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Phototropin Encoded by a Single-Copy Gene MediatesChloroplast Photorelocation Movements in theLiverwort Marchantia polymorpha1[W]

Aino Komatsu, Mika Terai, Kimitsune Ishizaki2, Noriyuki Suetsugu, Hidenori Tsuboi3,Ryuichi Nishihama, Katsuyuki T. Yamato4, Masamitsu Wada, and Takayuki Kohchi*

Graduate School of Biostudies, Kyoto University, Kyoto 606–8502, Japan (A.K., M.T., K.I., N.S., R.N., K.T.Y.,T.K.); and Faculty of Sciences, Kyushu University, Fukuoka 812–8581, Japan (N.S., H.T., M.W.)

Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoidphotodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplastmovements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspectsof signal transduction common to land plants are still unknown. Here, we show that the liverwortMarchantia polymorpha exhibits theaccumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark.Moreover, in silico and Southern-blot analyses revealed that theM. polymorpha genome encodes a single PHOT gene,MpPHOT, andits knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates alltypes of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation bothin vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fernAdiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarilyconserved regulatory activities for chloroplast photorelocation movement.M. polymorpha offers a simple and versatile platform foranalyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.

Light is not only an energy source for photosynthesisbut it is also a signal that regulates numerous physio-logical responses for plants. Because chloroplasts are theimportant organelle for photosynthesis, most plantspecies possess a light-dependent mechanism to regulatethe intracellular position of chloroplasts (chloroplastphotorelocation movement). Intensive studies on chloro-plast photorelocation movement have been performedsince the 19th century (Böhm, 1856). Senn (1908) de-scribed the chloroplast distribution patterns under differ-ent light conditions in various plant species, includingalgae, liverworts, mosses, ferns, and seed plants, andrevealed the general responses of chloroplasts to in-tensity and direction of light. Under low-light conditions,

chloroplasts are positioned along the cell walls perpen-dicular to the direction of incident light (i.e. periclinalcell walls) to efficiently capture light for photosynthesis(the accumulation response). By contrast, under high-lightconditions, chloroplasts are stacked along the cell wallsparallel to the direction of incident light (i.e. anticlinal cellwalls) to minimize total light absorption and to avoidphotooxidative damage (the avoidance response). Thesechloroplast movements are induced primarily by bluelight in most plant species (Suetsugu and Wada, 2007a).In some plant species, such as several ferns includingAdiantum capillus-veneris, the moss Physcomitrella patens,and some charophycean green algae (Mougeotia scalarisand Mesotaenium caldariorum), red light is also effective toinduce chloroplast movement (Suetsugu and Wada,2007b). Analyses of chloroplast movement in response toirradiation with polarized light and/or a microbeamsuggest that the photoreceptor for chloroplast movementis localized on or close to the plasma membrane (Hauptand Scheuerlein, 1990; Wada et al., 1993). In addition,chloroplasts assume their specific positions in the dark(dark positioning), although the patterns vary amongplant species (Senn, 1908). For example, the chloroplastsare localized at the bottom of the cell in palisade cells ofArabidopsis (Arabidopsis thaliana; Suetsugu et al., 2005a)and on the anticlinal walls bordering neighboring cells inthe prothallial cells of A. capillus-veneris (Kagawa andWada, 1993; Tsuboi et al., 2007).

Molecular mechanisms for chloroplast photorelocationmovements have been revealed through molecular ge-netic analyses using Arabidopsis (Suetsugu and Wada,

1 This work was supported by Grants-in-Aid for Scientific Re-search on Innovative Areas from the Japan Society for the Promotionof Science (grant nos. 23120516, 25120716, and 25113009 to T.K.).

2 Present address: Graduate School of Science, Kobe University,Kobe 657–8501, Japan.

3 Present address: Faculty of Dental Science, Kyushu University,Fukuoka 812–8582, Japan.

4 Present address: Department of Biology-Oriented Science andTechnology, Kinki University, Kinokawa 649–6493, Japan.

* Address correspondence to [email protected] 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:Takayuki Kohchi ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.114.245100

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2012). The light-activated kinase phototropin was identi-fied as the blue-light receptor (Jarillo et al., 2001; Kagawaet al., 2001; Sakai et al., 2001). Phototropin consists of twofunctional regions: a photosensory domain at the N ter-minus and a Ser/Thr kinase domain at the C terminus(Christie, 2007). The N-terminal photosensory domaincontains two light, oxygen, or voltage (LOV) domains,which belong to the Per/ARNT/Sim domain superfam-ily. Each LOV domain binds to one FMN and functionsas a blue-light sensor (Christie et al., 1999). The LOV2domain is essential for blue-light-dependent regulation ofthe activation of the C-terminal kinase domain (Christieet al., 2002; Harper et al., 2003).

Arabidopsis has two phototropins: phot1 and phot2(Christie, 2007). Besides chloroplast photorelocationmovement, phototropin controls other photoresponsesto optimize the photosynthetic efficiency in plants andimproves growth responses such as phototropism,stomatal opening, and leaf flattening (Christie, 2007).Both phot1 and phot2 redundantly regulate the chlo-roplast accumulation response (Sakai et al., 2001), hy-pocotyl phototropism (Huala et al., 1997; Sakai et al.,2001), stomatal opening (Kinoshita et al., 2001), andleaf flattening (Sakai et al., 2001; Sakamoto and Briggs,2002). Rapid inhibition of hypocotyl elongation is spe-cifically mediated by phot1 (Folta and Spalding, 2001),whereas the chloroplast avoidance response (Jarillo et al.,2001; Kagawa et al., 2001) and palisade cell development(Kozuka et al., 2011) are mediated primarily by phot2.

It is thought that the phototropin-regulated pho-toresponses are mediated by mechanisms in whichgene expression is not involved primarily. For ex-ample, chloroplast photorelocation movement can beobserved even in enucleated fern cells (Wada, 1988),and phototropins show only a minor contribution toblue-light-induced gene expression in Arabidopsis (Jiaoet al., 2003; Ohgishi et al., 2004; Lehmann et al., 2011).Furthermore, both phot1 and phot2 are localized on theplasma membrane despite the absence of a transmem-brane domain (Sakamoto and Briggs, 2002; Kong et al.,2006). During chloroplast movement, phototropins, inparticular phot2, associate not only with the plasmamembrane but also with the chloroplast outer membrane(Kong et al., 2013b). In addition, phot1 shows blue-light-dependent internalization into the cytoplasm (Sakamotoand Briggs, 2002; Knieb et al., 2004; Wan et al., 2008;Kaiserli et al., 2009), whereas phot2 exhibits a blue-light-dependent association with the Golgi apparatus (Konget al., 2006).

PHOT genes have been identified from various greenplants and are indicated to be duplicated in respectivelineages such as seed plants, ferns, lycophytes, andmosses (Li et al., 2014). In the fern A. capillus-veneris,chloroplast accumulation and avoidance responses areinduced by both blue and red light (Yatsuhashi et al.,1985). This fern has three phototropin family proteins,two phototropins (Acphot1 and Acphot2; Kagawa et al.,2004), and one neochrome that possesses the chromophore-binding domain of phytochrome and complete photo-tropin domains (Nozue et al., 1998). Neochrome is the

red-light receptor that mediates chloroplast movement(Kawai et al., 2003) and possibly blue-light-induced chlo-roplast movement through its LOV domains (Kanegaeet al., 2006). Because the Acphot2 mutant is defective inthe chloroplast avoidance response and dark positioning(Kagawa et al., 2004; Tsuboi et al., 2007), similar to thephot2 mutant in Arabidopsis (Jarillo et al., 2001; Kagawaet al., 2001; Suetsugu et al., 2005a), the function of phot2 inthe regulation of chloroplast movement is highly con-served in these vascular plants. In the moss P. patens, inwhich chloroplast accumulation and avoidance responsesare induced by both blue and red light (Kadota et al.,2000), seven phototropin genes are present in the draftgenome sequences (Rensing et al., 2008). The phototropinsencoded by four of these genes (PpphotA1, PpphotA2,PpphotB1, and PpphotB2) function in the blue-light-induced chloroplast movement (Kasahara et al., 2004).Moreover, red-light-induced chloroplast movements aremediated by both conventional phytochromes (Mittmannet al., 2004; Uenaka and Kadota, 2007) and phototropins(Kasahara et al., 2004). Because the direct associationbetween phytochromes and phototropins is suggestedto be involved in red-light-induced chloroplast move-ment (Jaedicke et al., 2012), phototropins should be es-sential components in the chloroplast movement signalingpathway (Kasahara et al., 2004).

A single PHOT gene was isolated in a unicellulargreen alga, Chlamydomonas reinhardtii (Huang et al., 2002;Kasahara et al., 2002). When expressed in Arabidopsisphot1 phot2 double-mutant plants, C. reinhardtii photo-tropin rescued the defects in chloroplast photorelocationmovement in phot1 phot2 plants (Onodera et al., 2005),indicating that the initial step of the phototropin-mediated signal transduction mechanism for chloro-plast movements is conserved in the green plant lineage.Although the existence of only one PHOT gene is idealfor elucidation of phototropin-mediated responses,C. reinhardtii cells contain a single chloroplast and showno chloroplast photorelocation movement.

Liverworts represent the most basal lineage of extantland plants and offer a valuable experimental systemfor elucidation of various physiological responsescommonly seen in land plants (Bowman et al., 2007).Marchantia polymorpha has emerged as a model liver-wort because molecular biological techniques, such asgenetic transformation and gene-targeting technol-ogies, have been established for the species (Ishizakiet al., 2008, 2013a; Kubota et al., 2013; Sugano et al.,2014). Furthermore, an ongoing M. polymorpha genomesequencing project under the Community SequencingProgram at the Joint Genome Institute has indicatedthat many biological mechanisms found in other groupsof land plants are conserved in a much less complex form.Blue-light-induced chloroplast movement was briefly re-ported inM. polymorpha (Senn, 1908; Nakazato et al., 1999).However, information on chloroplast photorelocationmovement in liverworts, includingM. polymorpha, is verylimited.

In this study, we investigated chloroplast photo-relocation movement in detail in M. polymorpha and

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analyzed the molecular mechanism underlying the pho-toreceptor system through molecular genetic analysis ofM. polymorpha phototropin.

