Chloroplast Outer Envelope Protein CHUP1 Is Essential for … · movement (suchasJAC1,PMI1, PMI2,and PMI15)that localizes on the chloroplast envelope (Oikawa et al., 2003; Schmidt

Chloroplast Outer Envelope Protein CHUP1 Is Essentialfor Chloroplast Anchorage to the Plasma Membraneand Chloroplast Movement1[W][OA]

Kazusato Oikawa, Akihiro Yamasato, Sam-Geun Kong, Masahiro Kasahara, Masato Nakai,Fumio Takahashi2, Yasunobu Ogura, Takatoshi Kagawa, and Masamitsu Wada3*

National Institute for Basic Biology, Okazaki, Aichi 444–8585, Japan (K.O., A.Y., S.-G.K., M.K., F.T., Y.O., T.K.,M.W.); Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Nojihigashi, Kusatsu,Shiga 525–8577, Japan (M.K.); and Institute for Protein Research, Osaka University, Suita, Osaka 565–0871,Japan (M.N.)

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusualpositioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocationmovement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes aprotein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-lengthCHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at theoutermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localizedmainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP(NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. Asimilar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containingOEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plantsexpressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coilregion of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP onthe plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outerenvelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 preventschloroplast aggregation and participates in chloroplast relocation movement.

The intracellular distribution of organelles is essen-tial for optimizing metabolic activities in plant cells;hence, the mechanisms by which organelles move totheir proper positions have long been investigated(Wada and Suetsugu, 2004). Chloroplast movement forefficient light absorption is the most precisely studiedof these phenomena, because of the importance ofphotosynthesis (Zurzycki, 1955; Takemiya et al., 2005).Chloroplasts change their position dynamically ac-

cording to the ambient light intensity. Under weaklight conditions, chloroplasts gather at the plasmamembrane along the periclinal cell wall in palisadecells (the accumulation response) in order to receiveoptimal sunlight exposure for efficient photosynthesis.In contrast, under strong light conditions, chloro-plasts are positioned at the plasma membrane alongthe anticlinal cell walls (the avoidance response) toavoid photodamage to the photosynthetic machinery(Kagawa and Wada, 2000; Kasahara et al., 2002; Wadaet al., 2003). Hence, chloroplast movement is essentialfor plants to get energy safely and efficiently undervarious light conditions. Chloroplast positioning in thedark is also known, but the patterns vary with plantspecies and tissues (Suetsugu et al., 2005).

Light-induced chloroplast relocation movementhas been studied using physiological approaches invarious plant species, including green algae (Hauptet al., 1969; Kraml et al., 1988), mosses (Kagawa et al.,1996; Kadota et al., 2000; Sato et al., 2001), ferns(Yatsuhashi et al., 1985; Yatsuhashi and Kobayashi,1993; Kagawa and Wada, 1996), and angiosperms(Trojan and Gabrys, 1996; Kagawa and Wada, 2000;Takagi, 2003). Recently, genetic approaches usingArabidopsis (Arabidopsis thaliana) mutants have iden-tified the photoreceptors and genes involved in chlo-

1 This work was supported by the Ministry of Education, Culture,Sports, Science, and Technology, Japan (grant no. 17084006 to M.W.),the Japan Society for the Promotion of Science (grant no. 16107002 toM.W.), and a Research Fellowship for Young Scientists to K.O.

2 Present address: Graduate School of Life Sciences, TohokuUniversity, Sendai 980–8577, Japan.

3 Present address: Department of Biology, Faculty of Science,Kyushu University, Fukuoka 812–8581, Japan.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Masamitsu Wada ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.123075

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roplast movement (for review, see Suetsugu andWada, 2007). Phototropin1 (phot1) and phot2 areblue light photoreceptors with two chromophore bind-ing sites (light, oxygen, and voltage domains) wherean FMN attaches (Jarillo et al., 2001; Kagawa et al.,2001; Sakai et al., 2001). Other factors involved inchloroplast movement have been identified as keyfactors in the signaling cascade that leads to chloro-plast relocation movement, including CHLOROPLASTUNUSUAL POSITIONING1 (CHUP1; Oikawa et al.,2003), JAC1 (for auxilin-like J-domain protein; Suetsuguet al., 2005), PLASTID MOVEMENT IMPAIRED1(PMI1; DeBlasio et al., 2005), PMI2 (Luesse et al.,2006), and PMI15 (Luesse et al., 2006). However, itremains unclear how these factors regulate chloroplastrelocation.

The cytoskeleton has also been implicated in chlo-roplast relocation movement (for review, see Takagi,2003; Wada et al., 2003). Pharmacological approacheshave shown that actin filaments are the main media-tors of chloroplast relocation movement (Kadota andWada 1992a; Tlalka and Gabrys, 1993; Sato et al., 2001).Several different actin filament structures were ob-served around chloroplasts in some plants tested byindirect fluorescence methods (Kadota and Wada,1992b; Dong et al., 1996, 1998; Kandasamy andMeagher,1999; Sakurai et al., 2005; Kumatani et al., 2006). Al-though these findings suggest that the specific actinfilament structures around chloroplasts are importantfor chloroplast relocation movement, it is not wellunderstoodwhen and how the actin structures function.Cytoskeletal myosin has also been suggested to contrib-ute to chloroplast relocation movement (Haupt andScheuerlein, 1990). When Arabidopsis palisade meso-phyll cells are treated with inhibitors of myosin, such as2,3-butanedione monoxime (BDM), N-ethylmaleimide,and 1-(5-iodonapthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride, chloroplasts are defec-tive in the accumulation response but not in theavoidance response (Paves and Truve, 2007). Thissuggests that an actomyosin system is at least involvedin the chloroplast accumulation response.

