Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the Liverwort Marchantia polymorpha1[W] Aino Komatsu, Mika Terai, Kimitsune Ishizaki 2, Noriyuki Suetsugu, Hidenori Tsuboi 3, Ryuichi Nishihama, Katsuyuki T. Yamato 4, 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-lightirradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation 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 the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.

Light is not only an energy source for photosynthesis but it is also a signal that regulates numerous physiological responses for plants. Because chloroplasts are the important organelle for photosynthesis, most plant species possess a light-dependent mechanism to regulate the intracellular position of chloroplasts (chloroplast photorelocation movement). Intensive studies on chloroplast photorelocation movement have been performed since the 19th century (Böhm, 1856). Senn (1908) described the chloroplast distribution patterns under different light conditions in various plant species, including algae, liverworts, mosses, ferns, and seed plants, and revealed the general responses of chloroplasts to intensity and direction of light. Under low-light conditions,

1 This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas from the Japan Society for the Promotion of 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 and Technology, Kinki University, Kinokawa 649–6493, Japan. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.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

chloroplasts are positioned along the cell walls perpendicular to the direction of incident light (i.e. periclinal cell walls) to efficiently capture light for photosynthesis (the accumulation response). By contrast, under high-light conditions, chloroplasts are stacked along the cell walls parallel to the direction of incident light (i.e. anticlinal cell walls) to minimize total light absorption and to avoid photooxidative damage (the avoidance response). These chloroplast movements are induced primarily by blue light in most plant species (Suetsugu and Wada, 2007a). In some plant species, such as several ferns including Adiantum capillus-veneris, the moss Physcomitrella patens, and some charophycean green algae (Mougeotia scalaris and Mesotaenium caldariorum), red light is also effective to induce chloroplast movement (Suetsugu and Wada, 2007b). Analyses of chloroplast movement in response to irradiation with polarized light and/or a microbeam suggest that the photoreceptor for chloroplast movement is localized on or close to the plasma membrane (Haupt and Scheuerlein, 1990; Wada et al., 1993). In addition, chloroplasts assume their specific positions in the dark (dark positioning), although the patterns vary among plant species (Senn, 1908). For example, the chloroplasts are localized at the bottom of the cell in palisade cells of Arabidopsis (Arabidopsis thaliana; Suetsugu et al., 2005a) and on the anticlinal walls bordering neighboring cells in the prothallial cells of A. capillus-veneris (Kagawa and Wada, 1993; Tsuboi et al., 2007). Molecular mechanisms for chloroplast photorelocation movements have been revealed through molecular genetic analyses using Arabidopsis (Suetsugu and Wada,

Plant PhysiologyÒ, September 2014, Vol. 166, pp. 411–427, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved.

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2012). The light-activated kinase phototropin was identified as the blue-light receptor (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropin consists of two functional regions: a photosensory domain at the N terminus and a Ser/Thr kinase domain at the C terminus (Christie, 2007). The N-terminal photosensory domain contains two light, oxygen, or voltage (LOV) domains, which belong to the Per/ARNT/Sim domain superfamily. Each LOV domain binds to one FMN and functions as a blue-light sensor (Christie et al., 1999). The LOV2 domain is essential for blue-light-dependent regulation of the activation of the C-terminal kinase domain (Christie et al., 2002; Harper et al., 2003). Arabidopsis has two phototropins: phot1 and phot2 (Christie, 2007). Besides chloroplast photorelocation movement, phototropin controls other photoresponses to optimize the photosynthetic efficiency in plants and improves growth responses such as phototropism, stomatal opening, and leaf flattening (Christie, 2007). Both phot1 and phot2 redundantly regulate the chloroplast accumulation response (Sakai et al., 2001), hypocotyl phototropism (Huala et al., 1997; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and leaf flattening (Sakai et al., 2001; Sakamoto and Briggs, 2002). Rapid inhibition of hypocotyl elongation is specifically 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 photoresponses are mediated by mechanisms in which gene expression is not involved primarily. For example, chloroplast photorelocation movement can be observed even in enucleated fern cells (Wada, 1988), and phototropins show only a minor contribution to blue-light-induced gene expression in Arabidopsis (Jiao et al., 2003; Ohgishi et al., 2004; Lehmann et al., 2011). Furthermore, both phot1 and phot2 are localized on the plasma membrane despite the absence of a transmembrane domain (Sakamoto and Briggs, 2002; Kong et al., 2006). During chloroplast movement, phototropins, in particular phot2, associate not only with the plasma membrane but also with the chloroplast outer membrane (Kong et al., 2013b). In addition, phot1 shows blue-lightdependent internalization into the cytoplasm (Sakamoto and Briggs, 2002; Knieb et al., 2004; Wan et al., 2008; Kaiserli et al., 2009), whereas phot2 exhibits a blue-lightdependent association with the Golgi apparatus (Kong et al., 2006). PHOT genes have been identified from various green plants and are indicated to be duplicated in respective lineages such as seed plants, ferns, lycophytes, and mosses (Li et al., 2014). In the fern A. capillus-veneris, chloroplast accumulation and avoidance responses are induced 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 chromophorebinding domain of phytochrome and complete phototropin domains (Nozue et al., 1998). Neochrome is the 412