RESULTS

Chloroplast Photorelocation Movements Are Induced byBlue Light But Not Red Light in M. polymorpha

The chloroplast distribution pattern was analyzedusing wild-type gemmalings (the early growth stage ofthalli developing from gemmae) in M. polymorpha. Un-der our growth conditions with continuous white light,chloroplasts were located over the upper cell surface as aresult of the accumulation response (Fig. 1A). After ir-radiation with strong blue light (50 Wm22 for 120 min),the chloroplasts relocated from the periclinal to the an-ticlinal cell walls (Fig. 1B), indicating that M. polymorphashows a typical chloroplast avoidance response. Bycontrast, the chloroplasts remained on the periclinalsurface after irradiation with red light at 50 Wm22 for120 min (Fig. 1C), suggesting that red light is ineffectiveat inducing the avoidance response in M. polymorpha.After dark treatment for 3 d, the chloroplasts movedfrom the periclinal to the anticlinal cell walls (Fig. 1D),similar to their distribution under strong blue light.However, there was a difference between dark andstrong-blue-light conditions in the distribution patternsof the chloroplasts in the outermost cells of gemmalings.Under strong blue light, the chloroplasts moved to allanticlinal cell walls in both the outermost and inner cellsof gemmalings (Fig. 1B). In the dark, chloroplasts movedto all anticlinal cell walls in the inner cells, but chloro-plasts in the outermost cell layers were excluded fromthe outermost anticlinal cell wall and thus were dis-tributed only along the anticlinal walls associated withneighboring cells (Fig. 1D). This distribution pattern indarkness was defined as dark positioning inM. polymorpha.Thus, the distribution pattern of chloroplasts inM. polymorpha both in the light and the dark are similarto those in the prothallial cells of A. capillus-veneris(Kagawa andWada, 1993, 1995; Tsuboi et al., 2007) except

that red light effectively induces chloroplast move-ment in A. capillus-veneris but not in M. polymorpha.

To investigate chloroplast photorelocation movementin detail, the movements in the gemmaling cells wereinduced by microbeam irradiation with different fluencerates of blue or red light and analyzed by means of time-lapse images (Fig. 2). In response to weak blue light(10Wm22), chloroplasts outside the irradiated area movedtoward the microbeam area, and those inside it stayedwithin the irradiated area (Fig. 2, A and E). Under strongblue light (50 Wm22), chloroplasts in the irradiated areaescaped from the microbeam and remained outside of theirradiated area (Fig. 2, B and F). These behaviors are sim-ilar to those observed in Arabidopsis (Kagawa and Wada,2000) and A. capillus-veneris (Kagawa and Wada, 1999).

The fluence-rate-dependent responses of chloroplastphotorelocation movement are summarized in Table I.When irradiated with blue light, clear chloroplast accu-mulation movement was observed at 0.01 to 25 Wm22 ofmicrobeam. However, at 37.5 Wm22, a weak avoidanceresponse was induced in some experiments, indicatingthat the transition from accumulation movement toavoidance movement occurred at about 37.5 Wm22. Anobvious avoidance movement was observed at 50 Wm22

or higher intensity. By contrast, the red-light microbeamat all examined fluence rates did not induce chloroplastphotorelocation movement (Fig. 2, C, D, G, and H). Inthe case of the moss P. patens, protonemata growingunder continuous red light but not under white lightexhibited red-light-induced chloroplast photorelocation(Kadota et al., 2000). However, neither the accumulationresponse nor the avoidance response was induced evenin the gemmalings grown under continuous red light inM. polymorpha. These results further confirmed that thechloroplast photorelocation movement is controlled byblue light but not red light in M. polymorpha.

The PHOT Gene Is a Single-Copy Gene in M. polymorpha

As a candidate for the photoreceptor that mediatesthe blue-light-dependent chloroplast movements, we

Figure 1. Chloroplast distribution patterns under differentlight conditions in wild-typeM. polymorpha. Gemmalingsincubated under continuous white light for 3 d weresubjected to different light treatments. A, Before light ir-radiation. B, After 120min of irradiation with high-fluenceblue light (50 Wm22). C, After 120 min of irradiation withhigh-fluence red light (50 Wm22). D, After 3 d darktreatment. The outermost cell walls are indicated by ar-rowheads in B and D. Bars = 20 mm.

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amplified M. polymorpha phototropin gene fragments byreverse transcription-PCR with PHOT-specific degenerateprimers and identified only one complementary DNA(cDNA) species for PHOT. We identified P1-derived ar-tificial chromosome (PAC) clones for the PHOT genomicfragments using the cDNA as a probe. In silico analysis ofcurrent genome sequencing and transcriptome data fromdifferent tissues and conditions supported the conclusionthat M. polymorpha has only one PHOT gene. We alsoperformed Southern-blot analysis under low stringentconditions using M. polymorpha genomic DNA digestedwith restriction enzymes. The patterns of restrictionfragments were consistent with those predicted from thegene structure of the cloned PHOT gene, confirming thatthis PHOT gene is a single copy (Supplemental Fig. S1).Thus, the PHOT gene was named MpPHOT.

The MpPHOT gene has 24 exons interrupted by 23 in-trons. The number and positions of introns in the codingsequence were conserved among MpPHOT, ArabidopsisPHOT1, and PHOT2. MpPHOT encodes an 1,115-aminoacid protein that contains two LOV domains (LOV1 andLOV2) and a Ser/Thr kinase domain (Fig. 3A). The LOVdomains of Mpphot are highly conserved and contain allof the amino acid residues important for FMN bindingand photoactivation (Crosson and Moffat, 2001), sug-gesting that the LOV domains of Mpphot are photoactive.The N-terminal extension upstream of the LOV1 domainis highly variable among phototropins from various

plants, but some Ser residues that are phosphorylatedin Arabidopsis phot1 and phot2 (Inoue et al., 2008, 2011)are conserved in Mpphot (Supplemental Fig. S2). Similarto fern and moss phototropins (Kagawa et al., 2004;Kasahara et al., 2004), the N-terminal extension ofMpphotis considerably longer than that of seed plant phototropins(Fig. 3A). The Ser/Thr kinase domain of Mpphot is alsohighly similar to that of phototropins from other plants(Supplemental Fig. S2). Notably, two Ser residues in theactivation loop in the kinase domain, which are auto-phosphorylated and essential for full activity of phot1 and

Figure 2. Microbeam-induced chloroplast movement in wild-type M. polymorpha. A to D, Cells were irradiated for 80 minwith 10 Wm22 (A) or 50 Wm22 (B) of blue light, or 10 Wm22 (C) or 50 Wm22 (D) of red light. E to H, The tracks of chloroplastmovements in the cells are shown as lines; 10 Wm22 (E) or 50 Wm22 (F) of blue light, or 10 Wm22 (G) or 50 Wm22 (H) of redlight. The rectangles indicate the positions of the microbeam irradiation. Black circles indicate initial positions of each chlo-roplast before microbeam irradiation. These experiments were repeated at least three times in different cells with similar results.Bar = 20 mm.

Table I. Chloroplast photorelocation movement under blue or redlight

Chloroplast photorelocation movement was analyzed by microbeamirradiation with various fluence rates of blue or red light.

Light Intensity Blue Red

Wm22

0.001 No movement Not determined0.01 Accumulation Not determined0.1 Accumulation Not determined1.0 Accumulation No movement10 Accumulation No movement25 Accumulation Not determined37.5 Accumulation or

weak avoidanceNot determined

50 Avoidance No movement75 Avoidance No movement


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phot2 in Arabidopsis (Inoue et al., 2008, 2011), are con-served in Mpphot (Supplemental Fig. S2).In the phylogenetic analysis (Fig. 3B; Supplemental

Fig. S3), MpPHOT was placed in the bryophyte cladethat is sister to the PHOT1 and PHOT2 groups in theangiosperm lineage as reported in a recent large-scalephylogenetic analysis of PHOT genes (Li et al., 2014).

Li et al. (2014) recently reported that only one PHOTgene was identified in all of the seven liverwort speciesexamined, including M. polymorpha, which is consis-tent with our results (Supplemental Fig. S1). Thus, it islikely that liverworts have only one PHOT gene ingeneral and that multiple duplication of PHOT genesoccurred during land plant evolution.

Figure 3. Protein structure and phylogenetic tree of phototropins from M. polymorpha and other plants. A, Domain organi-zation of phototropins from M. polymorpha (MpPHOT), Arabidopsis (AtPHOT1 and AtPHOT2), and C. reinhardtii (CrPHOT).Black and gray regions indicate LOV domains (LOV1 and LOV2) and Ser/Thr kinase domains, respectively. The numbers in-dicate the positions of the start and end of each domain. B, Phylogenetic relationships of plant phototropins. A majorityconsensus phylogeny for 24 phototropins from green algae and land plants was reconstructed by Bayesian inference analysis ofan alignment of sequences corresponding to amino acid residues 699 to 1087 of Mpphot (Supplemental Fig. S3). TheOstreococcus tauri PHOT sequence was used as the outgroup. Posterior probabilities are indicated at the nodes. Atr, Amborellatrichopoda; At, Arabidopsis; Os, Oryza sativa; Ac, A. capillus-veneris; Sm, S. moellendorffii; Pp, P. patens; Ms, M. scalaris; Ot,Ostreococcus tauri; and Cr, C. reinhardtii. Bar = 0.1 substitutions per site.



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Generation of MpPHOT Knockout Lines andComplementation Lines

To investigate the function of Mpphot in chloroplastphotorelocation movement, we generated MpPHOTknockout lines (MpphotKO) using the established tar-geting method with homologous recombination inM. polymorpha (Ishizaki et al., 2013a). A 276-bp portionincluding exon3 of MpPHOT was replaced with an ex-pression cassette for the hygromycin phosphotransferasegene (Fig. 4A; Supplemental Fig. S4A), to disrupt a re-gion corresponding to a LOV1 portion that includes theCys residue essential for formation of the flavin C (4a)-cysteinyl adduct (Supplemental Fig. S2). By genomicPCR screening, we obtained two independent targetedlines (Supplemental Fig. S4B). The Southern-blot analysisof genomic DNAs revealed that one copy of the transgenewas integrated into the MpPHOT locus (SupplementalFig. S4C). Because the two lines showed essentially thesame phenotype for chloroplast photorelocation movement,we only present the data obtained for one line (MpphotKO 1),which did not show obvious growth defects under ourgrowth conditions (Supplemental Fig. S5). Immunoblotanalysis with anti-Mpphot antibody showed that theMpphot protein was not detected in the MpphotKO, in-dicating that MpphotKO is a null mutant. To substantiatethat the disruption of MpPHOT caused the defectivephenotypes in MpphotKO (see below), we introduced theMpPHOT expression construct controlled by its own pro-moter into the MpphotKO, generating complemented linesgenomic MpPHOT (gMpPHOT)/MpphotKO. The amount ofMpphot in gMpPHOT/MpphotKO was similar to that inthe wild type (Fig. 4B). These knockout and complementedlines were used in experiments described below.

