PLASTID MOVEMENT IMPAIRED1 and PLASTIDMOVEMENT IMPAIRED1-RELATED1 MediatePhotorelocation Movements of BothChloroplasts and Nuclei1[OPEN]

Noriyuki Suetsugu2,3, Takeshi Higa2,4, Sam-Geun Kong2,5,6, and Masamitsu Wada4*

Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812–8581, Japan

ORCID IDs: 0000-0002-3328-3313 (N.S.); 0000-0002-3196-2160 (T.H.); 0000-0003-3013-4707 (S.-G.K.); 0000-0001-6672-7411 (M.W.).

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmentalstress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response)and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplastphotorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTIDMOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocationmovement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsisgenome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvementof PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocationmovements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in bothchloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. Inpavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, butpmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nucleicompletely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cellsand that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.

In plants, organelles movewithin the cell and becomeappropriately positioned to accomplish their functions

and adapt to the environment (for review, see Wadaand Suetsugu, 2004). Light-induced chloroplast move-ment (chloroplast photorelocation movement) is one ofthe best characterized organelle movements in plants(Suetsugu and Wada, 2012). Under weak light condi-tions, chloroplasts move toward light to capture lightefficiently (the accumulation response; Zurzycki, 1955).Under strong light conditions, chloroplasts escape fromlight to avoid photodamage (the avoidance response;Kasahara et al., 2002; Sztatelman et al., 2010; Davis andHangarter, 2012; Cazzaniga et al., 2013). In most greenplant species, these responses are induced primarily bythe blue light receptor phototropin (phot) in response toa range of wavelengths from UVA to blue light (ap-proximately 320–500 nm; for review, see Suetsugu andWada, 2012; Wada and Suetsugu, 2013; Kong andWada, 2014). Phot-mediated chloroplast movementhas been shown in land plants, such as Arabidopsis(Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al.,2001; Sakai et al., 2001), the fernAdiantum capillus-veneris(Kagawa et al., 2004), the moss Physcomitrella patens(Kasahara et al., 2004), and the liverwort Marchantiapolymorpha (Komatsu et al., 2014). Two phots in Arabi-dopsis, phot1 and phot2, redundantly mediate the ac-cumulation response (Sakai et al., 2001), whereas phot2primarily regulates the avoidance response (Jarilloet al., 2001; Kagawa et al., 2001; Luesse et al., 2010).

1 This work was supported by the Japan Society for the Promotionof Science (Grants-in-Aid for Scientific Research nos. 20870030 and26840097 to N.S., 25440140 to S.-G.K., and 20227001, 23120523,25120721, and 25251033 to M.W.).

2 These authors contributed equally to the article.3 Present address: Graduate School of Biostudies, Kyoto Univer-

sity, Kyoto 606–8502, Japan.4 Present address: Department of Biological Sciences, Graduate

School of Science and Engineering, Tokyo Metropolitan University,Tokyo 192–0397, Japan.

5 Present address: Division of Structural Biology, Medical Instituteof Bioregulation, Kyushu University, Fukuoka 812–8582, Japan.

6 Present address: Research Center for Live-Protein Dynamics,Kyushu University, Fukuoka 812–8582, Japan.

* Address correspondence to masamitsu.wada@gmail.com.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Masamitsu Wada (masamitsu.wada@gmail.com).

N.S. conceived the project; N.S., T.H., and S.-G.K. designed andperformed the experiments; N.S., T.H., S.-G.K., and M.W. analyzedthe data; N.S. and S.-G.K. wrote the article with contributions of allthe authors; M.W. supervised the project.

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M. polymorpha has only one phot that mediates both theaccumulation and avoidance responses (Komatsu et al.,2014), although two or more phots mediate chloroplastphotorelocationmovement inA. capillus-veneris (Kagawaet al., 2004) and P. patens (Kasahara et al., 2004). Thus,duplication and functional diversification of PHOT geneshave occurred during land plant evolution, and plantshave gained a sophisticated light sensing system forchloroplast photorelocation movement.

In general, movements of plant organelles, includingchloroplasts, are dependent on actin filaments (for re-view, see Wada and Suetsugu, 2004). Most organellescommon in eukaryotes, such as mitochondria, peroxi-somes, and Golgi bodies, use the myosinmotor for theirmovements, but there is no clear evidence that chloro-plast movement is myosin dependent (for review, seeSuetsugu et al., 2010a). Land plants have innovated anovel actin-based motility system that is specialized forchloroplast movement as well as a photoreceptor sys-tem (for review, see Suetsugu et al., 2010a; Wada andSuetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found inArabidopsis, are short actin filaments specifically lo-calized around the chloroplast periphery at the inter-face between the chloroplast and the plasmamembrane(Kadota et al., 2009). Strong blue light induces the rapiddisappearance of cp-actin filaments and then, theirsubsequent reappearance preferentially at the front re-gion of the moving chloroplasts. This asymmetric dis-tribution of cp-actin filaments is essential for directionalchloroplast movement (Kadota et al., 2009; Kong et al.,2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions ofchloroplasts becomes, the faster the chloroplasts move,in which the magnitude of the difference is determinedby fluence rate (Kagawa andWada, 2004; Kadota et al.,2009; Kong et al., 2013a). Strong blue light-induceddisappearance of cp-actin filaments is regulated in aphot2-dependent manner before the intensive polymer-ization of cp-actin filaments at the front region occurs(Kadota et al., 2009; Ichikawa et al., 2011; Kong et al.,2013a). This phot2-dependent response contributes tothe greater difference in the amount of cp-actin fila-ments between the front and rear regions of chloro-plasts. Similar behavior of cp-actin filaments has alsobeen observed in A. capillus-veneris (Tsuboi and Wada,2012) and P. patens (Yamashita et al., 2011).