red-light receptor that mediates chloroplast movement (Kawai et al., 2003) and possibly blue-light-induced chloroplast movement through its LOV domains (Kanegae et al., 2006). Because the Acphot2 mutant is defective in the chloroplast avoidance response and dark positioning (Kagawa et al., 2004; Tsuboi et al., 2007), similar to the phot2 mutant in Arabidopsis (Jarillo et al., 2001; Kagawa et al., 2001; Suetsugu et al., 2005a), the function of phot2 in the regulation of chloroplast movement is highly conserved in these vascular plants. In the moss P. patens, in which chloroplast accumulation and avoidance responses are induced by both blue and red light (Kadota et al., 2000), seven phototropin genes are present in the draft genome sequences (Rensing et al., 2008). The phototropins encoded by four of these genes (PpphotA1, PpphotA2, PpphotB1, and PpphotB2) function in the blue-lightinduced chloroplast movement (Kasahara et al., 2004). Moreover, red-light-induced chloroplast movements are mediated by both conventional phytochromes (Mittmann et al., 2004; Uenaka and Kadota, 2007) and phototropins (Kasahara et al., 2004). Because the direct association between phytochromes and phototropins is suggested to be involved in red-light-induced chloroplast movement (Jaedicke et al., 2012), phototropins should be essential components in the chloroplast movement signaling pathway (Kasahara et al., 2004). A single PHOT gene was isolated in a unicellular green alga, Chlamydomonas reinhardtii (Huang et al., 2002; Kasahara et al., 2002). When expressed in Arabidopsis phot1 phot2 double-mutant plants, C. reinhardtii phototropin rescued the defects in chloroplast photorelocation movement in phot1 phot2 plants (Onodera et al., 2005), indicating that the initial step of the phototropinmediated signal transduction mechanism for chloroplast movements is conserved in the green plant lineage. Although the existence of only one PHOT gene is ideal for elucidation of phototropin-mediated responses, C. reinhardtii cells contain a single chloroplast and show no chloroplast photorelocation movement. Liverworts represent the most basal lineage of extant land plants and offer a valuable experimental system for elucidation of various physiological responses commonly seen in land plants (Bowman et al., 2007). Marchantia polymorpha has emerged as a model liverwort because molecular biological techniques, such as genetic transformation and gene-targeting technologies, have been established for the species (Ishizaki et al., 2008, 2013a; Kubota et al., 2013; Sugano et al., 2014). Furthermore, an ongoing M. polymorpha genome sequencing project under the Community Sequencing Program at the Joint Genome Institute has indicated that many biological mechanisms found in other groups of land plants are conserved in a much less complex form. Blue-light-induced chloroplast movement was briefly reported in M. polymorpha (Senn, 1908; Nakazato et al., 1999). However, information on chloroplast photorelocation movement in liverworts, including M. polymorpha, is very limited. In this study, we investigated chloroplast photorelocation movement in detail in M. polymorpha and Plant Physiol. Vol. 166, 2014

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analyzed the molecular mechanism underlying the photoreceptor system through molecular genetic analysis of M. polymorpha phototropin. RESULTS Chloroplast Photorelocation Movements Are Induced by Blue Light But Not Red Light in M. polymorpha

