differential interference contrast optics with a Zeiss Axio Imager M2 multifunctional microscope outfitted with a Q­imaging MP5 color camera. Immunofluorescence la­beling of MCD1 and MinD1 was performed using protoplasts as previ­ously described (Wilson et al., 2011). Rabbit anti­MCD1 and anti­MinD1 (Nakanishi et al., 2009), and Alexa Fluor 488­conjugated anti­rabbit sec­ondary antibodies (Pierce; A11034, lot 1616933) were used at a dilution of 1:500. The slides were mounted using ProLong Diamond Antifade solution (Life Technologies). A Zeiss Axio Imager M2 multifunctional microscope equipped with an Andor Zyla monochrome CCD and filter cubes for YFP, CFP, and GFP was used for all fluorescence micrograph capture. Images were processed and arranged using Fiji image software (National Institutes of Health) and Adobe Photoshop (Adobe Systems).

Two-Hybrid Analysis

The constructs pGAD­ARC6N, pGAD­PARC6N, pGAD­ARC6C, pGAD­

PARC6C, pGBK­FtsZ1, pGBK­FtsZ2, and pGAD­ARC3 used in this study were described previously (Glynn et al., 2008; Schmitz et al., 2009; Zhang et al., 2013, 2016). The coding sequence for amino acids 53 to 114 of MCD1 was amplified using primer pair A074/A075 and cloned into pGB­KT7 using the NdeI and EcoRI sites to create pGBK­MCD1N. The coding sequence for amino acids 141 to 349 of MCD1 was amplified using primer pair C148/C149 and individually cloned into pGADT7 and pGBKT7 us­ing the NdeI and XmaI sites, yielding pGAD­MCD1C and pGBK­MCD1C. pGBK­MCD1­C(∆277­314) was generated by PCR­based mutagenesis using primer pair C159/C160. pGAD­MinE1 was generated by the primer set C111/C112. Yeast strain Y2HGold (Clontech) was cultured and transformed

The Role of MCD1 in Regulating Z­Ring Positioning 1821

as recommended by the manufacturer using standard synthetic dropout medium (Clontech) as indicated.

Pull-Down Assays

To express recombinant His­ARC6N and MBP­ARC6N in Escherichia coli, the coding region for amino acids 154 to 504 of ARC6 was amplified using primer pairs C165/C166 or C209/C190. These two PCR products were digested with NcoI/BgllI and NdeI/SalI and inserted into the NcoI/BamHI site of pHIS8­3 and the NdeI/SalI site of pMAL­c5x, respectively. For the His­ARC6C and MBP­ARC6C constructs, the coding region for amino acids 637 to 801 of ARC6 was amplified using primer set C246/C247 or C244/C245, and then two PCR products were cloned into the NcoI/SalI­digested pHIS8­3 and the NdeI/BamHI­digested pMAL­c5x, respectively. The coding regions of amino acids 53 to 114 and 141 to 349 of MCD1 were PCR amplified using primer pairs C267/C268 and C155/C156 and separately inserted into the pGEX­6p­1 vector after digestion, resulting in GST­MCD1N and GST­MCD1C. GST­MCD1C(∆277­314) was gen­erated by PCR­based mutagenesis using primer set C159/C160. For the recombinant MBP­PARC6N­His, the coding region of amino acids 77 to 573 of PARC6 was amplified using primer pair C001/C248 and cloned into pMAL­c5x after the NdeI/BamHI digestion. The FtsZ2 fragment with­out the first 48 amino acids was amplified using primer set C175/C174, digested with SapI/XhoI, and cloned into the SapI/XhoI­digested pTB146 vector (Cho et al., 2011), resulting in the His­SUMO­FtsZ2 construct. All of these constructs and the pGEX­6p­1, pMAL­c5X, and pTB146 empty vectors were expressed in Rosetta (DE3) E. coli (Novagen). Cells ex­pressing His­ARC6N or His­SUMO­FtsZ2 at OD600 of 0.7 were treated with 0.4 mM IPTG and incubated for 12 to 16 h at 18°C. Cells har­boring the other vectors were induced with 0.6 mM IPTG when the cell density reached an OD600 of 0.6 and incubated for 4 h at 37°C. Total protein was extracted from the induced cells by sonication in lysis buf­fer (50 mM Tris­HCl, pH 7.8, 300 mM NaCl, and 10 mM imidazole). After centrifugation at 100,000g for 20 min at 4°C, the supernatants were collected and analyzed by SDS­PAGE, and each recombinant protein in the soluble crude extract was quantified based on a comparison with 1 µg BSA on Coomassie­stained gels. His­SUMO­FtsZ2 and His­SUMO were purified using a HiTrap TALON column (GE Healthcare) and eluted with a gradient of imidazole. MBP and MBP­ARC6N were purified us­ing an MBP­Trap HP column (GE Healthcare) and eluted with 10 mM maltose.