Receptors on the surface of each organelle are es-sential regulators of the organelle response to stimuliand are linked to the cellular components that medi-ate organelle transport (Bretscher, 2003). In buddingyeast, for example, a complex made of mitochondrialmembrane proteins such as Mmm1p, Mdm10p, andMdm12p is located on the mitochondrial outer mem-brane and links the mitochondria to the actin cytoskel-eton during fission (Boldogh et al., 2003). Mitochondria

Figure 1. Immunoblot analysis of CHUP1. A, Diagram of the CHUP1protein representing the regions used as antigens for the production ofpolyclonal antibodies. Anti-CHUP1 antibodies targeting the N termi-nus (aH) and C terminus (aT) were produced against the amino acidsequences between positions 200 to 320 and positions 700 to 1,004,respectively. Both regions are shown as black lines. FABR, F-actin-binding region; PRR, Pro-rich region; CtR, C-terminal region. B and C,Total protein (20 mg each) extracted from leaves (B) and isolatedchloroplasts (C) of wild-type (WT) and chup1 plants were analyzed by

immunoblotting with the two anti-CHUP1 antibodies. The asterisksindicate the major CHUP1 bands (approximately 150 kD). D, Theprotease sensitivity of CHUP1 shows that its localization is similar tothat of Toc159, which was used as a control chloroplast outer envelopeprotein. Toc75 is also an outer envelope protein deeply embeddedin the membrane. Tic110 is an inner envelope protein. cpHsp70 isabundant in the stroma.

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use the Arp2/3 complex, which polymerizes actinmonomers at the rear side of mitochondria, as a forcegenerator to move in a manner similar to the intracel-lular movement of bacteria such as Listeria monocyto-genes and Shigella flexneri in animal cells (Gouin et al.,2005). Rab27 on the melanosome surface regulates amotor protein for melanosome movement (Wu et al.,2002). These examples suggest that the key proteins forchloroplast relocation movement may also exist on thechloroplast surface. Previously, we isolated the chup1mutant (Oikawa et al., 2003), which shows aggregationof chloroplasts at the bottom of cells and lacks chloro-plast relocation responses to any light conditions. TheCHUP1 gene encodes a protein with several putativefunctional regions that are related to actin polymeriza-tion and might be involved in chloroplast relocationmovement. CHUP1 is thought to be the only proteinamong the recently found proteins related to chloroplastmovement (such as JAC1, PMI1, PMI2, and PMI15) thatlocalizes on the chloroplast envelope (Oikawa et al.,

2003; Schmidt von Braun and Schleiff, 2008). However,the actual localization of full-length CHUP1 remainsunclear. Furthermore, it is also not clear whether thesepredicted functional regions of CHUP1 actually func-tion physiologically to regulate chloroplast relocationdownstream of the photoreceptor signal cascade.

In this study, we focused on CHUP1 function fromthe viewpoint of its localization. We found that full-length CHUP1 localizes on the outer envelope ofchloroplasts and that this localization is essential forCHUP1 function. Furthermore, we found that theCHUP1 protein consists of three functional regions: achloroplast translocation signal at the N terminus, aregion that anchors the chloroplast to the plasmamembrane and has a coiled-coil character, and acytoskeleton-associated region. Here, we report thatCHUP1 is targeted to chloroplasts and has the novelphysiological function of regulating chloroplast local-ization by anchoring chloroplasts to the plasma mem-brane and forming a bridge to the actin cytoskeleton.

Figure 2. Subcellular localization of CHUP1-GFPand GFP-CHUP1. A, Diagram showing the GFP-CHUP1 and CHUP1-GFP constructs used in theexperiments. GFP-CHUP1 and CHUP1-GFPgenes under the control of the CaMV 35S pro-moter were introduced into chup1 mutant plantsfor expression. For each construct, a representa-tive line is shown amongmultiple lines examined.B, Immunoblot analysis of CHUP1 proteinsin wild-type (WT), chup1, GFP-CHUP1, andCHUP1-GFP lines with aH (anti-CHUP1 anti-body against CHUP1200-320). The top gel showstotal leaf results, and the bottom gel shows resultsfrom isolated chloroplasts. C, Distribution pat-terns of chloroplasts in wild-type, chup1, GFP-CHUP1, and CHUP1-GFP lines after adaptationto different light conditions. Detached leaves ofthese plants were put on an agar surface in a petridish and incubated in the dark (D), under weakwhite light (W), or under strong white light (S) for3 h before observation by light microscopy. Bar =10 mm. D, Fluorescence images of GFP-CHUP1and CHUP1-GFP in transgenic plants. Subcellularlocalization of GFP fluorescence from GFP-CHUP1 and CHUP1-GFP in palisade mesophyllcells was observed with a confocal laser scanningmicroscope. Green indicates fluorescence fromGFP, and red indicates fluorescence from chloro-phyll in chloroplasts. Bar = 10 mm.

Functional Analyses of CHUP1

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RESULTS

Detection of CHUP1 in an Isolated Chloroplast Fraction

To determine the subcellular localization of the full-length CHUP1 biochemically, we performed immuno-blot analyses of whole leaves and isolated chloroplastsusing two different polyclonal antibodies, one againstthe N-terminal (head) 200 to 320 amino acids (aH) andthe other against the C-terminal (tail) 700 to 1,004amino acids (aT) of CHUP1 (Fig. 1A). A specificCHUP1 band of approximately 150 kD was detectedwith both anti-CHUP1 antibodies in whole leaf ex-tracts of wild-type plants but not in extracts of chup1plants (Fig. 1B). The CHUP1 signal was also detectedin the purified chloroplast fraction from wild-typeplants (Fig. 1C). Interestingly, CHUP1 protein was notdetected after treatment of isolated chloroplasts withthe protease thermolysin (Fig. 1D). The transportprotein Toc159, which is also sensitive to thermolysin,is localized on the chloroplast outer envelope andknown to project into the cytoplasm (Soll and Schleiff,2004), suggesting that CHUP1 may also be located onthe chloroplast surface.

Transgenic Plants Expressing Fusion Proteins of CHUP1

with GFP

To confirm the subcellular localization of CHUP1,we generated transgenic plants stably expressing GFPfused to the N terminus (GFP-CHUP1) or C terminus(CHUP1-GFP) of CHUP1 under the control of the

cauliflower mosaic virus (CaMV) 35S promoter on thechup1 background (Fig. 2A). An immunoblot analysisusing whole leaf extracts of transgenic plants con-firmed that CHUP1-GFP and GFP-CHUP1 accumu-lated to the levels of endogenous CHUP1 in the wildtype, although the GFP-CHUP1 and CHUP1-GFPwere detected as different sizes (Fig. 2B, top). GFP-CHUP1 was not detected in an isolated chloroplastfraction (Fig. 2B, bottom). Analyses of intracellularchloroplast distribution under various light conditionsrevealed that the transgenic lines expressing CHUP1-GFP showed normal chloroplast relocation, but GFP-CHUP1 could not complement the chup1 phenotype(Fig. 2C). Although the GFP fluorescence of CHUP1-GFP and GFP-CHUP1 was faint, CHUP1-GFP wasobserved at the periphery of chloroplasts and GFP-CHUP1 was not (Fig. 2D). The localization observed isconsistent with the results of the immunoblot analyses(Figs. 1 and 2, B and D).