The chloroplast distribution pattern was analyzed using wild-type gemmalings (the early growth stage of thalli developing from gemmae) in M. polymorpha. Under our growth conditions with continuous white light, chloroplasts were located over the upper cell surface as a result of the accumulation response (Fig. 1A). After irradiation with strong blue light (50 Wm22 for 120 min), the chloroplasts relocated from the periclinal to the anticlinal cell walls (Fig. 1B), indicating that M. polymorpha shows a typical chloroplast avoidance response. By contrast, the chloroplasts remained on the periclinal surface after irradiation with red light at 50 Wm22 for 120 min (Fig. 1C), suggesting that red light is ineffective at inducing the avoidance response in M. polymorpha. After dark treatment for 3 d, the chloroplasts moved from the periclinal to the anticlinal cell walls (Fig. 1D), similar to their distribution under strong blue light. However, there was a difference between dark and strong-blue-light conditions in the distribution patterns of the chloroplasts in the outermost cells of gemmalings. Under strong blue light, the chloroplasts moved to all anticlinal cell walls in both the outermost and inner cells of gemmalings (Fig. 1B). In the dark, chloroplasts moved to all anticlinal cell walls in the inner cells, but chloroplasts in the outermost cell layers were excluded from the outermost anticlinal cell wall and thus were distributed only along the anticlinal walls associated with neighboring cells (Fig. 1D). This distribution pattern in darkness was defined as dark positioning in M. polymorpha. Thus, the distribution pattern of chloroplasts in M. polymorpha both in the light and the dark are similar to those in the prothallial cells of A. capillus-veneris (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007) except

that red light effectively induces chloroplast movement in A. capillus-veneris but not in M. polymorpha. To investigate chloroplast photorelocation movement in detail, the movements in the gemmaling cells were induced by microbeam irradiation with different fluence rates of blue or red light and analyzed by means of timelapse images (Fig. 2). In response to weak blue light (10 Wm22), chloroplasts outside the irradiated area moved toward the microbeam area, and those inside it stayed within the irradiated area (Fig. 2, A and E). Under strong blue light (50 Wm22), chloroplasts in the irradiated area escaped from the microbeam and remained outside of the irradiated area (Fig. 2, B and F). These behaviors are similar to those observed in Arabidopsis (Kagawa and Wada, 2000) and A. capillus-veneris (Kagawa and Wada, 1999). The fluence-rate-dependent responses of chloroplast photorelocation movement are summarized in Table I. When irradiated with blue light, clear chloroplast accumulation movement was observed at 0.01 to 25 Wm22 of microbeam. However, at 37.5 Wm22, a weak avoidance response was induced in some experiments, indicating that the transition from accumulation movement to avoidance movement occurred at about 37.5 Wm22. An obvious avoidance movement was observed at 50 Wm22 or higher intensity. By contrast, the red-light microbeam at all examined fluence rates did not induce chloroplast photorelocation movement (Fig. 2, C, D, G, and H). In the case of the moss P. patens, protonemata growing under continuous red light but not under white light exhibited red-light-induced chloroplast photorelocation (Kadota et al., 2000). However, neither the accumulation response nor the avoidance response was induced even in the gemmalings grown under continuous red light in M. polymorpha. These results further confirmed that the chloroplast photorelocation movement is controlled by blue 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 mediates the blue-light-dependent chloroplast movements, we Figure 1. Chloroplast distribution patterns under different light conditions in wild-type M. polymorpha. Gemmalings incubated under continuous white light for 3 d were subjected to different light treatments. A, Before light irradiation. B, After 120 min of irradiation with high-fluence blue light (50 Wm22). C, After 120 min of irradiation with high-fluence red light (50 Wm22). D, After 3 d dark treatment. The outermost cell walls are indicated by arrowheads in B and D. Bars = 20 mm.

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Figure 2. Microbeam-induced chloroplast movement in wild-type M. polymorpha. A to D, Cells were irradiated for 80 min with 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 chloroplast movements 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 red light. The rectangles indicate the positions of the microbeam irradiation. Black circles indicate initial positions of each chloroplast before microbeam irradiation. These experiments were repeated at least three times in different cells with similar results. Bar = 20 mm.