Chloroplast Photorelocation Movement in MpphotKO

Is Impaired

To examine whether Mpphot is the blue-light re-ceptor for chloroplast photorelocation movement inM. polymorpha, we analyzed blue-light-induced chloro-plast movements in cells of MpphotKO and gMpPHOT/MpphotKO. Under continuous white light, the chloroplastslocalized sparsely on the upper cell surface in MpphotKO

(Fig. 5A), whereas the chloroplasts covered the wholecell surface in the wild type (Fig. 1A), indicating thatMpphotKO is defective in the chloroplast accumulationresponse. After blue-light irradiation (at 50 Wm22 for120 min), the chloroplasts did not change their positions(Fig. 5A). These results indicate that MpphotKO is defec-tive in both accumulation and avoidance responses ofchloroplasts. In addition, MpphotKO was deficient in darkpositioning of chloroplasts. After 3 d of dark adaptation, asignificant number of chloroplasts in MpphotKO wereobserved along the anticlinal walls without neighboringcells (Fig. 5A), whereas no chloroplasts localized on theoutermost anticlinal walls in the wild type (Fig. 1D).Furthermore, more chloroplasts localized on the peri-clinal wall in MpphotKO compared with the wild type.Measurement of the ratio of the area occupied with

chloroplasts to the area of whole cell surface furtherconfirmed no chloroplast movement after dark treatmentin MpphotKO (Supplemental Fig. S6). These defectsin MpphotKO under the different light conditions werecompletely rescued in gMpPHOT/MpphotKO (Fig. 5B;Supplemental Fig. S6). Thus, these results indicate thatMpphot is necessary for both accumulation and avoidanceresponses as well as the dark positioning of chloroplasts.

To further confirm the essential role of Mpphot inchloroplast photorelocation movement, we performedmicrobeam irradiation experiments withMpphotKO andgMpPHOT/MpphotKO. Under weak blue light (10 Wm22),the chloroplasts did not move toward the light-irradiatedarea, which was indicative of the defective accumulationresponse in MpphotKO (Fig. 6, A and E). Under strongblue light (50 Wm22), the avoidance response was notobserved in MpphotKO and the chloroplasts that hadresided in the light-irradiated area did not move outof this area (Fig. 6, B and F). Under both conditions, in

Figure 4. Targeted disruption of MpPHOT by homologous recombi-nation. A, Schematic diagram of targeted disruption of the MpPHOTlocus by homologous recombination. Filled rectangles indicate exons, andintervening thick lines indicate introns. B, MpPHOT protein accumulationin the wild type,MpphotKO, and gMpPHOT/MpphotKO. Proteins wereimmunodetected with an anti-Mpphot antibody. Coomassie Brilliant Bluestaining of RBCL is shown as a loading control. Plants were grown undercontinuous white light for 7 d. Each lane contains 20 mg of total proteins.DEn, 39 Part of the maize (Zea mays) En element; hpt, hygromycinphosphotransferase; RBCL, Rubisco large subunit; WT, wild type.


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MpphotKO chloroplasts were still distributed sparselyon the periclinal and anticlinal walls regardless of thelight conditions, indicating thatMpphotKO is defective inboth the accumulation and avoidance responses. IngMpPHOT/MpphotKO, both the accumulation and avoid-ance responses to weak and strong blue light, respec-tively, were restored (Fig. 6, C and D).Collectively, these results indicated that loss of chloro-

plast photorelocation movement in MpphotKO was causedbyMpPHOT disruption and, therefore, that Mpphot is thephotoreceptor for chloroplast photorelocation movementin M. polymorpha.

Mpphot Is Localized on the Plasma Membrane

To investigate the expression pattern and the intracel-lular localization of Mpphot, we generated transgeniclines expressing Mpphot translationally fused to the Cit-rine reporter protein at the C terminus under the controlof the MpPHOT promoter in the MpphotKO background(gMpPHOT-Citrine/MpphotKO). These lines showed nor-mal chloroplast photorelocation movement, indicatingthat Mpphot-Citrine is functional (Supplemental Fig. S7).Fluorescence microscopy showed that the fluorescence

from Mpphot-Citrine was observed throughout the en-tire body of the gemmalings (Fig. 7A). Mpphot-Citrineappeared to be localized predominantly on the plasmamembrane under higher magnification (Fig. 7B).To confirm the plasma membrane localization of

Mpphot, cytosolic andmembrane fractions were prepared

and subjected to immunoblot analysis (Fig. 7C). Successfulfractionation was verified using two antibodies againstcytosolic UDP-Glc pyrophosphorylase (UGPase) andplasma-membrane localized H+-ATPase (Maudoux et al.,2000). Immunoblot analysis with anti-Mpphot antibodyspecifically detected the Mpphot protein of 123 kD only inthe membrane fraction, similar to H+-ATPase (Fig. 7C).Mpphot and H+-ATPase were not detected in the cyto-solic fraction, in which UGPase was enriched. Togetherwith the microscopic results mentioned above, the similarpartitioning profiles for Mpphot and H+-ATPase indicatethat the Mpphot protein is predominantly localized to theplasma membrane.

Mpphot Exhibits Blue-Light-DependentAutophosphorylation Activity in Vitro and in Vivo

Given that the LOV domains and the Ser/Thr kinasedomain of Mpphot are highly conserved (SupplementalFig. S2), it is plausible that blue light activates auto-phosphorylation activity similar to phototropins fromother species (Christie, 2007). To examine the blue-light-stimulated kinase activity of Mpphot, the maltose bind-ing protein (MBP) fusion protein of full-length Mpphot(MBP-Mpphot) was expressed in Escherichia coli and af-finity purified. MBP-Mpphot was incubated with [g-32P]ATP under dark or blue light (18 Wm22) for 30 min. TheMBP-Mpphot protein was detected as a band of ap-proximately 170 kD in all lanes in SDS-PAGE (Fig. 8A,

Figure 5. Chloroplast distribution patterns of MpphotKO

and gMpPHOT/MpphotKO under different light conditions.A, MpphotKO. B, gMpPHOT/MpphotKO. Gemmalings in-cubated under continuouswhite light for 3 dwere used forthis analysis. Gemmalings were irradiated with white light(top), high-fluence blue light (50 Wm22 for 120 min;middle), and 3-d dark treatment (bottom). The outermostcell walls are indicated by arrowheads. Bars = 20 mm.



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left), consistent with the predicated molecular mass ofMBP-Mpphot (167 kD). Darkness induced only a weakautoradiography signal at the position of MBP-Mpphot(Fig. 8A, right), suggesting that Mpphot has low auto-phosphorylation activity in darkness. Conversely, bluelight induced a strong autoradiography signal and sig-nificant mobility shift, although the amount of MBP-Mpphot was unchanged regardless of light condition.This result indicated that blue light enhanced the kinaseactivity of Mpphot. Autophosphorylation of MBP-Mpphotwas detected after 1 min of irradiation with blue light andincreased progressively over the course of 30 min irradia-tion (Fig. 8B). The Asp residue in subdomain VII of theC-terminal kinase domain of Arabidopsis phot1 and phot2is functionally essential, and the replacement of the residuewith Asn results in loss of kinase activity (Suetsugu andWada, 2013). To substantiate that Mpphot is autophos-phorylated, we also analyzed the kinase-negative Mpphotprotein in which the corresponding Asp-922 residue wassubstituted with Asn (MBP-MpphotD922N). In MBP-MpphotD922N, signals were not detected in the dark-treatedcontrol or blue-light-irradiated samples, although theamount of MBP-MpphotD922N was comparable to that ofMBP-Mpphot during the experiment (Fig. 8A). These re-sults indicate that Mpphot possesses blue-light-dependentkinase activity and is autophosphorylated in vitro.

To investigate the blue-light-dependent autophos-phorylation activity of Mpphot in vivo, we examinedthe blue-light-dependent mobility shift of Mpphot

through immunoblot analysis with the anti-Mpphotantibody. Dark-adapted wild-type plants were irradiatedwith blue light (33 Wm22) for 10 min (Fig. 8C). After1 min of blue light irradiation, a slight mobility shift ofMpphot was observed and the band shift of Mpphot wassaturated after 3 min. When the 10-min-irradiated sam-ples were returned to the dark, the mobility of Mpphotgradually shifted to that of the nonirradiated sample(Fig. 8D). These results indicate that blue light inducedautophosphorylation of Mpphot and the phosphorylatedMpphot was dephosphorylated in darkness. Thus, theseresults suggest that Mpphot shows blue-light-regulatedkinase activity similar to phototropins in other plants.

Mpphot Can Function to Regulate Chloroplast AvoidanceResponse in Adiantum capillus-veneris

To investigate whether the function of Mpphot isconserved and Mpphot is functional in other plants inwhich multiple phototropins mediate chloroplast photo-relocation movement, we expressed Mpphot transientlyin the phot2mutant of the fern A. capillus-veneris, which isdefective in the avoidance response induced by strongblue light (Kagawa et al., 2004), and examined whetherMpphot can regulate the phot2-mediated chloroplastavoidance response in the heterologous system. Usingan assay system developed for assessing the function ofphototropins in A. capillus-veneris (Kagawa et al., 2004;

Figure 6. Chloroplast relocation in blue-light-irradiated cells of MpphotKO and gMpPHOT/MpphotKO. A to D, Chloroplastphotorelocation movement was analyzed in response to microbeam irradiation with different fluence rates of blue-light for80 min. 10 Wm22 (A and C) or 50 Wm22 (B and D) was irradiated to MpphotKO cells (A and B) and gMpPHOT/MpphotKO cells(C and D). E to H, The tracks of chloroplast movements in the cells for each light condition. See the legend of Figure 2 for details.These experiments were repeated at least three times in different cells with high reproducibility. Bar = 20 mm.


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Kong et al., 2013a), prothallial cells of the phot2 mutantwere cotransfected withMpPHOT and GFP cDNAs, bothof which were driven by the Cauliflower mosaic virus 35S(35S) promoter, using particle bombardment. GFP fluo-rescence was used as a marker to identify transfectedcells. An MpPHOT-transfected cell (i.e. GFP-positive cell)and an adjacent nontransfected cell were irradiated witha strong blue-light microbeam. After 60 min of irradiationwith strong blue light, the chloroplasts in the transgeniccells moved away from the light-irradiated area, whereasthe chloroplasts in an adjacent nontransfected mutant cellaccumulated in the irradiated area because of the defec-tive avoidance response (Fig. 9), indicating that the tran-sient expression of Mpphot was able to complement thedeficiency of the A. capillus-veneris phot2mutant in theavoidance movement. This result suggests that Mpphotis functional in regulation of the avoidance responses inA. capillus-veneris and that phototropin of M. polymorphahas the ability to transduce the blue-light signal to medi-ate the avoidance movement in A. capillus-veneris.