Correlation between the N-Terminal Hydrophobic

Region of CHUP1 and Localization of CHUP1 at thePeriphery of Chloroplasts

To investigate the chloroplast targeting region ofCHUP1, we used particle bombardment to transientlyexpress various fragments of CHUP1 fused to GFP inleaf cells of wild-type plants. Two types of fluorescencepatterns were observed, one surrounding chloroplastsand the other cytosolic (Fig. 3A). CHUP1 fragmentscontaining the N-terminal hydrophobic region (NtHR)

Figure 3. Transient expression analysis of GFP fusionproteins with truncated CHUP1 and the N-terminalregion of CHUP1. A, Each fragment obtained fromvarious parts of CHUP1 was fused to the N terminusof GFP (CHUP1n-GFP, where n = 1–25, 1–100,1–300, or 1–500) or to the C terminus of GFP (GFP-CHUP1n, where n = 1–25, 500–1,004, 750–1,004, or950–1,004) and expressed transiently under the con-trol of the CaMV 35S promoter. B, Various parts ofNtHR were fused to the N terminus of GFP (NtHRn-GFP, where n = 1–20, 1–15, 1–10, 1–5, 5–25, 10–25,15–25, or 20–25) and expressed transiently under thecontrol of the CaMV 35S promoter. GFP fluorescenceof each construct was observed and analyzed usingfive cells in each experiment. Green indicates fluo-rescence from GFP, and red indicates fluorescencefrom chlorophyll in chloroplasts. Numbers at top andbottom of the photographs are the amino acid posi-tions within CHUP1n. Bars = 20 mm.

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were fused to the N terminus of GFP to formCHUP11-25-GFP, CHUP11-100-GFP, CHUP11-300-GFP,and CHUP11-500-GFP. All were detected at the periph-ery of the chloroplasts, consistent with the distributionof CHUP1-GFP in Figure 2D. CHUP1 fragments werethen fused to the C terminus of GFP to form GFP-CHUP11-25, GFP-CHUP1500-1004, GFP-CHUP1750-1004,and GFP-CHUP1950-1004; all were detected in cytosol(Fig. 3A). This result was consistent with the distribu-tion of GFP-CHUP1 in stably expressed transgenic

lines shown in Figure 2D, but neither GFP-CHUP1 norCHUP1-GFP fluorescence was observed in the tran-siently expressing plants (data not shown). To clarifywhich parts or amino acids among 25 amino acids inNtHR were necessary for the localization, we con-structed various deletion constructs deleted from ei-ther the N-terminal or C-terminal side of NtHR fusedwith GFP and transiently expressed them in leafpalisade mesophyll cells. Almost all 25 amino acidstested were necessary for targeting NtHR-GFP frag-ments to chloroplasts (Fig. 3B).

Next, we compared the amino acid sequence of theArabidopsis NtHR with those of CHUP1 orthologsfrom rice (Oryza sativa), fern (Adiantum capillus-veneris),and moss (Physcomitrella patens). We found that theNtHR of all of the orthologs contained large hydro-phobic residues and some charged and polar aminoacids, such as Arg and Ser, as consensus amino acids(Fig. 4A). When NtHR-GFPs of the CHUP1 orthologswere expressed transiently in Arabidopsis leaf cells,GFP fluorescence was detected at the peripheryof chloroplasts for OsCHUP11-25 (rice CHUP1) andPpCHUP1A1-25 (moss CHUP1) but not AcCHUP1A1-25(fern CHUP1; Fig. 4B). To investigate the importance ofthe consensus amino acids Arg-4, Ser-12, and Arg-20,each modified NtHR sequence fused to GFP wastransiently expressed in Arabidopsis leaf cells. GFPfluorescence was found at the periphery of chloro-plasts when one of these amino acids in the NtHR waschanged to Ala, whereas the fluorescence was found inthe cytosol when two or all three of these amino acidswere changed (Fig. 4B).

Transgenic Lines Expressing NtHR-GFP Show Abnormal

Distribution of Chloroplasts

To address the correlation between NtHR and chlo-roplast distribution, NtHR-GFP was expressed stablyunder the control of the CaMV 35S promoter in wild-type cells of Arabidopsis (Fig. 5A). Transgenic lineswere classified into three groups according to theexpression level of NtHR-GFP (Fig. 5B). In the highlyexpressing lines, endogenous CHUP1 was detected asthe same level as in wild-type plants (Fig. 5C). GFPfluorescence was found at the periphery of chloro-plasts in all transgenic lines (Fig. 5D); however, chlo-roplasts in these transgenic lines showed abnormalpositioning and aggregation even under weak lightconditions (Fig. 5D). The chloroplast distribution be-came more abnormal as the NtHR-GFP expressionlevel increased, and chloroplast positioning in thehighly expressing line was similar to that in thechup1 mutant (Fig. 5D). These results suggest thatexcessively expressed NtHR-GFP disturbs the func-tion of CHUP1 by competing with authentic CHUP1 totarget chloroplasts; that is, it has a dominant-negativeeffect. To confirm this hypothesis, we performed asimilar experiment with CHLOROPLAST OUTERENVELOPE PROTEIN7 (AtOEP7), which inserts itsN terminus into the outer envelope and exposes its C

Figure 4. Hydrophilic amino acids in NtHR are critical for targetingCHUP1 to the chloroplast periphery. A, Multiple alignment of CHUP1-like proteins. The sequences were aligned using the ClustalW programat the European Bioinformatics Institute Web site. AtCHUP1, Arabi-dopsis CHUP1 (AB087408); OsCHUP1, Oryza sativa CHUP1(NM001072463.1); AcCHUP1A and AcCHUP1B, Adiantum capillus-veneris CHUP1A (AB444611) and CHUP1B (AB444612); PpCHUP1Aand PpCHUP1B, Physcomitrella patens CHUP1A (AB292414) andCHUP1B (AB292415). Blue shading indicates hydrophobic aminoacids, and red shading indicates hydrophilic amino acids replacedwith Ala for the experiments shown in B. B, Transient expression of GFPfusions of NtHR from AtCHUP1 and CHUP1-like proteins such asOsCHUP1, AcCHUP1A, and PpCHUP1A. C, Effects of amino acidsubstitutions in the NtHR of AtCHUP1 on the chloroplast targetingsignal. Amino acids Arg-4, Ser-12, and Arg-20, shown in red in A, werereplaced with Ala (red letters). GFP fluorescence patterns of eachconstruct were observed and analyzed using more than five cells ineach experiment. Green indicates fluorescence from GFP, and redindicates fluorescence from chlorophyll in chloroplasts. Bars = 20 mm.