Like chloroplasts, nuclei also show light-mediatedmovement and positioning (nuclear photorelocationmovement) in land plants (for review, see Higa et al.,2014b). In gametophytic cells ofA. capillus-veneris, weaklight induced the accumulation responses of bothchloroplasts and nuclei, whereas strong light inducedavoidance responses (Kagawa and Wada, 1993, 1995;Tsuboi et al., 2007). However, in mesophyll cells ofArabidopsis, strong blue light induced both chloroplastand nuclear avoidance responses, but weak blue lightinduced only the chloroplast accumulation response(Iwabuchi et al., 2007, 2010; Higa et al., 2014a). InArabidopsis pavement cells, small numbers of tiny

plastids were found and showed autofluorescence un-der the confocal laser-scanning microscopy (Iwabuchiet al., 2010; Higa et al., 2014a). Hereafter, the plastid inthe pavement cells is called the pavement cell plastid.Strong blue light-induced avoidance responses ofpavement cell plastids and nuclei were induced in aphot2-dependent manner, but the accumulation re-sponse was not detected for either organelle (Iwabuchiet al., 2007, 2010; Higa et al., 2014a). In bothArabidopsisand A. capillus-veneris, phots mediate nuclear photo-relocation movement, and phot2 mediates the nuclearavoidance response (Iwabuchi et al., 2007, 2010; Tsuboiet al., 2007). The nuclear avoidance response is depen-dent on actin filaments in both mesophyll and pave-ment cells of Arabidopsis (Iwabuchi et al., 2010).Recently, it was shown that the nuclear avoidance re-sponse relies on cp-actin-dependent movement ofpavement cell plastids, where nuclei are associatedwith pavement cell plastids of Arabidopsis (Higa et al.,2014a). In mesophyll cells, nuclear avoidance responseis likely dependent on cp-actin filament-mediatedchloroplast movement, because the mutants deficientin chloroplast movement were also defective in nuclearavoidance response (Higa et al., 2014a). Thus, photsmediate both chloroplast (and pavement cell plastid)and nuclear photorelocation movement by regulatingcp-actin filaments.

Molecular genetic analyses of Arabidopsis mutantsdeficient in chloroplast photorelocation movementhave identified many molecular factors involved insignal transduction and/or motility systems as well asthose involved in the photoreceptor system for chloro-plast photorelocation movement (and thus, nuclear pho-torelocation movement; for review, see Suetsugu andWada, 2012; Wada and Suetsugu, 2013; Kong andWada,2014). CHLOROPLAST UNUSUAL POSITIONING1(CHUP1; Oikawa et al., 2003) and KINESIN-LIKEPROTEIN FOR ACTIN-BASED CHLOROPLASTMOVEMENT (KAC; Suetsugu et al., 2010b) are keyfactors for generating and/or maintaining cp-actin fil-aments. Both proteins are highly conserved in landplants and essential for the movement and attachmentof chloroplasts to the plasmamembrane in Arabidopsis(Oikawa et al., 2003, 2008; Suetsugu et al., 2010b),A. capillus-veneris (Suetsugu et al., 2012), and P. patens(Suetsugu et al., 2012; Usami et al., 2012). CHUP1 islocalized on the chloroplast outer membrane and bindsto globular and filamentous actins and profilin in vitro(Oikawa et al., 2003, 2008; Schmidt von Braun andSchleiff, 2008). Although KAC is a kinesin-like protein,it lacks microtubule-dependent motor activity but hasfilamentous actin binding activity (Suetsugu et al.,2010b). An actin-bundling protein THRUMIN1 (THRUM1)is required for efficient chloroplast photorelocationmovement (Whippo et al., 2011) and interacts withcp-actin filaments (Kong et al., 2013a). chup1 and kacmutant plants were shown to lack detectable cp-actinfilaments (Kadota et al., 2009; Suetsugu et al., 2010b;Ichikawa et al., 2011; Kong et al., 2013a). Similarly,cp-actin filaments were rarely detected in thrum1

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mutant plants (Kong et al., 2013a), indicating thatTHRUM1 also plays an important role in maintainingcp-actin filaments.Other proteins J-DOMAIN PROTEIN REQUIRED

FORCHLOROPLASTACCUMULATIONRESPONSE1(JAC1; Suetsugu et al., 2005), WEAK CHLOROPLASTMOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodamaet al., 2010), and PLASTID MOVEMENT IMPAIRED2(PMI2; Luesse et al., 2006; Kodama et al., 2010) are in-volved in the light regulation of cp-actin filaments andchloroplast photorelocation movement. JAC1 is anauxilin-like J-domain protein that mediates the chloro-plast accumulation response through its J-domain func-tion (Suetsugu et al., 2005; Takano et al., 2010). WEB1and PMI2 are coiled-coil proteins that interact witheach other (Kodama et al., 2010). Although web1 andpmi2 were partially defective in the avoidance re-sponse, the jac1 mutation completely suppressed thephenotype of web1 and pmi2, suggesting that theWEB1/PMI2 complex suppresses JAC1 function (i.e.the accumulation response) under strong light con-ditions (Kodama et al., 2010). Both web1 and pmi2showed impaired disappearance of cp-actin filamentsin response to strong blue light (Kodama et al., 2010).However, the exact molecular functions of theseproteins are unknown.In this study, we characterizedmutant plants deficient

in the PMI1 gene and two homologous genes PLASTIDMOVEMENT IMPAIRED1-RELATED1 (PMIR1) andPMIR2. PMI1 was identified through molecular geneticanalyses of pmi1 mutants that showed severe defects inchloroplast accumulation and avoidance responses(DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang andAravind, 2010), but its roles and those of PMIRs incp-actin-mediated chloroplast and nuclear photo-relocation movements remained unclear. Thus, weanalyzed chloroplast and nuclear photorelocationmovements in the single, double, and triple mutantsof pmi1, pmir1, and pmir2.

RESULTS

PMI1 Is Essential for Chloroplast PhotorelocationMovement in Mesophyll Cells

We screened mutants using a band assay to identifythose deficient in chloroplast photorelocation move-ment (Kagawa et al., 2001; Oikawa et al., 2003; Suetsuguet al., 2005; Kodama et al., 2010). We isolated a mutantwith severe defects in chloroplast movement, andrough mapping and sequencing of candidate genesrevealed a mutation in its PMI1 gene (Fig. 1). The de-fect in chloroplast movement was complemented byPMI1pro::PMI1-GFP (see below). This mutant allele wasnamed pmi1-5, because pmi1-1, pmi1-2, pmi1-3, andpmi1-4 alleles have already been reported (DeBlasioet al., 2005; Rojas-Pierce et al., 2014). A 37-bp deletion(G172–T208 from start codon) was found in the PMI1exon 1 of pmi1-5 (Fig. 1A). The pmi1-5 mutation is

presumed to produce a premature stop codon. pmi1-5was characterized in detail in this study.