For the GST pull­down assays, 50 μL samples of a 50% slurry of Glutathione­Sepharose 4B beads (GE Healthcare) were equilibrated in LT buffer (lysis buffer including 0.1% Triton X­100). The slurries were mixed with 1 mL of crude cell extract with 10 µg of GST or GST­fusion proteins and incubated for 1.5 h at 4°C on a nutator. The beads were washed three times with 1 mL of LT buffer and nutated with 1 mL of total protein extracts containing 5 µg His­ARC6N, His­ARC6C or MBP­PARC6N­His, or with 5 µg of purified His­SUMO­FtsZ2, His­SUMO, MBP, or MBP­tagged protein in a final volume of 1 mL in LT buffer for 4 h at 4°C. The beads were washed three times with 1 mL of LT buffer, followed by the addition of 1× SDS loading buffer for immunoblot anal­ysis. For the MBP pull­down assays, 1 mL of cell crude extract including 10 µg of MBP or MBP­ARC6N was incubated with 50 μL of equilibrated amylose resin beads (NEB) for 2 h at 4°C on a nutator. After three washes, the samples were incubated with 5 µg of purified His­SUMO­FtsZ2 or His­SUMO protein in a final volume of 1 mL in LT buffer for 4 h at 4°C on a nutator, followed by treatment as described above for the GST pull­down assays. All negative controls were performed in parallel. The proteins were resolved on 8 to 16% or 10% precast protein gels (Bio­Rad) and subjected to immunoblot analysis using antibodies specific for the His tag (Easybio; EB2067), GST tag (Abcam; ab3614), and MBP tag (NEB; E8038, lot 0091304).

Protein Level Analysis in Plants

Proteins were extracted from 2­week­old seedlings of various geno­types. Fresh leaf samples (50 mg) were combined with 50 μL of 2× SDS loading buffer, and 4 μL of protein extract was loaded onto each lane of an SDS­PAGE gel. After SDS­PAGE, the proteins were blotted onto a PVDF membrane. ARC6­eGFP and FtsZ1­CFP were detected using a 1:2500 dilution of anti­GFP monoclonal antibody (Clontech; JL­8); MCD1 and Actin were detected using a 1:2500 dilution of anti­MCD1 polyclonal antibody and a 1:10,000 dilution of anti­Actin mouse monoclonal anti­body (Sigma­Aldrich; A0480, lot 055M4866V), respectively. Anti­mouse and anti­rabbit HRP secondary antibodies (Pierce Biotechnology) were diluted 1:10,000. The PVDF blots were developed using a SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Pierce) and imaged using an Amersham Imager 600 UV (GE Healthcare).

Accession Numbers

Sequence data from this study can be found in The Arabidopsis Infor­mation Resource (TAIR) database under the following accession num­bers: MCD1 (At1g20830), FtsZ1 (At5g55280), FtsZ2 (At2g36250), ARC6 (At5g42480), PARC6 (At3g19180), MinE1 (ARC12, At1g69390), MinD1 (ARC11, At5g24020), and ARC3 (At1g75010). Germplasm identification numbers of alleles used in this work are mcd1 (SALK_015389), arc6 (SAIL_693_G04), parc6 (SALK_100009), arc12 (TAIR CS16472), arc11 (TAIR CS281), and arc3 (SALK_057144).

Supplemental Data

Supplemental Figure 1. Trypsin protection assays of RecATP­

eYFP­ARC6 and MCD1­eYFP fusion proteins in the presence of detergent.

Supplemental Figure 2. Trypsin protection assays of ARC6­eCFP fusion protein.

Supplemental Figure 3. MCD1 fused to the C terminus of eYFP is guided into the chloroplast by the RecA transit peptide and is localized to the chloroplast envelope membrane in the protoplast.

Supplemental Figure 4. The FtsZ1pro­FtsZ1­CFP construct is fully functional in plants.

Supplemental Figure 5. Localization patterns of MinD1 in wild­type, mcd1, arc3, and arc12 chloroplasts.

Supplemental Figure 6. Yeast two­hybrid assays showing that MCD1 does not interact with ARC3 or MinE1.

Supplemental Table 1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Thomas G. Bernhardt for providing the SUMO­tag vector, Shin­ya Miyagishima for providing the Arabidopsis MCD1 and MinD1 antibodies, and the ABRC for the T­DNA insertional mutants mcd1, arc6, arc3, arc11, and arc12. We also appreciate the suggestions and comments from the editors and three anonymous reviewers for im­proving the quality of this manuscript. This work was supported by the National Science Foundation of China (Grant 31470296), the Beijing Natural Science Foundation (Grant 5152003), and the Youth Innovative Research Team of Capital Normal University (Grant 010175300900) to M.Z.

AUTHOR CONTRIBUTIONS

M.Z. conceived the project, designed experiments, and wrote the man­uscript. L.C., B.S., W.G., Q.­y.Z., H.Y., and M.Z. performed experiments and analyzed the data.

1822 The Plant Cell

Received April 24, 2018; revised June 8, 2018; accepted June 23, 2018; published July 2, 2018.

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DOI 10.1105/tpc.18.00189; originally published online July 2, 2018; 2018;30;1807-1823Plant Cell

Li Chen, Bing Sun, Wei Gao, Qi-yang Zhang, Huan Yuan and Min ZhangChloroplast Division

MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide

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