Mpphot Is Able to Regulate Both ChloroplastAccumulation and Avoidance Responses in Arabidopsis

To examine the conserved function of Mpphot tomediate the accumulation response in addition to the

avoidance response, Mpphot was introduced into theArabidopsis phot1 phot2 double mutant, which is de-fective in both the accumulation and avoidance re-sponses (Sakai et al., 2001). MpPHOT was expressedunder the control of the 35S promoter in phot1 phot2double mutants (MpPHOT/phot1 phot2). To characterizechloroplast photorelocation movement in the MpPHOTtransgenic lines, the changes in leaf transmittancecaused by chloroplast photorelocation movement wasanalyzed using a plate reader system (Wada and Kong,2011). In the wild type, leaf transmittance was changedin response to weak and strong blue-light irradiation.Weak light induced a decrease in transmittance as aresult of the accumulation response, whereas stronglight induced an increase as a result of the avoidanceresponse (Fig. 10, A and B). These changes were notdetected in the phot1 phot2 double mutants because the

Figure 8. Blue-light-dependent phosphorylation of Mpphot in vitro andin vivo. A, Autophosphorylation activity of Mpphot andMpphotD922N in vitro.Purified MBP-Mpphot and MBP-MpphotD922N were incubated in the dark(lanes D) or under blue light (18 Wm22, lanes BL) for 30 min with [g-32P]ATP. Asterisks indicate full-length MBP-Mpphot. A Coomassie Brilliant Blue-stained image is shown on the left, whereas an autoradiograph is presentedon the right. WT, Wild type. B, Time dependency of Mpphot autophos-phorylation in vitro. Blue light was irradiated for the indicated periods.C, Blue-light-induced phosphorylation of Mpphot in vivo. The Mpphotprotein from the blue-light-irradiated plants (0, 1, 3, 5, and 10 min) wasdetected with anti-Mpphot antibody. D, Dephosphorylation of Mpphot invivo. Dark-treated plants (lane D) were irradiated with blue light (33 Wm22)for 10 min (lane BL). After blue-light irradiation, plants were kept in the darkfor 60, 120, 180, or 360 min. Mpphot protein was detected as in C.

Figure 7. Expression pattern and intracellular localization of Mpphotin M. polymorpha. A and B, Confocal microscopic images of Citrinefluorescence in cells of gMpPHOT-Citrine/MpphotKO. A 3-d-oldgemmaling was observed. C, Immunoblot analysis of the total proteinfraction (T), cytosolic fraction (C), and membrane fraction (M) from thewild type. The blots were probed with specific antibodies againstMpphot, H+-ATPase, and UGPase. Bar = 50 mm in A; 10 mm in B.



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mutants lacked chloroplast photorelocation movement(Fig. 10, A and B). On the other hand, MpPHOT/phot1phot2 showed partial but significant changes in leaftransmittance under both weak and strong blue light(Fig. 10, A and B).

Furthermore, we confirmed the function of Mpphotin the mediation of chloroplast photorelocation move-ments by observing chloroplast movement in mesophyllcells of MpPHOT/phot1 phot2 in response to microbeamirradiation (Fig. 10C). When irradiated with weak bluelight (1 Wm22), the chloroplasts outside of the irradiatedarea moved toward the light-irradiated area. Conversely,when irradiated with strong blue light (100 Wm22), thelight-irradiated chloroplasts escaped from the irradiatedarea (Fig. 10C).

In conclusion, these results indicate that Mpphot wasable to regulate both accumulation and avoidance re-sponses that were impaired in the Arabidopsis phot1 phot2double mutant and possesses the functions of phot1and phot2 in Arabidopsis with respect to the chloro-plast photorelocation.

DISCUSSION

Phototropin-Mediated Chloroplast PhotorelocationMovements in M. polymorpha

Although intensive observation of chloroplast pho-torelocation movement has been performed in diverseplant groups such as green algae, mosses, ferns, andseed plants since the 19th century (Senn, 1908; Suetsuguand Wada, 2012), knowledge of chloroplast photo-relocation movement in liverworts is relatively limited.In this study, we observed light-induced movements ofchloroplasts in M. polymorpha in detail. Previous pre-liminary results suggested that blue light is effective inthe induction of chloroplast movement in M. polymorpha(Nakazato et al., 1999). We confirmed that chloroplastmovement was induced exclusively by blue light similarto most land plant species. At blue-light fluence rates inthe range of 0.01 to 25Wm22, the accumulation responsewas induced (Fig. 2; Table I). Conversely, the avoidanceresponse was induced at fluence rates of 50 Wm22 orstronger. AlthoughM. polymorpha is as sensitive to weakblue light to induce the accumulation response as otherplants are, the light intensity at which the responsechanges from accumulation to avoidance was muchhigher than that for vascular plants. At 10 Wm22 of bluelight or stronger, the chloroplasts escape from the light-irradiated area in Arabidopsis and A. capillus-veneris(Kagawa and Wada, 1999, 2000), whereas chloro-plasts accumulate toward the light-irradiated area inM. polymorpha (Fig. 2). In the case of P. patens, which isalso a bryophyte, the accumulation response is inducedat about 30 Wm22 of blue light, and stronger blue light(more than about 100 Wm22) is required to induce theavoidance response (Kadota et al., 2000; Sato et al.,2001). Thus, the light intensity necessary for the in-duction of avoidance response in M. polymorpha was

higher than that in vascular plants, and this propertymight be common in bryophytes.

In P. patens, red-light-induced chloroplast movementwas observed in red-light-grown protonema (Kadotaet al., 2000). This response was mediated by both con-ventional phytochromes (Mittmann et al., 2004; Uenakaand Kadota, 2007) and phototropins (Kasahara et al.,2004), suggesting that phototropins are an essentialcomponent for transmitting signals in the chloroplastmovement signaling pathway (Kasahara et al., 2004;Jaedicke et al., 2012). Because M. polymorpha has bothphytochrome and phototropin, we also examined chlo-roplast movements in red-light-grown plants in additionto white-light-grown plants of M. polymorpha. Similarto the plants grown under white light (Figs. 1C, and 2,C and D), the red-light-grown plants showed no red-light-induced chloroplast photorelocation move-ment. Red-light-induced chloroplast photorelocationmovements have not been observed in some mossesexamined, such as Funaria hygrometrica (Zurzycki, 1967)and Ceratodon purpureus (Kagawa et al., 1997), and most

Figure 9. Heterologous expression of MpPHOT in A. capillus-venerisphot2 mutant cells. An MpPHOT-expressing cell and its neighboringuntransfected cell in the phot2 prothallus were simultaneously irradiatedwith a microbeam of 100Wm22 blue light. Areas surrounded with brokenlines indicate the position of the microbeam irradiation. Bar = 20 mm.


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other land plants. Because the red-light-induced chlo-roplast photorelocation movement has been observedonly in P. patens among the bryophytes examined so far,it is suggested that chloroplast movement is not in-duced by red light in most bryophytes.

In addition to the blue-light-induced chloroplastmovement, we showed the specific distribution patternof chloroplasts in darkness in M. polymorpha (Fig. 1D).Patterns of dark positioning vary among plant species(Senn, 1908). Similar to that in A. capillus-veneris prothalli(Tsuboi et al., 2007), the chloroplasts moved only to theanticlinal walls with neighboring cells but not to theperipheral walls that lack neighboring cells after darktreatment. This dark positioning differed from that ofArabidopsis, in which chloroplasts accumulate at thebottom of the cell in darkness (Suetsugu et al., 2005a). InMpphotKO, the chloroplasts were distributed randomly incells and did not accumulate along specific cell wallsafter dark treatment. The defects in dark positioning ofphot2 mutants were previously reported for Arabidopsis(Suetsugu et al., 2005a) and A. capillus-veneris (Tsuboiet al., 2007). Thus, these results indicate that regulationof dark positioning by phototropins is conserved in landplants.

Intriguingly, in M. polymorpha, only one phototropin(Mpphot) mediates all three types of chloroplastphotorelocation movement (i.e. dark positioning, thelow-light-induced accumulation response, and thehigh-light-induced avoidance response; Figs. 1 and 6).In most land plant species, two or more PHOT genesmediate chloroplast photorelocation movement. Al-though multiple phototropins show some degree offunctional redundancy to mediate chloroplast photo-relocation movement, they also exhibit functionaldivergence. For example, in Arabidopsis, althoughphot1 and phot2 redundantly mediate the accumula-tion response, phot1 contributes to the accumulationresponse at a much weaker blue-light intensity com-pared with phot2 (Sakai et al., 2001). The phot2 pro-tein primarily regulates the avoidance response anddark positioning, whereas the contribution of phot1to these responses is negligible (Suetsugu et al., 2005a;Luesse et al., 2010). Similar to Arabidopsis, phot2 spe-cifically mediates the avoidance response and darkpositioning in the fern A. capillus-veneris (Kagawaet al., 2004; Tsuboi et al., 2007). The moss P. patenshas seven PHOT genes categorized into two groups,namely four PpPHOTAs and three PpPHOTBs (Rensinget al., 2008), of which PpphotAs contribute more tothe avoidance response than PpphotBs do (Kasaharaet al., 2004). Thus, during evolution, land plantsacquired multiple functionally differentiated pho-totropins with which to fine-tune chloroplast move-ments under the fluctuating natural light conditions.This study, together with the work by Li et al. (2014),proves that at least some liverworts have only asingle PHOT gene. Thus, liverworts may occupyan ancestral position in the evolution of the pho-toreceptor system for chloroplast photorelocationmovement.

Figure 10. Heterologous expression of MpPHOT in the Arabidopsisphot1 phot2 double mutant. A, Changes in leaf transmittance causedby chloroplast photorelocation movement. The graph shows rep-resentative data from three independent experiments. After 10 minin darkness, leaves were irradiated with blue light at 0.8, 5.3, and13.2 Wm22 sequentially at 10, 70, and 110 min, respectively,as indicated by the arrowheads. Irradiation ceased at 150 min(arrow). Dotted, gray, and black lines indicate the transmittance ofthe wild type, phot1 phot2, and MpPHOT/phot1 phot2, respec-tively. B, Rate of leaf transmittance change over 2 to 6 min afterblue light irradiation. Data are the means of three independentexperiments. Bars indicate the SE. White, gray, and black rectan-gles indicate the transmittance of the wild type, phot1 phot2, andMpPHOT/phot1 phot2, respectively. C, Chloroplast relocationmovement induced by continuous microbeam irradiation withblue light in MpPHOT/phot1 phot2. The white circle indicates thelight-irradiated area. Cells were irradiated with low-intensity bluelight (1 Wm22) for the accumulation response (left) or with high-intensity blue light (100 Wm22) for the avoidance response (right).Bar = 25 mm.