Chloroplast photorelocation movement in the wildtype, pmi1-5, and pmi1-2 (a transfer DNA [T-DNA] in-sertion mutant described in DeBlasio et al. [2005]; Fig.1A) was analyzed by measuring changes in leaf trans-mittance. Both chloroplast accumulation and avoidanceresponses (a weak light-induced decrease and a stronglight-induced increase in leaf transmittance, respec-tively) were severely impaired in pmi1-5 (Fig. 1, B andC; Supplemental Table S1). These impaired responseswere similar to those described previously for pmi1-1,a strong pmi1 allele (DeBlasio et al., 2005; Fig. 1A).Compared with pmi1-5, pmi1-2 showed weaker defectsin chloroplast photorelocation movement (Fig. 1, B andC; Supplemental Table S1), similar to the previous re-port that pmi1-2 was weaker than pmi1-1 (DeBlasioet al., 2005). Although pmi1-1 and pmi1-5 were severelyimpaired in chloroplast photorelocation movement,they retained partial chloroplast movement. Becausethere are two PMI1-like genes in the Arabidopsis ge-nome (At5g20610 and At5g26160 designated as PMIR1and PMIR2, respectively; DeBlasio et al., 2005), we as-sumed a possibility that the subtle chloroplast photo-relocation movement in pmi1 could be caused byPMIR1 and PMIR2. We obtained T-DNA insertion linesfor each gene (Fig. 1A) and generated double and triplemutants of pmi1 and pmir mutants. Contrary to our ex-pectations, the pmir1-1pmir2-1 double mutant exhibitedstronger chloroplast photorelocation movement com-pared with the wild type. The pmi1pmir1pmir2 triplemutants showed similar chloroplast photorelocationmovement to that of pmi1 single mutants (both pmi1-2and pmi1-5; Fig. 1, B and C; Supplemental Table S1).Between wild-type and pmi1mutant plants, we did notobserve any clear difference in leaf morphology, leafcolor, and chloroplast distribution pattern in dark-adapted cells as described previously (DeBlasio et al.,2005). Indeed, initial transmittance in dark-adaptedleaves was similar, and the slight differences in theinitial transmittance did not correlate with the differ-ences in the transmittance changes among genotypes(Supplemental Fig. S1). These results indicated thatPMI1 plays the major role in chloroplast movementcompared with PMIR1 and PMIR2. Hereafter, all ex-periments were performed using pmi1-5, pmir1-1, andpmir2-1 alleles.

Genetic Interaction between pmi1 and Other MutantsPartially Defective in Chloroplast PhotorelocationMovement in Mesophyll Cells

To elucidate the function of PMI1 in chloroplastphotorelocation movement, we analyzed the geneticinteraction between PMI1 and PHOT1, PHOT2, JAC1,WEB1, and PMI2 (and its homolog PMI15; Luesse et al.,2006; Fig. 2). For each gene, pmi1-5, phot1-5, phot2-1,jac1-2, web1-2, pmi2-2, and pmi15-1 alleles were used(Huala et al., 1997; Kagawa et al., 2001; Suetsugu et al.,

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2005; Luesse et al., 2006; Kodama et al., 2010). Althoughphot1 was partially defective in the accumulation re-sponse (Fig. 2A; Sakai et al., 2001), the avoidance re-sponse in phot1 was enhanced under certain conditions(Fig. 2A; Ichikawa et al., 2011). phot2 was severely de-fective in the avoidance response but not the accumu-lation response (Fig. 2A; Jarillo et al., 2001; Kagawaet al., 2001). pmi1phot2 showed a weak accumulationresponse similar to that of pmi1 and an impaired avoid-ance response similar to that of phot2 (Fig. 2A;Supplemental Table S1). However, there was a syner-gistic genetic interaction between the pmi1 and phot1mutations. pmi1phot1 showed a very weak avoidanceresponse (Fig. 2A; Supplemental Table S1). This resultindicated that PMI1 is necessary for phot2-mediatedchloroplast movements, especially the avoidance re-sponse, in the absence of phot1. jac1 was shown to beseverely defective in the accumulation response andpartially defective in the avoidance response (Suetsuguet al., 2005; Kodama et al., 2010). Like phot1pmi1, thepmi1jac1 double mutant was severely impaired in boththe accumulation and avoidance responses, similar tothe phot2jac1 double mutant (Suetsugu et al., 2005; Fig.2B). Thus, PMI1 has an important role in the phot2 sig-naling pathway that regulates the avoidance response.

We evaluated the genetic interaction between PMI1 andWEB1/PMI2 by analyzing pmi1web1 and pmi1pmi2pmi15.PMI15 is homologous to PMI2. The defect in chloro-plast movement was slightly stronger in pmi2pmi15

than in the pmi2 single mutant (Luesse et al., 2006; Fig.2B). Interestingly, the defect in the accumulation re-sponse of pmi1 was partially suppressed by web1 andpmi2pmi15mutations. Thus, the accumulation responseswere greater in pmi1web1 and pmi1pmi2pmi15 than inpmi1 (Fig. 2B; Supplemental Table S1). However, theavoidance response was greatly impaired in pmi1web1and pmi1pmi2pmi15, especially at 50 mmol m22 s21

(Fig. 2B; Supplemental Table S1). Superficially, the phe-notypes of pmi1web1 and pmi1pmi2pmi15were similar tothat of phot2. The enhanced accumulation response inpmi1web1 and pmi1pmi2pmi15 was suppressed by jac1mutation. pmi1web1jac1 and pmi1pmi2pmi15jac1 exhibi-ted similar phenotypes to that of pmi1jac1 (that is, thesevere attenuation of both the accumulation and avoid-ance responses; Fig. 2, C and D; Supplemental Table S1).These findings indicated that the suppression of the weakaccumulation response in pmi1 by the web1 or pmi2pmi15mutations depends on JAC1 activity.

PMI1 Is Localized Mainly in the Cytoplasm in BothMesophyll and Pavement Cells

The previous results (DeBlasio et al., 2005) and anal-yses of large-scale transcriptome (Zimmermann et al.,2004;Winter et al., 2007) and translatome data (Mustrophet al., 2009) indicated that PMI1 was preferentially ex-pressed in leaf tissues (Supplemental Fig. S2, A and B).