amplified M. polymorpha phototropin gene fragments by reverse transcription-PCR with PHOT-specific degenerate primers and identified only one complementary DNA (cDNA) species for PHOT. We identified P1-derived artificial chromosome (PAC) clones for the PHOT genomic fragments using the cDNA as a probe. In silico analysis of current genome sequencing and transcriptome data from different tissues and conditions supported the conclusion that M. polymorpha has only one PHOT gene. We also performed Southern-blot analysis under low stringent conditions using M. polymorpha genomic DNA digested with restriction enzymes. The patterns of restriction fragments were consistent with those predicted from the gene structure of the cloned PHOT gene, confirming that this 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 introns. The number and positions of introns in the coding sequence were conserved among MpPHOT, Arabidopsis PHOT1, and PHOT2. MpPHOT encodes an 1,115-amino acid protein that contains two LOV domains (LOV1 and LOV2) and a Ser/Thr kinase domain (Fig. 3A). The LOV domains of Mpphot are highly conserved and contain all of the amino acid residues important for FMN binding and photoactivation (Crosson and Moffat, 2001), suggesting that the LOV domains of Mpphot are photoactive. The N-terminal extension upstream of the LOV1 domain is highly variable among phototropins from various 414

plants, but some Ser residues that are phosphorylated in Arabidopsis phot1 and phot2 (Inoue et al., 2008, 2011) are conserved in Mpphot (Supplemental Fig. S2). Similar to fern and moss phototropins (Kagawa et al., 2004; Kasahara et al., 2004), the N-terminal extension of Mpphot is considerably longer than that of seed plant phototropins (Fig. 3A). The Ser/Thr kinase domain of Mpphot is also highly similar to that of phototropins from other plants (Supplemental Fig. S2). Notably, two Ser residues in the activation loop in the kinase domain, which are autophosphorylated and essential for full activity of phot1 and Table I. Chloroplast photorelocation movement under blue or red light Chloroplast photorelocation movement was analyzed by microbeam irradiation with various fluence rates of blue or red light. Light Intensity Wm

0.001 0.01 0.1 1.0 10 25 37.5 50 75

Blue

Red

No movement Accumulation Accumulation Accumulation Accumulation Accumulation Accumulation or weak avoidance Avoidance Avoidance

Not determined Not determined Not determined No movement No movement Not determined Not determined

22

No movement No movement

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phot2 in Arabidopsis (Inoue et al., 2008, 2011), are conserved in Mpphot (Supplemental Fig. S2). In the phylogenetic analysis (Fig. 3B; Supplemental Fig. S3), MpPHOT was placed in the bryophyte clade that is sister to the PHOT1 and PHOT2 groups in the angiosperm lineage as reported in a recent large-scale phylogenetic analysis of PHOT genes (Li et al., 2014).

Li et al. (2014) recently reported that only one PHOT gene was identified in all of the seven liverwort species examined, including M. polymorpha, which is consistent with our results (Supplemental Fig. S1). Thus, it is likely that liverworts have only one PHOT gene in general and that multiple duplication of PHOT genes occurred during land plant evolution.

Figure 3. Protein structure and phylogenetic tree of phototropins from M. polymorpha and other plants. A, Domain organization 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 indicate the positions of the start and end of each domain. B, Phylogenetic relationships of plant phototropins. A majority consensus phylogeny for 24 phototropins from green algae and land plants was reconstructed by Bayesian inference analysis of an alignment of sequences corresponding to amino acid residues 699 to 1087 of Mpphot (Supplemental Fig. S3). The Ostreococcus tauri PHOT sequence was used as the outgroup. Posterior probabilities are indicated at the nodes. Atr, Amborella trichopoda; 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. Plant Physiol. Vol. 166, 2014

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Generation of MpPHOT Knockout Lines and Complementation Lines

To investigate the function of Mpphot in chloroplast photorelocation movement, we generated MpPHOT knockout lines (MpphotKO) using the established targeting method with homologous recombination in M. polymorpha (Ishizaki et al., 2013a). A 276-bp portion including exon3 of MpPHOT was replaced with an expression cassette for the hygromycin phosphotransferase gene (Fig. 4A; Supplemental Fig. S4A), to disrupt a region corresponding to a LOV1 portion that includes the Cys residue essential for formation of the flavin C (4a)cysteinyl adduct (Supplemental Fig. S2). By genomic PCR screening, we obtained two independent targeted lines (Supplemental Fig. S4B). The Southern-blot analysis of genomic DNAs revealed that one copy of the transgene was integrated into the MpPHOT locus (Supplemental Fig. S4C). Because the two lines showed essentially the same phenotype for chloroplast photorelocation movement, we only present the data obtained for one line (MpphotKO 1), which did not show obvious growth defects under our growth conditions (Supplemental Fig. S5). Immunoblot analysis with anti-Mpphot antibody showed that the Mpphot protein was not detected in the MpphotKO, indicating that MpphotKO is a null mutant. To substantiate that the disruption of MpPHOT caused the defective phenotypes in MpphotKO (see below), we introduced the MpPHOT expression construct controlled by its own promoter into the MpphotKO, generating complemented lines genomic MpPHOT (gMpPHOT)/MpphotKO. The amount of Mpphot in gMpPHOT/MpphotKO was similar to that in the wild type (Fig. 4B). These knockout and complemented lines were used in experiments described below. Chloroplast Photorelocation Movement in MpphotKO Is Impaired