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Molecular Mechanisms of Phototropin-MediatedChloroplast Photorelocation Movement

Our physiological analyses ofMpphotKO indicate thatMpphot regulates three types of chloroplast movement.How does Mpphot mediate these different responses?The kinase activity of phototropins is essential for theregulation of chloroplast photorelocation movement(Kagawa et al., 2004; Kong et al., 2006, 2007; Sullivanet al., 2008; Suetsugu et al., 2013). In particular, auto-phosphorylation in the activation loop of the kinasedomain is necessary for induction of chloroplast photo-relocation movement (Inoue et al., 2008, 2011; Suetsuguet al., 2013). As shown in Supplemental Figure S2, thekinase domain is highly conserved in phot ofM. polymorpha.Two Ser residues in the activation loop that wereidentified as the essential autophosphorylation sites inArabidopsis (Inoue et al., 2008, 2011) are conserved inMpphot. The kinase activity and autophosphorylationof Mpphot is likely to be required for chloroplast pho-torelocation movement in M. polymorpha as shown inother plants.

In this study, we demonstrated the blue-light-induced autophosphorylation activity of Mpphot invitro and in vivo (Fig. 8). Because the activity wasnullified in MpphotD922N, which carried a kinase-negative amino acid substitution, the kinase activity ofMpphot is necessary for the autophosphorylation. Thus,Mpphot is a blue-light-regulated kinase, similar to otherphototropins. Mpphot was slightly autophosphorylatedin darkness in vitro, as reported for other phototropins(Matsuoka and Tokutomi, 2005; Jones et al., 2007; Jonesand Christie, 2008; Aihara et al., 2012). Autophosphor-ylation activity may be involved in chloroplast darkpositioning, which is mediated by Acphot2 (Tsuboiet al., 2007), Atphot2 (Suetsugu et al., 2005a), andMpphot. After blue-light-irradiated plants werereturned to the dark, autophosphorylated Mpphotwas gradually dephosphorylated. Dephosphoryla-tion of Arabidopsis phot2 is implicated in the fine-tuning of phot2 activity during blue-light-mediatedresponses (Tseng et al., 2012). The A1 subunit ofSer/Thr protein phosphatase2A (PP2AA1) interactswith phot2 and mediates phot2 dephosphoryla-tion (Tseng et al., 2012). A PP2AA1 homolog is pre-sent in the M. polymorpha genome sequences andthus protein phosphatase2A might mediate Mpphotdephosphorylation.

Furthermore, the kinase domains of phot1 andphot2 in Arabidopsis are responsible for localization ofthe proteins on the plasma membrane and specific light-induced internalization from the plasma membrane(Kong et al., 2006, 2013b; Kaiserli et al., 2009). Similar toother phototropins, Citrine-fusion proteins of Mpphotlocalized on the plasma membrane (Fig. 7), indicatingthat Mpphot functions on the plasma membrane. Pho-totropins of many plant species, including Mpphot, haveno transmembrane regions. Thus, it is assumed thatphototropins may be anchored to the plasma membraneby interacting with another factor localized in the plasma

membrane. The mechanism by which phototropinlocalizes on the plasma membrane may be common toland plants.

Evolution of Phototropin Genes

In our phylogenetic analysis (Fig. 3B), Mpphot wassister to the clade of land plant phototropins. Recentextensive cloning and phylogenetic analysis revealedthat duplications of PHOT genes occurred indepen-dently in different lineages (i.e. in seed plants as wellas in ferns and mosses), and that many liverwort spe-cies have a single PHOT (Li et al., 2014), as is the case forM. polymorpha. Mpphot may have retained the ancestralfunctions of phototropin that were gained before theevolutionary diversification of PHOT genes in landplants. If the ancestral PHOT originated as a single-copygene, it should have possessed the ability to mediateboth the accumulation and avoidance responses. Indeed,the transient expression of Mpphot in the A. capillus-veneris phot2 mutant rescued the defects in the avoid-ance response (Fig. 9), and the expression of Mpphot inthe Arabidopsis phot1 phot2 double mutant rescued thedefects in both accumulation and avoidance responses(Fig. 10). Importantly, the green alga C. reinhardtii has asingle PHOT gene and, similar to Mpphot, the expres-sion of Crphot in the Arabidopsis phot1 phot2 doublemutant rescued the defects in both accumulation andavoidance responses (Onodera et al., 2005). Thus, Crphotand Mpphot are phot2-like in that they can mediate boththe accumulation and avoidance responses. The chloro-plast avoidance response is essential for plant survivalunder sunlight (Kasahara et al., 2002). During early landplant evolution, the chloroplast avoidance response mayhave made a greater contribution to plant survival thanthe accumulation response, because there was no densecanopy to intercept light and the ancestral land plantswere directly exposed to sunlight. However, after theexplosive evolution and diversification of trees, manyplants had to live in shade and thus needed to use theweak light levels under the dense canopy. Duplication ofPHOT genes and subsequent acquisition of a weak-light-specific phototropin, such as the seed plant phot1, is onestrategy for adaptation to weak light. Another strategywould be integration of phototropin signaling with thephytochrome system; the acquisition of chimeric photo-receptor neochromes, which consist of a phytochromechromophore-binding domain and phototropin in fernsand M. scalaris (Nozue et al., 1998; Kawai et al., 2003;Schneider et al., 2004; Suetsugu et al., 2005b; Li et al.,2014), and the direct interaction between phytochromeand phototropin are implicated in the phytochrome-signaling pathway (Kasahara et al., 2004; Jaedickeet al., 2012). We observed that in M. polymorpha chloro-plast movement is not induced by red light at any flu-ence rate examined (Table I) and no neochrome has beenidentified in the M. polymorpha genome. Thus, the liver-wort M. polymorpha is indicated to have the most simplephotoreceptor system for chloroplast photorelocation


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movement among the model land plants. Evolution ofthe photoreceptor system for chloroplast photorelocationmovement (i.e. diversification of PHOT genes) mighthave helped to facilitate the explosive evolution of landplants under the fluctuating light environment on land.

MATERIALS AND METHODS

Culture and Growth Conditions ofMarchantia polymorpha

Male and female accessions of M. polymorpha, Takaragaike (Tak)-1 andTak-2, respectively, were asexually maintained as previously described(Ishizaki et al., 2008). Plants were incubated on one-half-strength Gamborg’sB5 agar medium (Gamborg et al., 1968) at 22°C under continuous white light(approximately 20 Wm22).

Observation of Chloroplast Photorelocation Movement inM. polymorpha

For observation of chloroplast distribution, the marginal region of 3-d-oldgemmalings was observed. A single or a few cell layers were present on themarginal region. Plants were cultured on one-half-strength Gamborg’s B5 agarmedium at 22°C for 3 d under continuous white light, and then incubatedunder the different light conditions indicated in the text. A blue LED illumi-nator (MIL-B18; SANYO Electric), red LED illuminator (MIL-R18; SANYOElectric), and neutral density filter (Smoke 20; Sumitomo 3M) were used. Forobservation of chloroplast dark positioning, plants were incubated in darknessfor 3 d before observation.

For microbeam irradiation, gemmalings were transferred to a custom-madecuvette (25 mm in diameter and 5 mm in height) that consisted of two sets of asteel ring and a round glass with a silicone-rubber ring spacer in the dark (Wadaet al., 1983). The cuvette containing the gemmaling was placed on the sample stageof a microbeam irradiator (Yatsuhashi and Wada, 1990; Tsuboi et al., 2006; Wada,2007). Microbeam irradiation was performed as previously described (Yatsuhashiand Wada, 1990; Tsuboi et al., 2006; Wada, 2007) with some modifications. Thegemmalings were irradiated with a microbeam at different intensities of red or bluelight. Chloroplast movement was observed using infrared light. Samples wereprepared under a dim green safe light. The paths of chloroplast movement inresponse to a red or blue microbeam were traced by taking photographs at 1-minintervals under infrared light. The size of the light-irradiated area was 50 mm 310 mm. The resulting images were processed and analyzed with ImageJ version1.45s software (http://rsbweb.nih.gov/ij/). The fluence of the red- and blue-light microbeams at 1 Wm22 used in these experiments was approximately 5.5and 3.8 mmol photons m22 s21, respectively.

Genomic DNA Preparation

Total genomic DNA was extracted from approximately 5 g fresh weight ofTak-1 thalli, which were grown for 2 weeks under white light and an additional2 d in the dark using a cetyltrimethylammonium bromide method with somemodifications as previously described (Ishizaki et al., 2013a). The extractedgenomic DNA was used for Southern-blot analysis.

Cloning of MpPHOT

A partial MpPHOT cDNA was obtained using degenerate primers as previ-ously described (Kagawa et al., 2004). Using primers designed on the basis of thepartial cDNA sequence, we searched our PAC genome library (Okada et al., 2000)for the PHOT gene by PCR and identified three PAC clones carryingMpPHOT.By sequence analysis of the PAC clones, the MpPHOT genomic sequence wasdetermined.

Southern-Blot Analysis

Approximately 4 mg of genomic DNA was digested overnight with XbaI, PstI,or BamHI. The digested DNAs were separated by gel electrophoresis and blottedonto a positively charged nylon membrane (Biodyne A; PALL). The LOV2 probe(1050 bp) for copy-number analysis and three probes (A, B, and C; 902, 1182, and1000 bp, respectively) for gene-targeting analysis (Supplemental Fig. S4) were

amplified by PCR. The membranes were hybridized in Church hybridizationbuffer (Church and Gilbert, 1984) at 50°C with the probes labeled with [g-32P]dCTP by random priming using the Random Primer Labeling Kit Version 2(Takara Bio). Washing and autoradiography were performed as previously de-scribed (Chiyoda et al., 2008).