Figure 1. Gene structure of PMI1, PMIR1,and PMIR2 and chloroplast photoreloca-tion movement in mesophyll cells of pmi1and pmir1 pmir2 mutants. A, Gene struc-ture and mutation sites of PMI1, PMIR1,and PMIR2 genes. Rectangles indicate exons(gray rectangles indicate 59 or 39 untrans-lated region), and intervening bars indicateintrons. The gray bar in PMI1 shows thepromoter region used in PMI1pro::PMI1-GFP. LB, Left border of T-DNA. B, Changesin leaf transmittance caused by chloroplastphotorelocation movement. After transmit-tance measurement started, dark-adaptedsamples were kept in darkness for an addi-tional 10 min. Then, samples were sequen-tially irradiatedwith continuous blue light at3, 20, and 50 mmol m22 s21 for 60, 40, and40 min indicated by white, sky blue, andblue arrows, respectively. Light was turnedoff at 150 min (black arrow). Mean valuesfrom three independent experiments areshown. Error bars indicate SEs. C, Changes inleaf transmittance rates from 2 to 6 min afterchanges in light fluence rate (3, 20, and50 mmol m22 s21) are indicated as per-centage transmittance change over 1 min.Mean values from three independent ex-periments are shown. Error bars indicateSEs. WT, Wild type.

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PMIR1 was ubiquitously expressed in various tissues,although the expression level of PMIR1 was lower thanthat of PMI1 in leaf tissues. No expression data wereavailable for PMIR2, because there was no microarrayprobe set for PMIR2. The proteome data (Joshi et al.,2011) indicated that PMI1 protein was expressed invarious organs. Compared with the PMI1 peptide, amuch smaller amount of PMIR1 peptide was detectedin leaves, and no PMIR2 was detected in leaves(Supplemental Fig. S2C).To investigate the subcellular localization of PMI1,

we generated transgenic pmi1 lines expressing thePMI1-GFP fusion protein under the control of the pu-tative PMI1 promoter (Fig. 3). Transgenic lines withapproximately three-quarters gentamycin resistancewere selected from the T2 generation; these lines con-tained a single copy of the transgene. Chloroplastphotorelocation movement was examined in T3 ho-mozygous siblings. Most of the transgenic lines exam-ined were complemented by PMI1pro::PMI1-GFP,indicating that PMI1-GFP was a functional protein(Supplemental Fig. S3). When confocal microscopicanalysis was performed using the fully rescued

PMI1pro::PMI1-GFP transgenic lines, PMI1-GFP fluo-rescence was consistently detected in the cytosol ofmesophyll cells and the thin layer of cytoplasm in thepavement cells without specific localization on themembrane or organelles (Fig. 3A).

To determine the possible effects of the pmi1 muta-tion on the abundance and fractionation profiles ofphot1, phot2, JAC1, KAC, and CHUP1, we performedimmunoblot analyses on fractionated proteins fromwild-type and pmi1 rosette leaves (Fig. 3B). phot1,phot2, and CHUP1 were enriched in the microsomalfraction, and KAC was detected mainly in the solublefraction as described previously (Suetsugu et al.,2010b). JAC1 was detected exclusively in the micro-somal fraction, although a previous transient expres-sion analysis of GFP-JAC1 suggested that JAC is asoluble protein (Suetsugu et al., 2005). The proteinlevels and fractionation patterns of these proteins inpmi1 were the same as those in wild-type plants. Thus,the defects in the chloroplast photorelocation move-ment of pmi1 were not caused by impaired protein ex-pression or altered localization of these proteins thatregulate chloroplast photorelocation movement.

Figure 2. Changes in leaf transmittancerates in mesophyll cells of mutants crossedbetween pmi1 and phot, jac1, web1, orpmi2. Changes in leaf transmittance ratesfrom2 to 6min after changes in light fluencerate (3, 20, and 50 mmol m22 s21). A, Ge-netic interaction between PMI1 and PHOTgenes. B, Genetic interaction between PMI1and JAC1, WEB1, and PMI2 (and PMI15)genes. C, Genetic interaction betweenPMI1, JAC1, and WEB1 genes. D, Geneticinteraction between PMI1, JAC1, and PMI2(and PMI15) genes. For details, see Figure1C. Mean values from three independentexperiments are shown. Error bars indicateSEs. WT, Wild type.

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PMI1 Is Involved in Regulating Cp-Actin Filaments inMesophyll Cells

To examine the role of PMI1 on the regulation of cp-actinfilaments, we observed the dynamics of actin filamentsvisualized with GFP-talin using confocal laser-scanning

microscopy (for details, see “Materials and Methods”;Kong et al., 2013a). Inwild-type cells (Fig. 4; SupplementalMovie S1), a small amount of cp-actin filaments was de-tectable around the entire rims of chloroplasts before bluelight irradiation (Fig. 4A,white arrows).After irradiationwith strong blue light, cp-actin filaments rapidly dis-appeared from the irradiated area (Fig. 4A,white arrowsat 2 min 4 s [02:04]). Thereafter, an asymmetric distri-bution of cp-actin filaments was established with theaccumulation of cp-actin filaments at the front regions ofmoving chloroplasts (Fig. 4A, yellow arrows), and thechloroplasts moved to the nonirradiated area. However,in pmi1 mutant cells, chloroplasts did not move awayfrom the strong light-irradiated area (Fig. 4B; SupplementalMovie S1). Also, cp-actin filaments were not detectable onthe chloroplasts (Fig. 4B).

However, when the pmi1 mutant cells were incu-bated in the dark for 4 min (4 min) after a 30-s irradia-tion with blue light (30 s), cp-actin filaments weredetected in these cells as in wild-type cells, althoughthere was a smaller amount of cp-actin filaments inpmi1 mutant cells than in wild-type cells (Fig. 5). Afterirradiation with strong blue light, cp-actin filamentsdisappeared more rapidly from pmi1 cells than fromwild-type cells but reappeared after an additional4-min dark incubation (4 min; Fig. 5, A and B). It shouldbe noted here that any significant difference was notdetected in the cortical actin filament patterns in wild-type and pmi1 mutant cells (Figs. 4 and 5A), indicatingthat the defect of pmi1 was not the cause of any possi-bility, such as differential photobleach of the fluores-cent protein. These findings suggested that the cp-actinfilaments were unstable in the pmi1 mutant cells. We,therefore, speculated that the imaging blue laser (488nm) used to detect GFP likely caused the disappearanceof cp-actin filaments in pmi1 cells. To address this pos-sibility, we examined the chloroplast avoidance re-sponse with an imaging laser of 516 nm, which is out ofthe absorption spectra of phots (Sakai et al., 2001). Thechloroplast avoidance responsewas effectively inducedin the pmi1mutant cells by the 458-nm stimulating laserwhen the 516-nm laser was set for imaging (Fig. 5, CandD; SupplementalMovie S2). This resultwas consistentwith the partial chloroplast photorelocation movementdetected by measuring the change in leaf transmittance,in which red light was used to read transmittance (Fig. 1,B and C). Collectively, these findings indicated that thedefects in chloroplast photorelocationmovement in pmi1result from the impaired regulation of cp-actinfilaments.