terminus to the cytosol (Lee et al., 2001). A construct ofthe OEP7 targeting signal fused to GFP (OEP71-50-GFP)was introduced into wild-type plants. The sequence ofthe targeting signal region of OEP7 (OEP71-50) showsvery low similarity to the NtHR sequence, althoughboth sequences have hydrophobic regions (Fig. 5E).Nevertheless, in the transgenic plants, chloroplastsaggregated and GFP fluorescence was observed at theperiphery of chloroplasts (Fig. 5F), consistent with thepattern seen with NtHR-GFP.

Requirement of the N-Terminal Targeting Signal for

CHUP1 Function

We next examined whether the amino acid sequenceof NtHR is essential for CHUP1 function by replacingthe NtHR of CHUP1 with OEP71-50. Genes encodingthe full-length CHUP1 or two CHUP1s modified at the

N terminus (CHUP1DNtHR and OEP71-50-CHUP1DNtHR)were expressed in chup1 mutant plants under thecontrol of the CaMV 35S promoter (Fig. 6A). Thetranscript of each construct in each transgenic line wasconfirmed by reverse transcription (RT)-PCR withspecific primers (Fig. 6B). Endogenous CHUP1, exog-enous CHUP1, and OEP71-50-CHUP1DNtHR were ex-pressed at their predicted sizes, as confirmed byimmunoblot analysis, but CHUP1DNtHR was not (Fig.6C). It is possible that CHUP1DNtHR was degradedbecause it was not localized to the correct position, thechloroplast outer envelope. We then tested whetherthe mutant phenotypes were complemented by theseconstructs under various light conditions. Chloro-plasts in transgenic lines expressing CHUP1 orOEP71-50-CHUP1DNtHR showed normal chloroplast re-location similar to that of the wild-type plants underall light conditions tested, but chloroplasts in the

Figure 5. Effects of NtHR-GFP on chloro-plast distribution in transgenic plants ofwild-type background. A, Diagram show-ing the construct used in this experiment.The construct was placed under the con-trol of the CaMV 35S promoter and intro-duced into wild-type plants. B, Relativefluorescence intensities of NtHR-GFP intransgenic plants. Transgenic lines wereclassified into three levels of expressionaccording to the fluorescence intensity:low, middle, and high. C, Immunoblotanalysis of endogenous CHUP1 proteinin wild-type (WT) and transgenic lines(GFP and High) using the anti-CHUP1antibody (aH). Experiments were repeatedthree times for confirmation. D, Effect ofNtHR-GFP on chloroplast aggregation inthe transgenic plants. Distribution patternsof chloroplasts were observed in eachtransgenic line with different expressionlevels under Nomarski optics (top) andfluorescence microscopy (bottom). Thecells of each transgenic line were adaptedto weak white light (10 mmol m22 s21) for3 h before observation. Bar = 20 mm. E,Comparison of amino acid sequences be-tween NtHR of CHUP1 and the chloro-plast targeting signal of OEP7. F, Effect ofOEP71-50-GFP on chloroplast aggregationin the transgenic plants under weak whitelight. Other details were the same as inFigure 2D. Bar = 20 mm.

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CHUP1DNtHR transgenic lines did not (Fig. 6D). Theseresults indicate that the presence of an N-terminalregion is essential for CHUP1 function, although thewild-type sequence can be replaced with OEP71-50.Thus, the localization of full-length CHUP1 at thechloroplast envelope is essential to its function in thechloroplast relocation machinery.

The Coiled-Coil Region of CHUP1 Anchors Chloroplaststo the Plasma Membrane

CHUP1 has various putative functional regions,including an N-terminal chloroplast targeting signalregion (N), a coiled-coil region (C), a filamentous actinbinding region (A), a Pro-rich region (P), and aC-terminal highly conserved region (Ct; Fig. 7A).However, the roles of these regions in chloroplast reloca-tion movement remain unknown. To examine whetherthese regions participate in the physiological regula-tion of chloroplast relocation, we conjugated varioustypes of truncated CHUP1 to GFP and stably ex-pressed these genes in chup1 plants under the controlof the CaMV 35S promoter (Fig. 7A). An immunoblotanalysis showed that these transgenes were expressedin each transgenic plant (Fig. 7B). However, the calcu-lated sizes of the accumulated CHUP1-GFP proteins inthe transgenic lines expressing CHUP11-300(N-C),CHUP11-500(N-C-A), and CHUP11-700(N-C-A-P) werelarger than the sizes estimated from the gene con-

structs (Fig. 7B). In contrast, the transgenic line ex-pressing CHUP11-50,500-1004(N-P-Ct) showed a specificsignal at the predicted size (Fig. 7B).

Next, we investigated the physiological effects ofthese constructs on chloroplast relocation under variouslight conditions by observing chloroplast positioning,focusing from top to bottom of the palisade mesophyllcells, with a microscope. In wild-type palisade meso-phyll cells, a few chloroplasts were localized to thesurfaces of cells, but mostly at the anticlinal position ifthey had been kept in the dark for 3 h. After 12 h in thedark, most chloroplasts were localized to the cell bot-tom, as reported previously (Suetsugu et al., 2005). Incontrast, chloroplasts accumulated along the periclinaland anticlinal sides of palisade mesophyll cells underweak and strong light conditions, respectively (Fig. 7C).On the other hand, at all light intensities, and even in thedark, the chloroplasts of N-C, N-C-A, and N-C-A-Ptransgenic plants were positioned along the whole partof anticlinal sides of palisade mesophyll cells, as ob-served for chloroplast positioning in the wild-typeplants under strong light conditions (Fig. 7C). TheN-P-Ct transgenic plant still showed the chup1 pheno-type (Fig. 7C). Since all of these constructs except forN-P-Ct contain the coiled-coil region ofCHUP1, the func-tion of the coiled-coil region must be responsible for thechloroplast distribution along the anticlinal cell walls.