To examine whether Mpphot is the blue-light receptor for chloroplast photorelocation movement in M. polymorpha, we analyzed blue-light-induced chloroplast movements in cells of MpphotKO and gMpPHOT/ MpphotKO. Under continuous white light, the chloroplasts localized sparsely on the upper cell surface in MpphotKO (Fig. 5A), whereas the chloroplasts covered the whole cell surface in the wild type (Fig. 1A), indicating that MpphotKO is defective in the chloroplast accumulation response. After blue-light irradiation (at 50 Wm22 for 120 min), the chloroplasts did not change their positions (Fig. 5A). These results indicate that MpphotKO is defective in both accumulation and avoidance responses of chloroplasts. In addition, MpphotKO was deficient in dark positioning of chloroplasts. After 3 d of dark adaptation, a significant number of chloroplasts in MpphotKO were observed along the anticlinal walls without neighboring cells (Fig. 5A), whereas no chloroplasts localized on the outermost anticlinal walls in the wild type (Fig. 1D). Furthermore, more chloroplasts localized on the periclinal wall in MpphotKO compared with the wild type. Measurement of the ratio of the area occupied with 416

Figure 4. Targeted disruption of MpPHOT by homologous recombination. A, Schematic diagram of targeted disruption of the MpPHOT locus by homologous recombination. Filled rectangles indicate exons, and intervening thick lines indicate introns. B, MpPHOT protein accumulation in the wild type, MpphotKO, and gMpPHOT/MpphotKO. Proteins were immunodetected with an anti-Mpphot antibody. Coomassie Brilliant Blue staining of RBCL is shown as a loading control. Plants were grown under continuous white light for 7 d. Each lane contains 20 mg of total proteins. DEn, 39 Part of the maize (Zea mays) En element; hpt, hygromycin phosphotransferase; RBCL, Rubisco large subunit; WT, wild type.

chloroplasts to the area of whole cell surface further confirmed no chloroplast movement after dark treatment in MpphotKO (Supplemental Fig. S6). These defects in MpphotKO under the different light conditions were completely rescued in gMpPHOT/Mpphot KO (Fig. 5B; Supplemental Fig. S6). Thus, these results indicate that Mpphot is necessary for both accumulation and avoidance responses as well as the dark positioning of chloroplasts. To further confirm the essential role of Mpphot in chloroplast photorelocation movement, we performed microbeam irradiation experiments with MpphotKO and gMpPHOT/MpphotKO. Under weak blue light (10 Wm22), the chloroplasts did not move toward the light-irradiated area, which was indicative of the defective accumulation response in MpphotKO (Fig. 6, A and E). Under strong blue light (50 Wm22), the avoidance response was not observed in MpphotKO and the chloroplasts that had resided in the light-irradiated area did not move out of this area (Fig. 6, B and F). Under both conditions, in Plant Physiol. Vol. 166, 2014

Phototropin-Mediated Chloroplast Movement in Liverwort

Figure 5. Chloroplast distribution patterns of MpphotKO and gMpPHOT/MpphotKO under different light conditions. A, MpphotKO. B, gMpPHOT/MpphotKO. Gemmalings incubated under continuous white light for 3 d were used for this 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 outermost cell walls are indicated by arrowheads. Bars = 20 mm.