PMI1 Alone Is Essential for the Nuclear AvoidanceResponse in Mesophyll Cells

We recently showed that cp-actin-dependent photo-relocationmovement of pavement cell plastids attachedto nuclei is required for the motive force generation fornuclear photorelocation movement in Arabidopsispavement cells and also, mesophyll cells (Higa et al.,2014a). We guessed that pmi1 single mutants but not

Figure 3. Subcellular localization of PMI1 and fractionation of proteinfactors regulating chloroplast movement in pmi1. A, Subcellular locali-zation of PMI1-GFP. Transverse sections of pavement cells andmesophyllcells were observed under a confocal laser-scanning microscope. Imageis false colored to indicate fluorescence of GFP (green) and chlorophyll(red). Arrows indicate PMI1-GFP fluorescence in the cytoplasm. B, Im-munoblot analysis of PHOT1, PHOT2, JAC1, CHUP1, and KAC proteinsin variousmutants. Total protein extracts (T)were fractionated into soluble(S) and microsomal (M) fractions by ultracentrifugation (100,000g for30 min at 4˚C). Immunoblotting was performed using indicated antisera(Suetsugu et al., 2010b). Numbers on the left indicate the molecularweight of protein markers in the far left lanes. Arrows indicate deducedfull-length bands of indicated proteins. The small arrow indicates thephot1 protein band recognized by phot2 antisera. WT, Wild type.

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pmir1pmir2 might be severely defective in the nuclearavoidance response in mesophyll cells, because pmi1but not pmir1pmir2 exhibited severe defects in chloroplastphotorelocation movement (Fig. 1). In both wild-typeand pmir1pmir2 plants, approximately 25% of nuclei indark-adapted plants were in the light position (i.e. ap-proximately 75% of nuclei in the dark position; Fig. 6).Strong blue light induced the nuclear avoidance re-sponse, and the response was saturated after 6 h (about60%–70% of nuclei were light positioned; Fig. 6). How-ever, pmi1 and pmi1pmir1pmir2 mutant plants showedalmost no nuclear avoidance response in mesophyllcells, and approximately 25% of nuclei were in the lightposition over the light irradiation period (Fig. 6). Theseresults showed that PMI1 is necessary for nuclear avoid-ance response as well as chloroplast photorelocationmovement in mesophyll cells.

PMI1 and PMIR1 Are Essential for the Nuclear AvoidanceResponse in Pavement Cells

In pavement cells of wild-type plants, most of nucleiwere positioned on the cell bottom in darkness (darkposition; Fig. 7A, dark) and moved to the anticlinalwalls in response to strong blue light (light position;Fig. 7A, blue light; Iwabuchi et al., 2007, 2010; Higaet al., 2014a). Wemeasured the percentage of pavementcells in which the nucleus was in the light positionduring the irradiation with strong blue light (Fig. 7,B–D). Inwild-type plants, approximately 30%of nuclei indark-adapted plants were in the light position (Fig. 7B),and thus, approximately 70% of nuclei were in the darkposition. Strong blue light induced the movement ofnuclei from the cell bottom to the anticlinal cell wall.This response was saturated after 9 h (about 70% ofnuclei were light positioned; Fig. 7B), reproducing theresults reported previously (Higa et al., 2014a). pmir1and pmir1pmir2 double mutant but not pmir2 similarlyshowed a slight impairment in strong light-inducednuclear movement. Although the population of nucleiin the light position sharply increased at 3 h after strongblue light irradiation in pmir1 and pmir1pmir2 like in thewild type, the light positioning was almost saturatedaround 60% at 6 h and even at 12 h after light irradia-tion, which was slightly less than that of the wild type(approximately 70%; Fig. 7B; Supplemental Table S1),indicating that PMIR1 but not PMIR2 is involved innuclear photorelocation movement in pavement cells.This result is consistent with the fact that PMIR2 is notexpressed in green parts (only very weak expression inroots; Supplemental Fig. S2). In pmi1, nuclear photo-relocation movement in pavement cells was greatlyimpaired; even after 12 h, only 57% of nuclei were in thelight position (Fig. 7, C and D; Supplemental Table S1).Notably, pmi1pmir1 double- and pmi1pmir1pmir2 triple-mutant plants lacked light-induced nuclear movement,and approximately 40% to 50% of nuclei were in thelight position, regardless of the light conditions (Fig. 7,C andD). The defective light-induced nuclearmovement

Figure 4. Observation of cp-actin filaments on moving chloroplastsin mesophyll cells of wild-type and pmi1 cells. A and B, Time-lapseimages of reorganization of cp-actin filaments in wild-type (A) andpmi1 (B) cells during chloroplast movement in response to strong bluelight (BL). Actin filaments were probed with GFP-mouse talin fusionprotein (green). Blue broken lines indicate the BL-irradiated area. Ir-radiation times (minutes-seconds) are shown at the top left corner.Note that cp-actin filaments rapidly reorganized on the rims of movingchloroplasts (numbers 1–6). White arrows indicate rapid disappear-ance of cp-actin filaments from the rear region of moving chloroplasts;yellow arrows indicate reappearance of cp-actin filaments in thefront region of moving chloroplasts. For the full time-lapse series, seeSupplemental Movie S1. Bar = 10 mm.