To further examine whether chloroplasts in thesetransgenic lines respond to light, we analyzed chloro-

Figure 6. Substitution of the NtHR of CHUP1 with the chloroplast envelope targeting signal sequence of OEP7. A, Diagramshowing CHUP1 constructs expressed in chup1 plants under the control of the CaMV 35S promoter. CHUP1DNtHR, NtHR-deletedCHUP1; OEP71-50-CHUP1DNtHR, NtHR-deleted CHUP1 with the chloroplast envelope targeting signal sequence of OEP7 (OEP71-50).Arrow sets show the primer regions used for RT-PCR. B, mRNA accumulation of each construct in the three transgenic lines. RT-PCRs were performed with the specific primers shown as (1), (2), and (3) in A. WT, Wild type. C, Immunoblotting of CHUP1. Totalprotein was extracted from leaves of each plant, and 20 mg of each was analyzed with an anti-CHUP1 antibody (aH). D,Distribution patterns of chloroplasts. Leaves of wild-type, chup1, and transgenic plants were adapted to different light conditions,dark (D), weak white light (W), or strong white light (S), and observed with a microscope. Bar = 20 mm.





plast relocation movement in N-C-A plants by time-lapse imaging after microbeam irradiation. In cells ofwild-type plants, chloroplasts moved toward the areairradiated with weak light but avoided the areas ofstrong light (Fig. 7D; Supplemental Movie S1). How-

ever, no chloroplast relocationmovement was observedin cells of the N-C-A transgenic lines, although thechloroplasts of the transgenic line looked firmly an-chored to the plasma membrane in any light condi-tions (Fig. 7D; Supplemental Movie S2). The fact that

Figure 7. Functional analysis of the coiled-coil region in CHUP1 protein. A, Diagrams showing each construct stably expressedin chup1 plants under the control of the CaMV 35S promoter. N, NtHR; C, coiled-coil region; A, F-actin-binding region (FABR); P,Pro-rich region (PRR); Ct, C-terminal region (CtR). Numbers under each construct are the amino acid numbers from the Nterminus. Transgenic lines expressing each construct are represented as N-C for CHUP11-300, N-C-A for CHUP11-500, N-C-A-P forCHUP11-720, and N-P-Ct for CHUP11-50,500-1004. B, Immunoblot analysis of transgenic lines. Immunoblotting was performedusing total protein from the transgenic lines and anti-CHUP1 antibodies (aH and aT). Asterisks show the specific band ofCHUP1-GFP detected by each antibody. C, Distribution patterns of chloroplasts in wild-type (WT), chup1, and transgenic lines.Leaves of wild-type, chup1, and transgenic plants were adapted to the dark (D), weak white light (W), or strong white light (S) for3 h and observed with a microscope. Bar = 20 mm. D, Time-lapse analyses of chloroplast relocation movement induced bymicrobeam irradiation in the cells of N-C-A and wild-type plants. A middle part of the cell surface was irradiated with amicrobeam of weak blue light (3 mmol m22 s21) for 60 min and then with strong blue light (30 mmol m22 s21) for 30 min. Imageswere taken every minute and stacked for a movie file (Supplemental Movies S1 and S2). Bar = 10 mm. E, Distribution patterns ofchloroplasts in each transgenic line in the presence of LatB and BDM. Leaves of transgenic lines were treatedwith 100 mM LatB or50 mM BDM under white light for 3 h. Bar = 20 mm. These experiments were repeated at least three times.

Oikawa et al.




CHUP11-300(N-C) does not contain the filamentousactin-binding region raises the question of whethercytoskeletal components are involved in this chloro-plast distribution. We tested whether treatment withcytoskeletal inhibitors would change the chloroplastdistribution pattern under weak light in the transgeniclines (Fig. 7E). The chloroplast distribution patternwas not affected at all by latrunculin B (LatB) andBDM in any line tested (Fig. 7E).

To address the function of the coiled-coil region forattaching chloroplasts to the plasma membrane, wetransiently expressed three truncated N-terminal re-gions of CHUP1 fused with GFP in wild-type cells(Fig. 8A). GFP fluorescence from CHUP125-322-GFP (i.e.the coiled-coil region with GFP) was detected at theplasma membrane in a pattern identical to that ofthe fluorescence of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridiniumdibromide (Fig. 8B), which was used as a plasmamem-brane marker (Bolte et al., 2004). However, the fluo-rescence of CHUP125-166-GFP (a truncated coiled-coilregion with GFP) was detected in the cytosol, similarto the pattern of GFP as a control (Fig. 8B). GFP fluo-rescence from CHUP125-500-GFP in palisade mesophyllcells was too weak to determine its subcellular localiza-tion (Fig. 8B). These results indicate that the coiled-coilregion of CHUP1 interacts with the plasma membrane,enabling CHUP1 to bridge the chloroplast and theplasma membrane to anchor chloroplasts on the anti-clinal side.

Effects of Cytoskeletal Inhibitors on ChloroplastAccumulation Movement

To investigate the role of the coiled-coil region ofendogenous CHUP1 in light-dependent chloroplastrelocation, we observed the distribution patterns ofchloroplasts in wild-type, chup1, phot1phot2, and N-C-Alines under weak light conditions after treatmentwith the cytoskeleton inhibitors LatB and BDM (Fig.9). When these plants were kept in the dark for 3 h,chloroplasts gathered at the cell bottom in chup1 cellsbut were located along the anticlinal plasma mem-brane in the wild-type, phot1phot2, and transgenicN-C-A plants (Fig. 9A). In the wild type, chloroplastaccumulation at the periclinal plasma membrane wasusually observed after transferring the plants to weaklight, but the relocation movement was not observedafter treatment with LatB or BDM (Fig. 9A). No chlo-roplast relocalization was observed in phot1phot2, N-C-A,and chup1 plants irrespective of the presence of cyto-skeletal inhibitors and even under weak light con-ditions (Fig. 9A). Finally, we observed chloroplastdistribution under strong light in these plants. Weobserved chloroplast relocation from the avoidance tothe accumulation positions only in wild-type plants,and this movement was prevented by the cytoskeletalinhibitors (Fig. 9B). These results indicate that chloro-plast distributions in phot1phot2 and N-C-A leaves aredetermined by the coiled-coil structure of endogenous

Figure 8. Transient expression analysis of the coiled-coil region ofCHUP1 in leaf cells. A, Diagrams showing the constructs used in thisexperiment. Each construct was transiently expressed in Arabidopsisleaf palisade mesophyll cells by particle bombardment. B, GFP fluo-rescence observed with a confocal laser scanning microscope in eachexpressed cell. Green indicates fluorescence from GFP, and red indi-cates fluorescence from chlorophyll in chloroplasts. N-(3-Triethyl-ammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridiniumdibromide (FM4-64) was used as a plasma membrane marker. Bar =20 mm.