MpphotKO chloroplasts were still distributed sparsely on the periclinal and anticlinal walls regardless of the light conditions, indicating that MpphotKO is defective in both the accumulation and avoidance responses. In gMpPHOT/MpphotKO, both the accumulation and avoidance responses to weak and strong blue light, respectively, were restored (Fig. 6, C and D). Collectively, these results indicated that loss of chloroplast photorelocation movement in MpphotKO was caused by MpPHOT disruption and, therefore, that Mpphot is the photoreceptor for chloroplast photorelocation movement in M. polymorpha.

and subjected to immunoblot analysis (Fig. 7C). Successful fractionation was verified using two antibodies against cytosolic UDP-Glc pyrophosphorylase (UGPase) and plasma-membrane localized H+-ATPase (Maudoux et al., 2000). Immunoblot analysis with anti-Mpphot antibody specifically detected the Mpphot protein of 123 kD only in the membrane fraction, similar to H+-ATPase (Fig. 7C). Mpphot and H+-ATPase were not detected in the cytosolic fraction, in which UGPase was enriched. Together with the microscopic results mentioned above, the similar partitioning profiles for Mpphot and H+-ATPase indicate that the Mpphot protein is predominantly localized to the plasma membrane.

Mpphot Is Localized on the Plasma Membrane

To investigate the expression pattern and the intracellular localization of Mpphot, we generated transgenic lines expressing Mpphot translationally fused to the Citrine reporter protein at the C terminus under the control of the MpPHOT promoter in the MpphotKO background (gMpPHOT-Citrine/MpphotKO). These lines showed normal chloroplast photorelocation movement, indicating that Mpphot-Citrine is functional (Supplemental Fig. S7). Fluorescence microscopy showed that the fluorescence from Mpphot-Citrine was observed throughout the entire body of the gemmalings (Fig. 7A). Mpphot-Citrine appeared to be localized predominantly on the plasma membrane under higher magnification (Fig. 7B). To confirm the plasma membrane localization of Mpphot, cytosolic and membrane fractions were prepared Plant Physiol. Vol. 166, 2014

Mpphot Exhibits Blue-Light-Dependent Autophosphorylation Activity in Vitro and in Vivo

Given that the LOV domains and the Ser/Thr kinase domain of Mpphot are highly conserved (Supplemental Fig. S2), it is plausible that blue light activates autophosphorylation activity similar to phototropins from other species (Christie, 2007). To examine the blue-lightstimulated kinase activity of Mpphot, the maltose binding protein (MBP) fusion protein of full-length Mpphot (MBP-Mpphot) was expressed in Escherichia coli and affinity purified. MBP-Mpphot was incubated with [g-32P] ATP under dark or blue light (18 Wm22) for 30 min. The MBP-Mpphot protein was detected as a band of approximately 170 kD in all lanes in SDS-PAGE (Fig. 8A, 417

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Figure 6. Chloroplast relocation in blue-light-irradiated cells of MpphotKO and gMpPHOT/MpphotKO. A to D, Chloroplast photorelocation movement was analyzed in response to microbeam irradiation with different fluence rates of blue-light for 80 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.

left), consistent with the predicated molecular mass of MBP-Mpphot (167 kD). Darkness induced only a weak autoradiography signal at the position of MBP-Mpphot (Fig. 8A, right), suggesting that Mpphot has low autophosphorylation activity in darkness. Conversely, blue light induced a strong autoradiography signal and significant mobility shift, although the amount of MBPMpphot was unchanged regardless of light condition. This result indicated that blue light enhanced the kinase activity of Mpphot. Autophosphorylation of MBP-Mpphot was detected after 1 min of irradiation with blue light and increased progressively over the course of 30 min irradiation (Fig. 8B). The Asp residue in subdomain VII of the C-terminal kinase domain of Arabidopsis phot1 and phot2 is functionally essential, and the replacement of the residue with Asn results in loss of kinase activity (Suetsugu and Wada, 2013). To substantiate that Mpphot is autophosphorylated, we also analyzed the kinase-negative Mpphot protein in which the corresponding Asp-922 residue was substituted with Asn (MBP-Mpphot D922N). In MBPMpphotD922N, signals were not detected in the dark-treated control or blue-light-irradiated samples, although the amount of MBP-MpphotD922N was comparable to that of MBP-Mpphot during the experiment (Fig. 8A). These results indicate that Mpphot possesses blue-light-dependent kinase activity and is autophosphorylated in vitro. To investigate the blue-light-dependent autophosphorylation activity of Mpphot in vivo, we examined the blue-light-dependent mobility shift of Mpphot 418

through immunoblot analysis with the anti-Mpphot antibody. Dark-adapted wild-type plants were irradiated with blue light (33 Wm22) for 10 min (Fig. 8C). After 1 min of blue light irradiation, a slight mobility shift of Mpphot was observed and the band shift of Mpphot was saturated after 3 min. When the 10-min-irradiated samples were returned to the dark, the mobility of Mpphot gradually shifted to that of the nonirradiated sample (Fig. 8D). These results indicate that blue light induced autophosphorylation of Mpphot and the phosphorylated Mpphot was dephosphorylated in darkness. Thus, these results suggest that Mpphot shows blue-light-regulated kinase activity similar to phototropins in other plants.