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in the pmi1pmir2 double- and pmi1pmir1pmir2 triple-mutant plants was similar to that in the pmi1 single- andpmi1pmir1 double-mutant plants (Fig. 7D; SupplementalTable S1).When light-adapted plantswere transferred todark conditions, the nuclei moved from the anticlinalwalls to the cell bottom, and it took approximately 20 hto complete the dark positioning (Supplemental Fig. S4).Although dark positioning occurred in pmi1, pmir1pmir2,

and pmi1pmir2, there was no detectable dark positioningin pmi1pmir1 and pmi1pmir1pmir2, mirroring the defec-tive light-induced nuclear movement in these mutants(Supplemental Fig. S4). Importantly, clear blue light-induced avoidance movement of pavement cell plastidsoccurred in the wild type (8 of 11 examined plastids) andpmi1 (5 of 13 examined plastids) but not in pmi1pmir1pmir2(0 of 7 examined plastids; Supplemental Movie S3). These

Figure 5. Reorganizations of cp-actin filaments in mesophyll cells under different light conditions. A, Light-dependent reorgani-zation of cp-actin filaments. Cells ofwild-type (WT) and pmi1 leaveswere irradiatedwith serial scans of a 458-nm laser for 30 s (bluelight [BL] 30 s) and then incubated in the dark for 4min (dark [D] 4min). Next, 3-min serial scanswith 458- and 488-nm lasers (BL 3min) were carried out to induce disappearance of cp-actin filaments. Finally, cells were incubated in the dark for 4 min (D 4 min).Images are false colored to show GFP (green) and chlorophyll (red) fluorescence. Note that cp-actin filaments disappeared after BLirradiation and reappeared after 4 min of adaptation in the dark in both the wild type and pmi1. Bar = 5 mm. B, BL-induced dis-appearance of cp-actin filaments inwild-type andpmi1mutant cells. Fluorescence intensities of cp-actin filamentsweremeasured atchloroplast edges inwild-type and pmi1mutant cells, representing changes in the amount of cp-actin filaments during BL irradiationfor 3min after the 4-min dark adaption. Values aremean6 SD (n=5 squares) in arbitrary units. C andD, Effect of 488- (C) and516-nm(D) imaging lasers on avoidance response in pmi1mutant cells. Time-lapse images were collected at approximately 30-s intervalswith two different imaging lasers (488 and 516 nm) for 15min and 8 s. The blue rectangular regions (region of interest [roi], 103 20mm) were irradiated with a stimulating laser (458 nm) during intervals between the image acquisitions of chlorophyll fluorescenceimageswith the imaging lasers. Chlorophyll fluorescence is false colored in red. Right showsmoving paths of individual chloroplasts(a–d). For the full time-lapse series, see Supplemental Movie S2. Bars = 10 mm.

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results indicated that, in pavement cells, PMI1 andPMIR1 redundantly mediate the avoidance responsesof nuclei and pavement cell plastids.

DISCUSSION

Although PMI1 was identified through the analysisof a mutant deficient in chloroplast photorelocationmovement a decade ago (DeBlasio et al., 2005), the rolesof PMI1 and its homologous proteins PMIR1 and PMIR2in not only chloroplast photorelocation movement butalso nuclear photorelocation movement remained to bedetermined. Therefore, we aimed to analyze the physi-ological and cellular functions of PMI1 and homologousPMIRproteins inArabidopsis. Ourfindings showed thatthe pmi1mutant plants are defective in both chloroplastaccumulation and the avoidance response and that thedefective chloroplast movement resulted from the im-paired regulation of cp-actin filaments in pmi1 mutantcells. Furthermore, our results revealed that PMI1 andPMIR1 are essential for the nuclear avoidance response.PMI1 is a plant-specific protein in the C2-domain

superfamily (DeBlasio et al., 2005; Zhang and Aravind,2010). The typical C2 domain of protein kinase C bindslipid in a calcium-dependent manner and thus, is in-volved in membrane targeting (Rizo and Südhof, 1998;Zhang and Aravind, 2010). PMI1 contains a C2 domainat the N terminus and a C-terminal conserved regionthat is found in plant PMI1 and PMIR proteins (DeBlasioet al., 2005). PMI1 is further classified into the N-terminal

C2 (NT-C2) family within the C2 superfamily (Zhangand Aravind, 2010). As its name suggests, the NT-C2family contains the C2 domain at the N terminus; thisfamily was recently identified as one of the four new C2subfamilies (Zhang and Aravind, 2010). Although theexact function of the C2 domain in NT-C2 family pro-teins has yet to be determined, the N-terminal conservedregion including the C2 domain of PMI1 might be es-sential for PMI1 function. pmi1-2 carries a T-DNA in-sertion that might result in a truncated PMI1 consistingof the entireN-terminal region, including theC2domain.The phenotype of pmi1-2 is weaker than that of pmi1-5.The sequence of pmi1-5 carries a premature stop codonthat might result in a PMI1 N-terminal fragment lackingthe intact conserved N-terminal region, suggesting thatthe N-terminal region including the C2 domain retainssome function of PMI1 if it is expressed.

Several NT-C2-domain family proteins contain adomain at the C terminus that is involved in regulatingactin filaments (for example, the Dilute- and Calponin-homologous domains; Zhang and Aravind, 2010),suggesting that NT-C2 family proteins might functionin regulating actin filaments. A previous study reportedthat the pmi1 mutant showed a normal pattern of cor-tical actin filaments (DeBlasio et al., 2005). However, wefound that the pmi1 mutant was defective in the regu-lation of cp-actin filaments, which are essential forphotorelocation movement and the attachment ofchloroplasts to the plasma membrane (Kadota et al.,2009; Kong et al., 2013a). These observations indicatedthat PMI1 mediates chloroplast photorelocation move-ment by the regulation of cp-actin filaments. Althoughour genetic analyses suggested that PMI1 functions pri-marily in the phot2 signaling pathway, the defects in cp-actin filaments differed between phot2 and pmi1. cp-actinfilament dynamics in the phot2 mutant cells were de-fective specifically in the process of depolymerization inresponse to strong blue light (Kadota et al., 2009; Konget al., 2013a). Although the fundamental processes of cp-actin filament dynamics, including actin polymerizationand depolymerization, were normal in pmi1 cells, theywere much more sensitive to blue light-dependent de-polymerization than wild-type cells. Consequently, theasymmetric distribution of cp-actinfilamentswas poorlyestablished in pmi1 cells, in which the 488-nm imaginglasermay have been sufficient to activate the phot signal.These results suggested that PMI1 is a downstreamsignaling factor that functions in the signaling pathwayfrom light perception to actin-based movement, includ-ing the regulation of cp-actin filaments.