CHUP1 that is located along the anticlinal plasmamembrane.

DISCUSSION

Proteins located on an organelle envelope, such asMmm1p, Mdm10p, and Mdm12p on mitochondria(Boldogh et al., 2003), Vac8 on vacuoles (Tang et al.,2003), Inp2 on peroxisomes (Fagarasanu et al., 2006),and Rab27 on melanosomes (Wu et al., 2002), havebeen reported to play a role in the intracellular local-ization of the organelles. We propose that CHUP1 hassimilarly an important function in chloroplast reloca-tion movement because it was found to localize on theouter envelope and also at the peripheral region ofchloroplasts in this work.

We detected full-length CHUP1 in the isolated chlo-roplast fraction by immunoblotting. Expression ofCHUP1-GFP in chup1 mutant plants complementedthe chup1 phenotype, and the GFP fluorescence wasdetected at the chloroplast periphery. In contrast, GFP-CHUP1 did not complement the chup1 phenotype, andits GFP fluorescence was mostly detected in the cyto-sol, probably because the NtHR of CHUP1 wasmasked by the GFP fused to the N terminus so thatCHUP1 could not be properly targeted to chloroplasts.To better define the targeting region of AtCHUP1, weinvestigated the targeting sequence of CHUP1 byusing GFP-fused constructs possessing deletions orsubstitutions within the NtHRs of CHUP1s fromArabidopsis and other species, such as rice, fern, and

moss. Our results showed that almost 25 amino acidsand two of the three consensus amino acids, Arg-4,Ser-12, and Arg-20, were important for targeting.However, the fluorescence of AcCHUP1A1-25-GFPwas not detected around the chloroplast periphery.AcCHUP1A1-25 lacks only one of the three consensusamino acids in the conserved hydrophobic region.This suggests that either the seven amino acids inlength between these consensus amino acids in thetargeting sequence is critical for the targeting mecha-nism or that the targeting sequence of AcCHUP1A islonger than that of CHUP1 from other species. Futurework will be designed to test these possibilities. Theimportance of NtHR for the distribution and func-tion of CHUP1 was further demonstrated in trans-genic plants expressing CHUP1 without the NtHR(CHUP1DNtHR), which did not complement the chup1phenotype. However, transgenic plants expressingOEP71-50-CHUP1DNtHR were able to restore the wild-type phenotype in chup1 mutants, suggesting thatOEP71-50 can substitute for NtHR. This finding meansthat the specific amino acid sequence of NtHR itself isnot critical for CHUP1 function, but the presence of anN-terminal targeting sequence and the subsequentlocalization of CHUP1 on the chloroplast outer enve-lope are essential. The OEP7 signal peptide inserts itsN terminus into the chloroplast outer envelope byrecognizing the specific lipid composition in the outerenvelope (Schleiff et al., 2001); therefore, NtHR mightalso be directly inserted into the chloroplast outerenvelope. Stable transgenic lines overexpressingOEP71-50-GFP or NtHR-GFP in wild-type plants (Fig.

Figure 9. Distribution pattern of chloroplasts under cytoskeletal inhibitor treatments. A, Plants of wild-type (WT), chup1,phot1phot2, and CHUP11-500-GFP lines were kept in the dark for 3 h (D). The plants were transferred to weak light (D-W) with orwithout LatB (+LatB) or BDM (+BDM) for 3 h. B, The plants were adaptedwith strong light (S) and then moved to weak light (S-W)with or without LatB or BDM for 3 h. Other details are the same as in Figure 2C. Bars = 20 mm.

Oikawa et al.




5, D and E) showed chloroplast aggregation similar tothe chup1 mutant phenotype (Lee et al., 2001; Oikawaet al., 2003). These results indicate that NtHR and thetargeting sequence of OEP7 occupy the same positionon the chloroplast outer envelope.The interpretation of the dominant-negative effect of

overexpressed NtHR-GFP or OEP71-50-GFP in wild-type cells is not clear. Because the structures of CHUP1and GFP are very different from each other, competi-tion between CHUP1 and GFP for the same bindingpartners is not plausible. One possible interpretation isthat CHUP1 competes with NtHR-GFP or OEP71-50-GFP for a nonspecific, three-dimensional space on theouter envelope. If the overexpressed NtHR-GFP orOEP71-50-GFP occupies the surface of the outer enve-lopemore rapidly than newly synthesized CHUP1, theexisting CHUP1 proteins might gradually be replacedby the GFP proteins, resulting in the release of CHUP1from the outer envelope and chloroplast aggregation.The sequences that target many chloroplast proteins

to the chloroplast outer envelope have been wellstudied (Hofmann and Theg, 2005; Inoue, 2007). How-ever, it has been difficult to predict the consensuspattern for the chloroplast outer envelope targetingsequence because of its variety and complexity (Inoue,2007). In fact, CHUP1 has not been recognized as achloroplast outer envelope protein in recent databasesand proteome analyses (Schleiff et al., 2003; van Wijk,2004). The amino acid sequence of NtHR, therefore, isa novel type of signal sequence for targeting a proteinto the chloroplast outer envelope, and as such, itprovides additional information for research on chlo-roplast outer envelope proteins.In this work, we studied the roles of the predicted

functional regions or domains of CHUP1 on chloro-plast relocationmovement using deletion constructs ofCHUP1 fused to GFP. In the transgenic lines express-ing CHUP11-300-GFP (abbreviated N-C), which lacks anactin-binding region, chloroplasts did not aggregatebut localized along the anticlinal plasma membraneunder any light intensity without a cytoskeletal sys-tem. These results suggested that the N-C region ofCHUP1 functions as a bridge connecting a chloroplastand the plasma membrane, with NtHR inserting intothe chloroplast outer membrane and the coiled-coilregion binding to plasma membrane proteins.One possible explanation for why chloroplasts in the

N-C line plants show an anticlinal but not a periclinaldistribution is that protein(s) interacting with thecoiled-coil region exists only in the anticlinal plasmamembrane of palisade mesophyll cells and not in thepericlinal membrane. However, a similar distributionpattern of chloroplasts was observed in the cellstreated with inhibitors of actin polymerization ormyosin function (Fig. 7). Furthermore, a chloroplastdistribution similar to that in N-C lines was alsoreported in transgenic plants overexpressing the ki-nase domain of phot2 (Kong et al., 2007) and in achloroplast relocation mutant, jac1, which is defectivein the accumulation response (Suetsugu et al., 2005).