Mpphot Can Function to Regulate Chloroplast Avoidance Response in Adiantum capillus-veneris

To investigate whether the function of Mpphot is conserved and Mpphot is functional in other plants in which multiple phototropins mediate chloroplast photorelocation movement, we expressed Mpphot transiently in the phot2 mutant of the fern A. capillus-veneris, which is defective in the avoidance response induced by strong blue light (Kagawa et al., 2004), and examined whether Mpphot can regulate the phot2-mediated chloroplast avoidance response in the heterologous system. Using an assay system developed for assessing the function of phototropins in A. capillus-veneris (Kagawa et al., 2004; Plant Physiol. Vol. 166, 2014

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avoidance response, Mpphot was introduced into the Arabidopsis phot1 phot2 double mutant, which is defective in both the accumulation and avoidance responses (Sakai et al., 2001). MpPHOT was expressed under the control of the 35S promoter in phot1 phot2 double mutants (MpPHOT/phot1 phot2). To characterize chloroplast photorelocation movement in the MpPHOT transgenic lines, the changes in leaf transmittance caused by chloroplast photorelocation movement was analyzed using a plate reader system (Wada and Kong, 2011). In the wild type, leaf transmittance was changed in response to weak and strong blue-light irradiation. Weak light induced a decrease in transmittance as a result of the accumulation response, whereas strong light induced an increase as a result of the avoidance response (Fig. 10, A and B). These changes were not detected in the phot1 phot2 double mutants because the

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

Kong et al., 2013a), prothallial cells of the phot2 mutant were cotransfected with MpPHOT and GFP cDNAs, both of which were driven by the Cauliflower mosaic virus 35S (35S) promoter, using particle bombardment. GFP fluorescence was used as a marker to identify transfected cells. An MpPHOT-transfected cell (i.e. GFP-positive cell) and an adjacent nontransfected cell were irradiated with a strong blue-light microbeam. After 60 min of irradiation with strong blue light, the chloroplasts in the transgenic cells moved away from the light-irradiated area, whereas the chloroplasts in an adjacent nontransfected mutant cell accumulated in the irradiated area because of the defective avoidance response (Fig. 9), indicating that the transient expression of Mpphot was able to complement the deficiency of the A. capillus-veneris phot2 mutant in the avoidance movement. This result suggests that Mpphot is functional in regulation of the avoidance responses in A. capillus-veneris and that phototropin of M. polymorpha has the ability to transduce the blue-light signal to mediate the avoidance movement in A. capillus-veneris. Mpphot Is Able to Regulate Both Chloroplast Accumulation and Avoidance Responses in Arabidopsis

To examine the conserved function of Mpphot to mediate the accumulation response in addition to the Plant Physiol. Vol. 166, 2014

Figure 8. Blue-light-dependent phosphorylation of Mpphot in vitro and in vivo. A, Autophosphorylation activity of Mpphot and MpphotD922N 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 Bluestained image is shown on the left, whereas an autoradiograph is presented on the right. WT, Wild type. B, Time dependency of Mpphot autophosphorylation in vitro. Blue light was irradiated for the indicated periods. C, Blue-light-induced phosphorylation of Mpphot in vivo. The Mpphot protein from the blue-light-irradiated plants (0, 1, 3, 5, and 10 min) was detected with anti-Mpphot antibody. D, Dephosphorylation of Mpphot in vivo. 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 dark for 60, 120, 180, or 360 min. Mpphot protein was detected as in C. 419

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mutants lacked chloroplast photorelocation movement (Fig. 10, A and B). On the other hand, MpPHOT/phot1 phot2 showed partial but significant changes in leaf transmittance under both weak and strong blue light (Fig. 10, A and B). Furthermore, we confirmed the function of Mpphot in the mediation of chloroplast photorelocation movements by observing chloroplast movement in mesophyll cells of MpPHOT/phot1 phot2 in response to microbeam irradiation (Fig. 10C). When irradiated with weak blue light (1 Wm22), the chloroplasts outside of the irradiated area moved toward the light-irradiated area. Conversely, when irradiated with strong blue light (100 Wm22), the light-irradiated chloroplasts escaped from the irradiated area (Fig. 10C). In conclusion, these results indicate that Mpphot was able to regulate both accumulation and avoidance responses that were impaired in the Arabidopsis phot1 phot2 double mutant and possesses the functions of phot1 and phot2 in Arabidopsis with respect to the chloroplast photorelocation.