Because the interface between chloroplasts and theplasma membrane is the important site for generationof cp-actin filaments and thus, the motive force forchloroplast movement (Kadota et al., 2009; Suetsuguet al., 2010a; Kong et al., 2013a), factors for chloroplastphotorelocationmovementmust be present in this area.CHUP1 and some phots (especially phot2) are localizedon the chloroplast outer envelope (Oikawa et al., 2008;Schmidt von Braun and Schleiff, 2008; Kong et al.,2013b), although most phots are localized on the

Figure 6. Distinct roles of PMI1 and PMIRs on nuclear photorelocationmovement in mesophyll cells. Time-course analysis of nuclear avoid-ance response in mesophyll cells of wild-type (WT), pmi1, pmir1pmir2double-mutant, and their triple-mutant plants. Nuclear avoidance re-sponse was induced by strong blue light (50 mmol m22 s21). The per-centage of cells in which the nucleus was in the light position isdepicted in mean 6 SD. Each data point was obtained from five leaves;100 cells were observed in each leaf.

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plasma membrane (Sakamoto and Briggs, 2002; Konget al., 2006). KAC proteins were present in both thesoluble andmicrosomal fractions, suggesting that someportion of KAC proteins is localized on the plasmamembrane (Suetsugu et al., 2010b). JAC1 was detectedin the microsomal fraction (Fig. 3B). PMI1-GFP fluo-rescence was detected mainly in the cytoplasm of me-sophyll cells (Fig. 3A). Although PMI1 proteins wereidentified in the proteome data for the plasma mem-brane protein (Nühse et al., 2003, 2004; Zhang and Peck,2011), we could not detect a specific association ofPMI1-GFP with the plasma membrane and/or organ-elles in the microscopic analysis.

A previous study identified PMI1 homologs inmonocot (rice [Oryza sativa] and corn [Zea mays]) andlegume species (soybean [Glycine max] and Medicagotrunculata; DeBlasio et al., 2005). Two Arabidopsisproteins (PMIR1 and PMIR2) distantly similar to PMI1(DeBlasio et al., 2005) were also identified. Detaileddatabase searches and phylogenetic analyses revealedthat PMI1/PMIR proteins are present in most landplants and the green alga Klebsormidium flaccidum(Supplemental Fig. S5). However, PMI1-clade proteinsare found only in seed plants, indicating that the sep-aration between PMI1 and PMIR clades occurred beforethe separation between gymnosperms and angiosperms.

Thus, it is plausible that ancestral PMI1/PMIR proteins(i.e. nonseed plant PMI1/PMIRproteins) have the abilityto regulate chloroplast photorelocation movement andthat the functional divergence between PMI1 and PMIRclades in seed plants occurred during the seed plantevolution in such a way of tissue-specific expression.

Although the involvement of PMIR1 and PMIR2 inchloroplast photorelocation movement is unclear inmesophyll cells, PMIR1 together with PMI1 are essentialfor the nuclear avoidance response in pavement cells(Supplemental Fig. S6). The nuclear avoidance responseis mediated by nucleus-attached pavement cell plastidsin a cp-actin filament-dependent manner (Higa et al.,2014a). The pmi1pmir1pmir2 plants were defective in theblue light-induced avoidance response of pavement cellplastids, although pmi1 retained the avoidance responseof pavement cell plastids (Supplemental Movie S3), in-dicating that PMI1 and PMIR1 redundantly mediate theblue light-induced avoidance response of pavement cellplastids. A tissue-specific translatome analysis showedthat PMIR1 was expressed preferentially in leaf pave-ment cells (Mustroph et al., 2009; Supplemental Fig.S2C), supporting the specific function of PMIR1 inpavement cells.

Although both PMI1 and PMIR1 were required forthe avoidance responses of pavement cell plastids and

Figure 7. Distinct roles of PMI1 and PMIRson nuclear photorelocation movement inpavement cells. A, Representative imagesshowing dark position (left) and light po-sition (right) of nuclei under the strong bluelight (BL) in pavement cells of wild-typeArabidopsis. Bar = 25 mm. B to D, Time-course analysis of nuclear avoidance re-sponse in pavement cells of wild-type (WT),pmi1, pmir1, pmir2 single, and their double-and triple-mutant plants. The other details arethe same as in Figure 6.

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nuclei in pavement cells, PMI1 alone was essential forchloroplast and nuclear avoidance responses in meso-phyll cells (Supplemental Fig. S6). Thus, defects in thephotorelocation movements of pavement plastids andchloroplasts were strongly correlated with the defectivenuclear avoidance response in both pavement andmesophyll cells, respectively. The chup1mutant showedimpaired chloroplast and nuclear avoidance responsesin mesophyll cells (Higa et al., 2014a). Furthermore, inthe jac1 mutant, chloroplasts and nuclei were localizedconstitutively on the anticlinal walls (Suetsugu et al.,2005; Higa et al., 2014a). Therefore, it is plausible thatlight-induced movement of chloroplasts is essential forthe nuclear avoidance response in mesophyll cells.However, there is no direct evidence for the chloroplast-mediated nuclear movement, because it is too difficult toanalyze the nuclear movement independent of chloro-plasts in mesophyll cells in which the nucleus is alwayssurrounded with many chloroplasts.In conclusion, our results showed that PMI1 plays an

important role in cp-actin-mediated chloroplast pho-torelocation movement in mesophyll cells and thatPMIR1 together with PMI1 are essential for cp-actin-mediated photorelocation movement of pavement cellplastids (Supplemental Fig. S6). Our results also showedthat PMI1- and PMI1/PMIR1-dependent photoreloca-tion movements of chloroplasts and pavement cellplastids are required for the motive force generation fornuclear photorelocation movement in mesophyll andpavement cells, respectively. Because cryptogamic landplants, such as bryophytes and lycophytes, have PMI1-like genes, it is plausible that PMI1 like is necessary forchloroplast and nuclear photorelocation movements inthese plants as well. Detailed analyses of PMI1/PMIR1in Arabidopsis and PMI1 orthologs in cryptogamic landplants are required to unravel the molecular mechanismof these responses.