Phylogenetic Analysis of Phototropins

A multiple alignment of amino acid sequences of phototropins was con-structed using the MUSCLE program (Edgar, 2004) implemented in Geneioussoftware (version 6.1.8; Biomatters; http://www.geneious.com/) with defaultparameters. Unaligned gaps were first removed from the resulting alignmentusing Gblocks (http://molevol.cmima.csic.es/castresana/Gblocks_server.html), and then the conserved region covering the Ja helix and the C-terminalSer/Thr kinase domain was extracted before the phylogenetic tree construc-tion, which was performed using Markov chain Monte Carlo simulations byMrBayes 2.0.9 (Huelsenbeck and Ronquist, 2001) implemented in the Gene-ious software. The parameters used were as follows: rate matrix, blosum; ratevariation, g; g categories, 4; chain length, 1,000,000; subsampling frequency,200; heated chains, 4; burn-in length, 250,000; and heated chain temperature,0.2. The Ostreococcus tauri PHOT sequence was used as the outgroup. Theaccession numbers of analyzed proteins are as follows: Arabidopsis (Arabidopsisthaliana), At_PHOT1 (AAC01753) and At_PHOT2 (AAC27293); Oryza sativa,Os_PHOT1a (BAA84780) and Os_PHOT1b (BAA84779); Amborella trichopoda,Atr_PHOT1 (XP_006828236) and Atr_PHOT2 (XP_006849852); Adiantum capillus-veneris, Ac_PHOT1 (BAA95669), Ac_PHOT2 (BAD16730); Selaginella moellendorffii,Sm_PHOT1-1 (EFJ32904), Sm_PHOT1-2 (EFJ15768), Sm_PHOT2-1 (EFJ27458), andSm_PHOT2-2 (EFJ07343); Physcomitrella patens, Pp_PHOTA1 (EDQ60892),Pp_PHOTA2 (EDQ60548), Pp_PHOTA3 (EDQ69871), Pp_PHOTA4 (EDQ71981),Pp_PHOTB1 (EDQ68737), Pp_PHOTB2 (EDQ49461), and Pp_PHOTB3 (EDQ79801);Mougeotia scalaris, Ms_PHOTA (AB206968) and Ms_PHOTB (AB206969);Chlamydomonas reinhardtii, Cr_PHOT (CAC94941); and Ostreococcus tauri,Ot_PHOT (CAL58288). The alignment used for phylogenetic analysis isshown in Supplemental Figure S3.

Targeted Gene Knockout of MpPHOT

To generate the MpPHOT-targeting vector, pJHY-TMp1 was used (Ishizakiet al., 2013a). The 59 and 39 homology arms (3492 and 3482 bp, respectively)were amplified from genomic DNA by PCR amplification using KOD FXNeo (Toyobo) with the following primer pairs: PHOT-5IF-L/PHOT-5IF-R(59-CTAAGGTAGCGATTAAGTGGTGGCAAACGAGGTAG-39/59-CCGGG-CAAGCTTTTACTGGAAAGAAGCGAGAGCAT-39) for the 59 homology arm,and PHOT-3IF-L/PHOT-3IF-R (59-AACACTAGTGGCGCGTCATCATCTAC-GTCGCTTCG-39/59-TTATCCCTAGGCGCGCGATGCTCTGCGAGACATTA-39)for the 39 homology arm. The PCR products of the 59 and 39 homology arms werecloned into the PacI and AscI sites of pJHY-TMp1, respectively, using the In-FusionHD Cloning Kit (Clontech).

Introduction of the targeting construct into M. polymorpha was performedwith Agrobacterium tumefaciens C58C1 GV2260 as previously described (Ishizakiet al., 2008, 2013a). F1 spores generated by crossing Tak-1 and Tak-2 were usedfor transformation. Isogenic lines (designated as G1 lines) were obtained byisolating gemmae, which develop from single cells, from independent T1 lines(Ishizaki et al., 2012) and were screened for gene-targeted lines (designated asMpphotKO) by genotyping using the method previously described (Ishizakiet al., 2013a) with minor modifications. The PCR program was 94°C for 2 min, fol-lowed by 40 cycles of 98°C for 10 s, and 68°C for 5 min. The following primer pairswere used: GT-L2/GT-R3, GT_L0/P1R, and HIF/GT_R5 (59-ATGGG-GAGTGCTGATGAAGA-39/59-TCCCTGGAGAAATCGACTGT-39, 59-GAATCTG-GCAAGGAGTTCCA-39/59-GAAGGCTTCTGATTGAAGTTTCCTTTTCTG-39 and59-GTATAATGTATGCTATACGAAGTTATGTTT-39/59-GGCCTAGGAAAGACAA-CACG-39, respectively).

After PCR screening, two independent MpPHOT knockout lines wereidentified. Plants grown from gemmae of G1 lines (the G2 generation) wereused for phenotypic analysis and protein blotting.

Complementation Lines of MpphotKO

To generate complementation lines of MpphotKO, a binary vector harboringa mALS mutated acetolactate synthase gene that confers chlorosulfuron re-sistance was used. For construction of a plasmid containing the MpPHOTgenomic fragment, the promoter and coding regions were amplified by



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PCR using the following primer pairs: PRO_L/PRO_R and Infusion_fw/Infusion_rv (59-CACCATCCAGCACCATGAGAAGTA-39/59-AAGCTTGGC-TCGTCCTGATTT-39 and 59-TCAGGACGAGCCAAGATGATGCCCTCCA-CGGATTC-39/59-CGCGCCCACCCTTCTGAATTTGACATCCTCCTAAG-39,respectively). The promoter fragment was cloned into pENTR/D-TOPO(Life Technologies). After digestion with HindIII, the amplified coding re-gion was cloned into the HindIII site of the plasmid carrying the promoterfragment with the In-Fusion HD Cloning Kit to generate the plasmidpAI019, which contained the MpPHOT genomic fragment from 5.0-kb up-stream of ATG to the 39 untranslated region (total 13 kb). The resultantMpPHOT cassette was cloned into a binary vector using LR Clonase II (LifeTechnologies) according to the manufacturer’s protocol. Complementationlines gMpPHOT/MpphotKO were generated by transformation of the resultingbinary plasmid into regenerating thalli of MpphotKO as previously described(Kubota et al., 2013). The several transformants were obtained through se-lection with chlorosulfuron and used as gMpPHOT/MpphotKO lines.

Construction of Mpphot Bacterial Expression Vectors

The cDNA fragments for the N-terminal region of Mpphot (aminoacids 522251) as the antigen and full-length Mpphot were amplified byPCR with oligonucleotide primers for the N-terminal region of Mpphot(59-TTTGGATCCTCAGCTGCGGAAGATGCCTTGG-39/59-TTTTCTAGAGG-ACGCGCGGCCGGTCGA-39) and for full-length Mpphot (59-TTTACGAGTAT-GATGGCCCTCCAC-39/59-TTTACGAGTTTAATATTCATCAAATGAGGC-39)using MpPHOT cDNA as the template. The kinase-negative mutant of Mpphot(MpphotD922N) was prepared by replacement of an Asp residue essential for ki-nase activity by Asn in subdomain VII of the kinase domain (Hanks and Hunter,1995). The amino acid substitutions were introduced by PCR with oligonucleotideprimers (59-AATTTCGACCTTTCCTTCTTGAC-39/59-AGTGAGCTGCACATGC-CCATCT-39). The fragments were cloned into the modified pMAL-c2 expressionvector (New England Biolabs), in which the factor Xa cleavage sequence wasreplaced by the recognition sequence for PreScission protease (GE Healthcare) andthe 6xHis tag sequence was added next to the SalI site.

Expression and Purification of Recombinant Proteins

For the expression of MBP-Mpphot(522251)-6xHis, MBP-Mpphot, andMBP-MpphotD922N, the Escherichia coli strain Rosetta2(DE3) was transformedwith the respective expression plasmid and induced with 1 mM isopropyl-b-D-thiogalactopyranoside for 24 h at 18°C. Cells were collected by centrif-ugation and were resuspended in a lysis buffer containing 20 mM Tris-HCl,150 mM NaCl, 10% (v/v) glycerol, 1 mM dithiothreitol (DTT), 0.1 mg mL21

lysozyme, and cOmplete EDTA-Free Protease Inhibitor (Roche). After thecells were lysed by sonication, the recombinant proteins in the supernatantswere purified by affinity chromatography using amylose resin (New Eng-land Biolabs). MBP-Mpphot and MBP-MpphotD922N were used for the invitro phosphorylation assay.

For the preparation of Mpphot antigen, the MBP moiety was removed fromMBP-Mpphot(522251)-6xHis with Turbo 3C Protease (Accelagen). Mpphot(522251)-6xHis was purified by affinity chromatography using cOmplete His-Tag Purification Resin (Roche) and was used for producing the rabbit poly-clonal antibody (KIWA Laboratory Animals).

In Vitro Phosphorylation Assay

In vitro kinase activity assay was performed as previously described (Okajimaet al., 2011) with some modifications. MBP-Mpphot and MBP-MpphotD922N wereincubated at 24°C in a kinase reaction buffer containing 30 mM Tris-HCl, pH 8.0,100 mM NaCl, 1 mM EGTA, 10% (v/v) glycerol, 10 mM MgCl2, 50 mM ATP, and200 kBq of [g-32P] ATP. Blue light was provided from a blue LED illuminator(MIL-B18; SANYO Electric). The samples were separated by SDS-PAGE. Phos-phorylation signals were detected with an image analyzer (FLA3000; Fujiflm). Forthe analysis of blue-light-induced autophosphorylation activity, MBP-Mpphotwas irradiated with blue light (18 Wm22) for the first 0, 1, 5, 10, 20, or 30 minof the total 30-min incubation time with [g-32P] ATP.

Immunoblotting and Membrane Fractionation

To prepare samples for immunoblot analysis, 7-d-old plants were incubatedfor 3 d in the dark. After various light treatments, plants were frozen and

crushed in a mortar. For the experiment in Figure 4B, the homogenates mixedwith equal volumes of a lysis buffer (1 mM EDTA, 1 mM DTT, 10 mM sodiumfluoride, 1 mM phenylmethylsulfonyl fluoride, 100 mM NaCl, 1% (v/v) TritonX-100, and 50 mM Tris-HCl, pH 7.4) were centrifuged at 16,000g for 20 min at4°C. For the experiments in Figure 8, C and D, the homogenates mixed with23 sample buffer (10% (v/v) 2-mercaptoethanol, 4% (w/v) SDS, 20% (v/v)glycerol, and 125 mM Tris-HCl, pH 6.8) were incubated at 95°C for 5 min andthen centrifuged at 16,000g for 20 min at room temperature. The supernatantswere subjected to immunoblot analysis with anti-Mpphot antibody as de-scribed below.