These findings suggest that chloroplast positioningalong the anticlinal plasma membrane is the defaultposition for chloroplasts when chloroplast relocationmovement becomes defective.

The F-actin-binding region and the C-terminal Pro-rich region of CHUP1 might be involved in actinpolymerization (Oikawa et al., 2003). It was reportedrecently that CHUP1 has the ability to bind to profilinand G-actin (Schmidt von Braun and Schleiff, 2008).These functional regions might be essential for chlo-roplasts to move from their primary position at theanticlinal to the periclinal plasma membrane for theaccumulation response or to the bottom of the cell fordark positioning. In this work, we identified two otherfunctional regions in CHUP1, the NtHR, which targetsCHUP1 to the chloroplast outer envelope, and thecoiled-coil region, which anchors the chloroplast to theplasma membrane. These four functional regions ofCHUP1 mediate the interaction between chloroplastsand the plasma membrane and are involved in thepolymerization of or interaction with actin filamentsinvolved in chloroplast relocation movement. Theoverall function of CHUP1 enables chloroplasts tospread over the cell surface to form a single layerwithout aggregation or to move in any direction tomaximize efficient photosynthesis.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of wild-type (accession Columbia, gl-1), chup1-2 (Oikawa et al., 2003),

and transgenic lines of Arabidopsis (Arabidopsis thaliana) were sown on the

surface of one-third-strength Murashige and Skoog medium solidified with

0.8% (w/v) agar containing 1% (w/v) Suc on plastic culture plates as

described previously (Kagawa and Wada, 2000), incubated at 4�C for 2 d,

and then grown under light conditions of 16 h of light (white fluorescent light

at 70 mmol m22 s21) and 8 h of dark at 22�C for 3 to 4 weeks in an incubator

(Biotron LH300-RPSMP; Nippon Medical and Chemical Instruments).

CHUP1 Antisera Preparations

Two different regions of the CHUP1 gene, corresponding to H1 (121 amino

acids of CHUP1 at the coiled-coil region; CHUP1200-320) and T1 (the 305 amino

acids of CHUP1 at the C-terminal region; CHUP1700-1004), were cloned into a

pET21d vector for expression in Escherichia coli BL21 (DE3) cells (Novagen) as

the His-tagged fusion proteins CHUP1-H1 and CHUP1-T1, respectively.

Expression was induced by the addition of 1 mM isopropyl-1-thio-b-D-

galactopyranoside for 4 h at 37�C. The cells were harvested and broken in a

commercial bacterial cell lysis buffer (CelLytic; Sigma-Aldrich). CHUP1-H1

was recovered in the soluble fraction, whereas CHUP1-T1 became insoluble.

CHUP1-T1 was solubilized with a buffer containing 7 M urea, 50 mM Tris-HCl

(pH 7.5), and 300 mM NaCl. The solubilized protein was purified with a Talon

metal affinity column (Clontech). The purified proteins were desalted with

NAP-10 columns (GE Healthcare) containing 0.01% Triton X-100 and used for

the generation of antibodies in rabbits, as described previously (Asakura et al.,

2004).

Immunoblotting

Rosette leaves of Arabidopsis were homogenized with phosphate-buffered

saline (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4)

containing 10% (w/v) glycerol. The homogenized samples were centrifuged

at 10,000g for 5 min. The supernatants (20 mg of protein) were mixed with an

equal volume of 23 sample buffer (50 mM Tris-HCl [pH 6.8], 2% [w/v] SDS,





6% [v/v] 2-mercaptoethanol, and 10% [w/v] glycerol). The solubilized

samples were subjected to SDS-PAGE separation on a 7.5% (w/v) acrylamide

gel. The resolved proteins were blotted onto a Hybond-P membrane (GE

Healthcare). The anti-CHUP1-H1 (aH) and anti-CHUP1-T1 (aT) antibodies

were diluted 1:2,000 and used to detect the CHUP1 proteins. An anti-rabbit

IgG conjugated with alkaline phosphatase (GE Healthcare) was used as a

secondary antibody for both the aH and aT primary antibodies. Finally, cross-

reactive protein bands were detected using a commercial alkaline phospha-

tase development kit (ProtoBlot II AP System with Stabilized Substrate Kit;

Promega).

To determine the CHUP1 localization in chloroplasts, immunoblot analysis

was performed with isolated chloroplasts treated with thermolysin, 100 or 200

mg mL21 for 20 min on ice. Toc159, Toc75, Tic110, and cpHsp70 were used as

known controls of chloroplast proteins (Asakura et al., 2004; Kikuchi et al., 2006).

Chloroplast Preparation

Rosette leaves of Arabidopsis (10 g fresh weight) were homogenized in 200

mL of a homogenizing medium (50 mM HEPES-KOH [pH 7.8], 330 mM

sorbitol, 2 mM EDTA, 1 mM MnCl2, 1 mM MgCl2, 1 mM phenylmethylsulfonyl

fluoride, and 5 mM sodium ascorbate) using a modified mixer equipped with

razors. The homogenate was filtered through a doubled rayon-polyester filter

(Miracloth; Calbiochem). The filtered samples were centrifuged at 1,500g for

5 min. The precipitate was gently resuspended in 10 mL of a washing medium

containing 50 mM HEPES-KOH (pH 7.8), 330 mM sorbitol, and a protease

inhibitor cocktail (Complete EDTA-free; Roche Diagnostics). This fraction was

loaded on layers of 40% and 80% Percoll medium (40% or 80% [v/v] Percoll

[GE Healthcare], 50 mM HEPES-KOH [pH 7.8], 330 mM sorbitol, and the

protease inhibitor cocktail). After centrifugation at 1,500g for 15 min, intact

chloroplasts banded at the interface between the 40% and 80% Percoll media

were carefully recovered. The isolated chloroplasts were washed with the

washing medium by centrifugation at 1,500g for 5 min. The precipitate was

resuspended in the washing medium and used as intact chloroplast samples.