MATERIALS AND METHODS

Plant Materials, Plant Growth, and Mutant Screening

Arabidopsis (Arabidopsis thaliana) seeds (Columbia-0, glabrous1) were sownon one-third-strength Murashige and Skoog culture medium containing 1%(w/v) Suc and 0.8% (w/v) agar.After incubation for 2 d at 4°C, the seedlingswerecultured under white light at approximately 100 mmol m22 s21 under a 16-h/8-hlight-dark cycle at 23°C in a growth chamber. Approximately 2-week-old seed-lings were used for mutant screening and analyses of chloroplast and nuclearphotorelocation movements. The band assay used to screen mutants and isolatethose deficient in chloroplast photorelocation movement has been describedpreviously (Kagawa et al., 2001;Oikawaet al., 2003; Suetsugu et al., 2005; Kodamaet al., 2010). The SALK T-DNA insertion lines (set of SALK T-DNA lines[CS27943]; pmi1-2 [SALK_141795; DeBlasio et al., 2005]; pmir1-1 [SALK_098762];and pmir2-1 [SALK_055706]) and the N7 nuclear marker line (Cutler et al., 2000)were provided by the Arabidopsis Biological Stock Center. According to previousreports (DeBlasio et al., 2005; Rojas-Pierce et al., 2014), our pmi1mutant line wasnamed pmi1-5. Double- and triple-mutant plants were generated by geneticcrossing. Mutant lines containing the N7 nuclear marker and GFP-mouse-talin(Kadota et al., 2009; Kong et al., 2013a) were generated by genetic crossing.

Generation of Transgenic Plants

To construct the PMI1pro::PMI1-GFP vector,GFP complementary DNAwascloned into the pPZP221/35S-nopaline synthase terminator (nosT) binary vector

(Hajdukiewicz et al., 1994) using the KpnI and SalI restriction sites, yieldingpPZP221/35S::GFP-nosT. A PMI1 gene fragment including the 2,817-bp 59 se-quence (before the start codon) and the gene body region including the openreading frame but lacking the stop codon were cloned into the KpnI site ofpPZP221/35S-GFP-nosT. The pmi1-5 mutants were transformed with pPZP221/PMI1pro::PMI1-GFP-nosT by the floral-dipping method using Agrobacteriumtumefaciens (GV3101::pMP90).

Analyses of Chloroplast Photorelocation Movement

Chloroplast photorelocationmovementwas analyzedbymeasuring changesin leaf transmittance as described previously (Kodama et al., 2010; Wada andKong, 2011). The third leaves were detached from 16-d-old seedlings andplaced on 1% (w/v) gellan gum in a 96-well plate. Samples were dark adaptedat least for 1 h before transmittance measurements.

Analyses of Nuclear Photorelocation Movement

Time-course experiments for nuclear photorelocation movement were per-formed as described previously (Higa et al., 2014a). For strong light-inducednuclear movement, 2-week-old plants were dark adapted for 24 h and irradi-ated with 50-mmol m22 s21 blue light for 12 h. The leaves were collected andfixed at 0, 3, 6, 9, and 12 h after light irradiation as described previously (Higaet al., 2014a). To analyze dark-induced nuclear movement, 2-week-old plantswere irradiatedwith 50-mmolm22 s21 blue light for 12 h and then dark adapted.The leaves were collected and fixed after 12, 16, 20, and 24 h of dark adaptation.

Immunoblot Analyses

Crude protein extracts were prepared from 2-week-old rosette leaves andfractionated as described previously. Immunoblot analysis was performed aspreviously described (Suetsugu et al., 2010b).

Confocal Laser-Scanning Microscopy

The subcellular localization of PMI1-GFP and cp-actin filaments and nuclearphotorelocation movement were observed under a confocal microscope (SP5;Leica Microsystems) as described previously (Kong et al., 2013a; Higa et al.,2014a). The multi-Ar laser was used at 488 nm for GFP and 458 nm (the outputlaser power of 2.8mW) for the chloroplast and nuclear avoidance responses. Thefluorescent signals were captured through the narrow bands of 500 to 550 nmfor GFP and 650 to 710 nm for chlorophyll autofluorescence.

Phylogenetic Analysis of PMI1 and PMIR Proteins

Multiple alignment, alignment curation, phylogenetic tree construction,and tree visualization were performed using MUSCLE (Edgar, 2004), Gblocks(Castresana, 2000), PhyML (Guindon andGascuel, 2003), and TreeDyn (Chevenetet al., 2006) outputs, respectively, according to a predefined pipeline at the Phy-logeny.fr server (Dereeper et al., 2008).

Sequence data from this article can be found in The Arabidopsis InformationResource (TAIR10) database under accession numbers PMI1 (At1g42550), PMIR1(At5g20610), and PMIR2 (At5g26160). Accession numbers and gene identifiers forgenes used in phylogenetic analyses are provided in Supplemental Figure S5.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Initial transmittance in leaves of dark-adaptedwild-type and pmi1/pmir mutant plants.

Supplemental Figure S2. Transcript and protein expression data of PMI1,PMIR1, and PMIR2 from Arabidopsis genome-wide transcriptome, trans-latome, and proteome database.

Supplemental Figure S3. Leaf transmittance changes indicative of chloro-plast photorelocation movement in mesophyll cells in PMI1pro::PMI1-GFP lines.

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Supplemental Figure S4. PMI1 and PMIR1, but not PMIR2, are essentialfor nuclear dark positioning in pavement cells.

Supplemental Figure S5. Phylogenetic tree of PMI1/PMIR proteins.

Supplemental Figure S6. Roles of PMI1/PMIR proteins.

Supplemental Table S1. Statistical tests for the data mentioned in the text.

Supplemental Movie S1. Reorganization of cp-actin filaments in wild-typeand pmi1 cells during strong blue light-induced chloroplast avoidanceresponse.

Supplemental Movie S2. Strong blue light-induced chloroplast avoidanceresponse in pmi1 mutant cells.

Supplemental Movie S3. Observation of pavement cell plastid irradiatedwith strong blue light in pmi1 and pmi1pmir1pmir2 pavement cells.

ACKNOWLEDGMENTS

We thank Atsuko Tsutsumi for laboratory assistance and the Arabidop-sis Biological Stock Center for T-DNA lines.

Received February 12, 2015; accepted August 27, 2015; published August 31,2015.

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Essential Factors for Plastid and Nuclear Movement

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