Membrane fractionation was performed as previously described (Ishizakiet al., 2013b) with some modifications. Plants were grown under continuouswhite light for 14 d and then incubated in darkness for 3 d. Seven g of planttissue were crushed in a mortar. The crushed tissues were mixed with 25 mLof homogenization buffer containing 500 mM Suc, 10% (v/v) glycerol, 20 mMEDTA, 20 mM EGTA, 50 mM sodium fluoride, 1% (w/v) polyvinylpyrrolidone,10 mM ascorbic acid, 2 mM DTT, 3 mM phenylmethylsulfonyl fluoride, cOm-plete Mini EDTA-Free Protease Inhibitor, and 50 mM Tris-MES, pH 8.0. Thehomogenates were filtered through a cell strainer (70-mm nylon; BD Biosci-ences). After centrifugation of the filtrate at 3,000g for 10 min at 4°C, the su-pernatant (total protein fraction) was further centrifuged at 100,000g for60 min at 4°C to separate the supernatant (cytosolic fraction) and the membrane-containing precipitate. The precipitate was washed two times in homogenizationbuffer and resuspended in 13 sample buffer (5% (v/v) 2-mercaptoethanol,2% (w/v) SDS, 10% (v/v) glycerol, 0.0025% (w/v) bromophenol blue, and62.5 mM Tris-HCl, pH 6.8) to generate the membrane fraction. Each fraction wassubjected to immunoblot analysis.

For immunoblot analyses, proteins were separated in the modified 8% (w/v)SDS-PAGE gels (acrylamide to N,N9-metylenebisacrylamide ratio of 29.8:0.2) forthe experiment in Figure 8, C and D, and in the standard 8% (w/v) SDS-PAGEgels for other experiments. The proteins were transferred onto polyvinylidenedifluoride membranes (Bio-Rad Laboratories), and detected with various anti-bodies. The primary antibodies were diluted as follows: 1:5,000 for anti-Mpphot,1:1,000 for anti-UGPase (AgriSera), and 1:2,000 for anti-H+-ATPase (AgriSera).Antirabbit IgG horseradish peroxidase-conjugated secondary antibody (GEHealthcare) was diluted to 1:10,000. Signals were detected using the ECL Pluswestern-blotting detection system (GE Healthcare) and the ImageQuant LAS-4010 digital imaging system (GE Healthcare).

Subcellular Localization Analysis

An entry clone carrying an MpPHOT fragment spanning from the promoter tothe last sense codon was generated as described above, using the Infusion_Cend_rvprimer (59-CGCGCCCACCCTTATATTCATCAAATGAGGCGG-39) instead ofthe Infusion_rv primer. The resultant MpPHOT cassette was used to generatea binary vector for fusion of the MpPHOT gene with Citrine at the C terminus. Thebinary plasmid was transformed into regenerating thalli of MpphotKO as describedabove. Fluorescence signals derived from Citrine were detected in 3-d-old gemmal-ings with a confocal laser scanning microscope (FV1000; Olympus) using an 515-nmlaser for excitation and a detection window in the range of 525 to 565 nm.

Analysis of Chloroplast Photorelocation Movement inMpPHOT-Transfected Cells of A. capillus-venerisphot2 Prothalli

MpPHOT cDNA amplified with primers (59-CACCATGATGCCCTCCAC-39/59-TTAATATTCATCAAATGAGGCGG-39) was cloned into a 35S gatewaydestination vector. The 35S:MpPHOT and 35S:GFP vectors were cotransfectedinto the phot2-deficient A. capillus-veneris prothalli by particle bombardment aspreviously described (Kagawa et al., 2004). GFP fluorescence was used as amarker to identify transfected cells. The MpPHOT-transfected cell (i.e. GFP-positive cells) and the adjacent nontransfected cell were irradiated with astrong blue-light microbeam. The chloroplast avoidance response induced byhigh-fluence blue light (15 Wm22) was recorded at 1-min intervals.

Analysis of Chloroplast Photorelocation Movement inMpPHOT-Transformed phot1 phot2Mutant of Arabidopsis

The MpPHOT cDNA was subcloned into the pGWB2 vector by the LRreaction of the Gateway system (Life Technologies). The construct was in-troduced into the Arabidopsis phot1 phot2 mutant (phot1-5 phot2-1) using


Komatsu et al.

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Agrobacterium-mediated transformation. Analysis of chloroplast movement us-ing leaf transmittance was performed as previously described (Wada and Kong,2011). The red light (650 nm) transmittance was automatically recorded every2 min using a microplate reader (VersaMax; Molecular Devices). The valuesshown are the mean 6 SE derived from three experiments.

Sequence data from this article can be found in the DNA Data Bank of Japan/GenBank/EMBL databases under accession numbers AB938187 (MpPHOT gene)and AB938188 (MpPHOT cDNA).

Supplemental Data

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

Supplemental Figure S1. Southern-blot analysis of the MpPHOT gene.

Supplemental Figure S2. Alignment of amino acid sequences of photo-tropins from M. polymorpha, Arabidopsis, and C. reinhardtii.

Supplemental Figure S3. Alignment of amino acid sequences of photo-tropins from a variety of plant species used for the phylogenetic analysisin Figure 3B.

Supplemental Figure S4. Strategy for targeted disruption of the MpPHOTlocus and analysis of homologous recombination events.

Supplemental Figure S5. Images of 20-d-old thalli of the wild type andMpphotKO.

Supplemental Figure S6. Comparison of the ratio of the area occupiedwith chloroplasts to the area of whole cell surface of the wild type,MpphotKO,and gMpPHOT/MpphotKO.

Supplemental Figure S7. Chloroplast distribution of gMpPHOT-Citrine/MpphotKO under various light conditions.

ACKNOWLEDGMENTS

We thank Akira Nagatani for Arabidopsis phototropin mutant seeds, andYukiko Yasui, Sakiko Ishida, Yuuki Sakai, Nozomi Kawamoto, Koji Okajima,Satoru Tokutomi, and Sam-Geun Kong for technical advice and helpful discussions.

Received June 12, 2014; accepted August 2, 2014; published August 5, 2014.

LITERATURE CITED

Aihara Y, Yamamoto T, Okajima K, Yamamoto K, Suzuki T, Tokutomi S,Tanaka K, Nagatani A (2012) Mutations in N-terminal flanking regionof blue light-sensing light-oxygen and voltage 2 (LOV2) domain disruptits repressive activity on kinase domain in the Chlamydomonas photo-tropin. J Biol Chem 287: 9901–9909

Böhm J (1856) Beiträge zur näheren Kenntnis des Chlorophylls. S B AkadWiss Wien Math-nat Kl 22: 479–498

Bowman JL, Floyd SK, Sakakibara K (2007) Green genes-comparativegenomics of the green branch of life. Cell 129: 229–234

Chiyoda S, Ishizaki K, Kataoka H, Yamato KT, Kohchi T (2008) Directtransformation of the liverwort Marchantia polymorpha L. by particlebombardment using immature thalli developing from spores. Plant CellRep 27: 1467–1473

Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58: 21–45Christie JM, Salomon M, Nozue K, Wada M, Briggs WR (1999) LOV (light,

oxygen, or voltage) domains of the blue-light photoreceptor phototropin(nph1): binding sites for the chromophore flavin mononucleotide. ProcNatl Acad Sci USA 96: 8779–8783

Christie JM, Swartz TE, Bogomolni RA, Briggs WR (2002) PhototropinLOV domains exhibit distinct roles in regulating photoreceptor function.Plant J 32: 205–219

Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad SciUSA 81: 1991–1995

Crosson S, Moffat K (2001) Structure of a flavin-binding plant photore-ceptor domain: insights into light-mediated signal transduction. ProcNatl Acad Sci USA 98: 2995–3000

Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy andhigh throughput. Nucleic Acids Res 32: 1792–1797

Folta KM, Spalding EP (2001) Unexpected roles for cryptochrome 2 andphototropin revealed by high-resolution analysis of blue light-mediatedhypocotyl growth inhibition. Plant J 26: 471–478

Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of sus-pension cultures of soybean root cells. Exp Cell Res 50: 151–158

Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein ki-nase superfamily: kinase (catalytic) domain structure and classification.FASEB J 9: 576–596

Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropinlight switch. Science 301: 1541–1544

Haupt W, Scheuerlein R (1990) Chloroplast movement. Plant Cell Environ13: 595–614

Huala E, Oeller PW, Liscum E, Han IS, Larsen E, Briggs WR (1997) ArabidopsisNPH1: a protein kinase with a putative redox-sensing domain. Science 278:2120–2123

Huang K, Merkle T, Beck CF (2002) Isolation and characterization of aChlamydomonas gene that encodes a putative blue-light photoreceptor ofthe phototropin family. Physiol Plant 115: 613–622

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference ofphylogenetic trees. Bioinformatics 17: 754–755

Inoue S, Kinoshita T, Matsumoto M, Nakayama KI, Doi M, Shimazaki K(2008) Blue light-induced autophosphorylation of phototropin is a primarystep for signaling. Proc Natl Acad Sci USA 105: 5626–5631

Inoue S, Matsushita T, Tomokiyo Y, Matsumoto M, Nakayama KI,Kinoshita T, Shimazaki K (2011) Functional analyses of the activationloop of phototropin2 in Arabidopsis. Plant Physiol 156: 117–128

Ishizaki K, Chiyoda S, Yamato KT, Kohchi T (2008) Agrobacterium-mediatedtransformation of the haploid liverwort Marchantia polymorpha L., anemerging model for plant biology. Plant Cell Physiol 49: 1084–1091

Ishizaki K, Johzuka-Hisatomi Y, Ishida S, Iida S, Kohchi T (2013a) Ho-mologous recombination-mediated gene targeting in the liverwortMarchantia polymorpha L. Sci Rep 3: 1532

Ishizaki K, Mizutani M, Shimamura M, Masuda A, Nishihama R, Kohchi T(2013b) Essential role of the E3 ubiquitin ligase nopperabo1 in Schizogenousintercellular space formation in the liverwort Marchantia polymorpha. PlantCell 25: 4075–4084

Ishizaki K, Nonomura M, Kato H, Yamato KT, Kohchi T (2012) Visuali-zation of auxin-mediated transcriptional activation using a common auxin-responsive reporter system in the liverwortMarchantia polymorpha. J Plant Res125: 643–651

Jaedicke K, Lichtenthäler AL, Meyberg R, Zeidler M, Hughes J (2012) Aphytochrome-phototropin light signaling complex at the plasma mem-brane. Proc Natl Acad Sci USA 109: 12231–12236

Jarillo JA, Gabrys H, Capel J, Alonso JM, Ecker JR, Cashmore AR (2001)Phototropin-related NPL1 controls chloroplast relocation induced byblue light. Nature 410: 952–954