All procedures were carried out at 4�C.

Plasmid Construction

To generate plasmids for transiently expressing fusion proteins of each

DNA fragment and GFP, the corresponding regions were amplified by PCR

using specific primers and a CHUP1 cDNA or OEP7 cDNA as a template

(Supplemental Table S1). The PCR products were cloned into the CaMV35-

sGFP(S65T) plasmid (Chiu et al., 1996; Niwa et al., 1999) at the SalI-NcoI site for

N-terminal GFP fusion and at the BsrGI-NotI site for C-terminal GFP fusion.

The resulting plasmids were named as described in Supplemental Table S1. To

generate plasmids for transformation of Arabidopsis, each DNA fragment

that had been cloned in the CaMV35-sGFP(S65T) plasmid for transient

expression of GFP fusion proteins as described above was introduced into a

binary vector, pPZP211, at the XbaI-SmaI site (Supplemental Table S1).

Transformation of Arabidopsis

Transformation was performed according to the Agrobacterium tumefaciens-

mediated floral dip method (Clough and Bent, 1998). T1 plants were selected

on a medium containing one-third-strength Murashige and Skoog salts and

1% Sucwith 0.8% agar and 25 mgmL21 kanamycin. On the basis of segregation

of kanamycin resistance, T2 lines that contained a single transgene locus were

selected, and homozygous T3 seeds were maintained for further experiments.

At least three homozygous lines were used for each transgenic plant in this

study. All of the transgenic plants were isolated independently.

Transient Expression

Arabidopsis leaves were bombarded with gold particles (1 mm in diameter)

coated with plasmid DNA using a gene delivery system (PDS-1000/He particle

delivery system; Bio-Rad) as described previously (Oikawa et al., 2003). The

samples were incubated at 22�C for 12 h before observation with a microscope.

mRNA Detection by RT-PCR

Total RNAwas isolated using an RNA isolation kit (RNeasy Plant Mini Kit;

Qiagen) from rosette leaves of transgenic plants. RT-PCR was performed

according to the instructions in a commercial RT-PCR kit (PrimeScript RT-PCR

Kit; TaKaRa). The primers used to identify each transcript from the transgenes

were the specific forward primers CHUP1 (5#-AAGTCGACATGGGAAAA-

ACTTCGGGA-3#), CHUP1DNtHR (5#-AAGTCGACATGTCCAAACCAAGCA-

AACCATCAG-3#), and OEP71-50-CHUP1DNtHR (5#-ATGGGAAAAACTTCG-

GGA-3#), and a common reverse primer, 5#-AACCATGGACTCTTTCT-

CAGCTTTCTCCAAGT-3#.

Observation of Chloroplast Relocation

Chloroplast relocation in palisade mesophyll cells of transformants was

analyzed with detached leaves incubated on agar plates under weak light

(10 mmol m22 s21), strong light (100 mmol m22 s21), or in the dark for 3 h

after adaptation in the dark for 6 h. Chloroplast positioning of each pali-

sade mesophyll cell was visually determined with a microscope (Axioplan2;

Zeiss) while adjusting focuses throughout the cell from top to bottom.

Fluorescence Microscopic Analysis

The subcellular localization of CHUP1-GFP, GFP-CHUP1, and NtHR-GFP

was determined using a fluorescence microscope (Axioplan2; Zeiss) or a laser

scanning microscope (LSM Meta 510; Zeiss). Before observation, detached

leaves were adapted to weak light for 3 h.

Time-Lapse Imaging of Transformed Cells

Chloroplast relocation movement of wild-type and transgenic lines was

observed as described previously (Kagawa and Wada, 2000). The individual

cell was irradiated with a microbeam of blue light with a spot size of 19 mm in

diameter. Images of observed cells were obtained at 30-s intervals. The

intensity of the blue light was 3 mmol m22 s21 for the weak light response and

30 mmol m22 s21 for the strong light response. Cells were incubated in the dark

for at least 12 h before use.

Measurement of Relative Fluorescence Intensity for

NtHR-GFP Accumulation

Fluorescence intensity was measured as a gray value with the public

domain software ImageJ (http://rsb.info.nih.gov/ij/). The average fluores-

cence intensities of GFP were obtained by measuring the fluorescence inten-

sity at the chloroplast periphery of 20 individual chloroplasts in both the GFP

and NtHR-GFP lines. The fluorescence intensity of each NtHR-GFP line was

expressed as fluorescence percentage normalized to the value of the GFP line

with SD.

Inhibitor Assay

LatB (Biomol) and BDM (Sigma-Aldrich) were used to disrupt the actin

filament and inhibit myosin function, respectively. LatB and BDM were

dissolved in dimethyl sulfoxide as stock solutions of 2 mM and 1 M, respec-

tively. Final concentrations of 100 mM LatB and 50 mM BDM were used in the

experiments. Detached leaves placed on agar plates were adapted to either

strong light or dark for 3 h. Then the leaves were submerged in medium

containing either inhibitor and incubated under weak light for 3 h. Chloro-

plast distribution was observed with a microscope as described previously

(Oikawa et al., 2003).

The cDNA sequences for AtCHUP1, OsCHUP1, AcCHUP1A, AcCHUP1B,

PpCHUP1A, and PpCHUP1B were deposited in the DNA Data Bank of Japan

under accession numbers AB087408, NM001072463.1, AB444611, AB444612,

AB292414, and AB292415, respectively. The cDNA sequence for AtOEP7 was

deposited in GenBank under accession number NP_190810.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table S1. Primer sets for the plasmids used in these

experiments.

Supplemental Movie S1. The accumulation response and avoidance

response of chloroplasts in wild-type cells.

Oikawa et al.




SupplementalMovie S2. The response does not occur in N-C-A transgenic

plants. For details, see the legend of Figure 7D.

ACKNOWLEDGMENTS

We thank M. Nishimura for the use of a microscope and M. Maeshima for

kindly providing several antibodies.

Received May 19, 2008; accepted August 4, 2008; published August 20, 2008.

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Chloroplast Outer Envelope Protein CHUP1 Is Essential for … · movement (suchasJAC1,PMI1, PMI2,and PMI15)that localizes on the chloroplast envelope (Oikawa et al., 2003; Schmidt