Jiao Y, Yang H, Ma L, Sun N, Yu H, Liu T, Gao Y, Gu H, Chen Z, Wada M,et al (2003) A genome-wide analysis of blue-light regulation of Arabi-dopsis transcription factor gene expression during seedling development.Plant Physiol 133: 1480–1493

Jones MA, Christie JM (2008) Phototropin receptor kinase activation byblue light. Plant Signal Behav 3: 44–46

Jones MA, Feeney KA, Kelly SM, Christie JM (2007) Mutational analysisof phototropin 1 provides insights into the mechanism underlying LOV2signal transmission. J Biol Chem 282: 6405–6414

Kadota A, Sato Y, Wada M (2000) Intracellular chloroplast photorelocationin the moss Physcomitrella patens is mediated by phytochrome as well asby a blue-light receptor. Planta 210: 932–937

Kagawa T, Kasahara M, Abe T, Yoshida S, Wada M (2004) Functionanalysis of phototropin2 using fern mutants deficient in blue light-inducedchloroplast avoidance movement. Plant Cell Physiol 45: 416–426

Kagawa T, Lamparter T, Hartman E, Wada M (1997) Phytochrome-mediated branch formation in protonemata of the moss Ceratodon pur-pureus. J Plant Res 110: 363–370

Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S,Okada K, Wada M (2001) Arabidopsis NPL1: a phototropin homolog con-trolling the chloroplast high-light avoidance response. Science 291: 2138–2141

Kagawa T, Wada M (1993) Light-dependent nuclear positioning in pro-thallial cells of Adiantum capillus-veneris. Protoplasma 177: 82–85

Kagawa T, Wada M (1995) Polarized light induces nuclear migration inprothallial cells of Adiantum capillus-veneris L. Planta 196: 775–780



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http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1http://www.plantphysiol.org/cgi/content/full/pp.114.245100/DC1

Kagawa T, Wada M (1999) Chloroplast-avoidance response induced byhigh-fluence blue light in prothallial cells of the fern Adiantum capillus-veneris as analyzed by microbeam irradiation. Plant Physiol 119: 917–924

Kagawa T, Wada M (2000) Blue light-induced chloroplast relocation inArabidopsis thaliana as analyzed by microbeam irradiation. Plant CellPhysiol 41: 84–93

Kaiserli E, Sullivan S, Jones MA, Feeney KA, Christie JM (2009) Domainswapping to assess the mechanistic basis of Arabidopsis phototropin1 receptor kinase activation and endocytosis by blue light. Plant Cell 21:3226–3244

Kanegae T, Hayashida E, Kuramoto C, Wada M (2006) A single chromo-protein with triple chromophores acts as both a phytochrome and aphototropin. Proc Natl Acad Sci USA 103: 17997–18001

Kasahara M, Kagawa T, Sato Y, Kiyosue T, Wada M (2004) Phototropinsmediate blue and red light-induced chloroplast movements in Phys-comitrella patens. Plant Physiol 135: 1388–1397

Kasahara M, Swartz TE, Olney MA, Onodera A, Mochizuki N, Fukuzawa H,Asamizu E, Tabata S, Kanegae H, Takano M, et al (2002) Photochemicalproperties of the flavin mononucleotide-binding domains of the phototropinsfromArabidopsis, rice, and Chlamydomonas reinhardtii. Plant Physiol 129: 762–773

Kawai H, Kanegae T, Christensen S, Kiyosue T, Sato Y, Imaizumi T,Kadota A, Wada M (2003) Responses of ferns to red light are mediatedby an unconventional photoreceptor. Nature 421: 287–290

Kinoshita T, Doi M, Suetsugu N, Kagawa T, Wada M, Shimazaki K (2001)Phot1 and phot2 mediate blue light regulation of stomatal opening.Nature 414: 656–660

Knieb E, Salomon M, Rüdiger W (2004) Tissue-specific and subcellularlocalization of phototropin determined by immuno-blotting. Planta 218:843–851

Kong SG, Kagawa T, Wada M, Nagatani A (2013a) A C-terminal mem-brane association domain of phototropin 2 is necessary for chloroplastmovement. Plant Cell Physiol 54: 57–68

Kong SG, Kinoshita T, Shimazaki K, Mochizuki N, Suzuki T, Nagatani A(2007) The C-terminal kinase fragment of Arabidopsis phototropin 2triggers constitutive phototropin responses. Plant J 51: 862–873

Kong SG, Suetsugu N, Kikuchi S, Nakai M, Nagatani A, Wada M (2013b)Both phototropin 1 and 2 localize on the chloroplast outer membranewith distinct localization activity. Plant Cell Physiol 54: 80–92

Kong SG, Suzuki T, Tamura K, Mochizuki N, Hara-Nishimura I,Nagatani A (2006) Blue light-induced association of phototropin 2 withthe Golgi apparatus. Plant J 45: 994–1005

Kozuka T, Kong SG, Doi M, Shimazaki K, Nagatani A (2011) Tissue-autonomous promotion of palisade cell development by phototropin 2in Arabidopsis. Plant Cell 23: 3684–3695

Kubota A, Ishizaki K, Hosaka M, Kohchi T (2013) Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha usingregenerating thalli. Biosci Biotechnol Biochem 77: 167–172

Lehmann P, Nöthen J, von Braun SS, Bohnsack MT, Mirus O, Schleiff E(2011) Transitions of gene expression induced by short-term blue light.Plant Biol (Stuttg) 13: 349–361

Li FW, Villarreal JC, Kelly S, Rothfels CJ, Melkonian M, Frangedakis E,Ruhsam M, Sigel EM, Der JP, Pittermann J, et al (2014) Horizontaltransfer of an adaptive chimeric photoreceptor from bryophytes to ferns.Proc Natl Acad Sci USA 111: 6672–6677

Luesse DR, DeBlasio SL, Hangarter RP (2010) Integration of Phot1, Phot2,and PhyB signalling in light-induced chloroplast movements. J Exp Bot61: 4387–4397

Matsuoka D, Tokutomi S (2005) Blue light-regulated molecular switch ofSer/Thr kinase in phototropin. Proc Natl Acad Sci USA 102: 13337–13342

Maudoux O, Batoko H, Oecking C, Gevaert K, Vandekerckhove J, Boutry M,Morsomme P (2000) A plant plasma membrane H+-ATPase expressed inyeast is activated by phosphorylation at its penultimate residue and bindingof 14-3-3 regulatory proteins in the absence of fusicoccin. J Biol Chem 275:17762–17770

Mittmann F, Brücker G, Zeidler M, Repp A, Abts T, Hartmann E, Hughes J(2004) Targeted knockout in Physcomitrella reveals direct actions ofphytochrome in the cytoplasm. Proc Natl Acad Sci USA 101: 13939–13944

Nakazato T, Kadota A, Wada M (1999) Photoinduction of spore germina-tion in Marchantia polymorpha L. is mediated by photosynthesis. PlantCell Physiol 40: 1014–1020

Nozue K, Kanegae T, Imaizumi T, Fukuda S, Okamoto H, Yeh KC,Lagarias JC, Wada M (1998) A phytochrome from the fern Adiantumwith features of the putative photoreceptor NPH1. Proc Natl Acad SciUSA 95: 15826–15830

Ohgishi M, Saji K, Okada K, Sakai T (2004) Functional analysis of eachblue light receptor, cry1, cry2, phot1, and phot2, by using combinatorialmultiple mutants in Arabidopsis. Proc Natl Acad Sci USA 101: 2223–2228

Okada S, Fujisawa M, Sone T, Nakayama S, Nishiyama R, Takenaka M,Yamaoka S, Sakaida M, Kono K, Takahama M, et al (2000) Construc-tion of male and female PAC genomic libraries suitable for identificationof Y-chromosome-specific clones from the liverwort, Marchantia poly-morpha. Plant J 24: 421–428

Okajima K, Matsuoka D, Tokutomi S (2011) LOV2-linker-kinasephosphorylates LOV1-containing N-terminal polypeptide substratevia photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett585: 3391–3395

Onodera A, Kong SG, Doi M, Shimazaki K, Christie J, Mochizuki N,Nagatani A (2005) Phototropin from Chlamydomonas reinhardtii is func-tional in Arabidopsis thaliana. Plant Cell Physiol 46: 367–374

Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H,Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, et al (2008) ThePhyscomitrella genome reveals evolutionary insights into the conquest ofland by plants. Science 319: 64–69

Sakai T, Kagawa T, Kasahara M, Swartz TE, Christie JM, Briggs WR,Wada M, Okada K (2001) Arabidopsis nph1 and npl1: blue light receptorsthat mediate both phototropism and chloroplast relocation. Proc NatlAcad Sci USA 98: 6969–6974

Sakamoto K, Briggs WR (2002) Cellular and subcellular localization ofphototropin 1. Plant Cell 14: 1723–1735

Sato Y, Wada M, Kadota A (2001) Choice of tracks, microtubules and/oractin filaments for chloroplast photo-movement is differentially con-trolled by phytochrome and a blue light receptor. J Cell Sci 114: 269–279

Schneider H, Schuettpelz E, Pryer KM, Cranfill R, Magallón S, Lupia R(2004) Ferns diversified in the shadow of angiosperms. Nature 428: 553–557

Senn G (1908) Die Gestalts- und Lageveränderung der Pflanzen-Chromatophoren,Leipzig, Germany, W. Engelmann

Suetsugu N, Kagawa T, Wada M (2005a) An auxilin-like J-domain protein,JAC1, regulates phototropin-mediated chloroplast movement in Arabi-dopsis. Plant Physiol 139: 151–162

Suetsugu N, Kong SG, Kasahara M, Wada M (2013) Both LOV1 and LOV2domains of phototropin2 function as the photosensory domain for hy-pocotyl phototropic responses in Arabidopsis thaliana (Brassicaceae). AmJ Bot 100: 60–69

Suetsugu N, Mittmann F, Wagner G, Hughes J, Wada M (2005b) A chi-meric photoreceptor gene, NEOCHROME, has arisen twice during plantevolution. Proc Natl Acad Sci USA 102: 13705–13709

Suetsugu N, Wada M (2007a) Chloroplast photorelocation movementmediated by phototropin family proteins in green plants. Biol Chem 388:927–935

Suetsugu N, Wada M (2007b) Phytochrome-dependent photomovementresponses mediated by phototropin family proteins in cryptogam plants.Photochem Photobiol 83: 87–93

Suetsugu N, Wada M (2012) Chloroplast photorelocation movement: asophisticated strategy for chloroplasts to perform efficient photosynthesis. InNajafpour MM, ed, Advances in Photosynthesis—Fundamental Aspects, Ri-jeka, Croatia, InTech Publishers, pp 215–

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