DISCUSSION Phototropin-Mediated Chloroplast Photorelocation Movements in M. polymorpha

Although intensive observation of chloroplast photorelocation movement has been performed in diverse plant groups such as green algae, mosses, ferns, and seed plants since the 19th century (Senn, 1908; Suetsugu and Wada, 2012), knowledge of chloroplast photorelocation movement in liverworts is relatively limited. In this study, we observed light-induced movements of chloroplasts in M. polymorpha in detail. Previous preliminary results suggested that blue light is effective in the induction of chloroplast movement in M. polymorpha (Nakazato et al., 1999). We confirmed that chloroplast movement was induced exclusively by blue light similar to most land plant species. At blue-light fluence rates in the range of 0.01 to 25 Wm22, the accumulation response was induced (Fig. 2; Table I). Conversely, the avoidance response was induced at fluence rates of 50 Wm22 or stronger. Although M. polymorpha is as sensitive to weak blue light to induce the accumulation response as other plants are, the light intensity at which the response changes from accumulation to avoidance was much higher than that for vascular plants. At 10 Wm22 of blue light or stronger, the chloroplasts escape from the lightirradiated area in Arabidopsis and A. capillus-veneris (Kagawa and Wada, 1999, 2000), whereas chloroplasts accumulate toward the light-irradiated area in M. polymorpha (Fig. 2). In the case of P. patens, which is also a bryophyte, the accumulation response is induced at about 30 Wm22 of blue light, and stronger blue light (more than about 100 Wm22) is required to induce the avoidance response (Kadota et al., 2000; Sato et al., 2001). Thus, the light intensity necessary for the induction of avoidance response in M. polymorpha was 420

Figure 9. Heterologous expression of MpPHOT in A. capillus-veneris phot2 mutant cells. An MpPHOT-expressing cell and its neighboring untransfected cell in the phot2 prothallus were simultaneously irradiated with a microbeam of 100 Wm22 blue light. Areas surrounded with broken lines indicate the position of the microbeam irradiation. Bar = 20 mm.

higher than that in vascular plants, and this property might be common in bryophytes. In P. patens, red-light-induced chloroplast movement was observed in red-light-grown protonema (Kadota et al., 2000). This response was mediated by both conventional phytochromes (Mittmann et al., 2004; Uenaka and Kadota, 2007) and phototropins (Kasahara et al., 2004), suggesting that phototropins are an essential component for transmitting signals in the chloroplast movement signaling pathway (Kasahara et al., 2004; Jaedicke et al., 2012). Because M. polymorpha has both phytochrome and phototropin, we also examined chloroplast movements in red-light-grown plants in addition to white-light-grown plants of M. polymorpha. Similar to the plants grown under white light (Figs. 1C, and 2, C and D), the red-light-grown plants showed no redlight-induced chloroplast photorelocation movement. Red-light-induced chloroplast photorelocation movements have not been observed in some mosses examined, such as Funaria hygrometrica (Zurzycki, 1967) and Ceratodon purpureus (Kagawa et al., 1997), and most Plant Physiol. Vol. 166, 2014

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Figure 10. Heterologous expression of MpPHOT in the Arabidopsis phot1 phot2 double mutant. A, Changes in leaf transmittance caused by chloroplast photorelocation movement. The graph shows representative data from three independent experiments. After 10 min in darkness, leaves were irradiated with blue light at 0.8, 5.3, and 13.2 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 of the wild type, phot1 phot2, and MpPHOT/phot1 phot2, respectively. B, Rate of leaf transmittance change over 2 to 6 min after blue light irradiation. Data are the means of three independent experiments. Bars indicate the SE . White, gray, and black rectangles indicate the transmittance of the wild type, phot1 phot2, and MpPHOT/phot1 phot2, respectively. C, Chloroplast relocation movement induced by continuous microbeam irradiation with blue light in MpPHOT/phot1 phot2. The white circle indicates the light-irradiated area. Cells were irradiated with low-intensity blue light (1 Wm22 ) for the accumulation response (left) or with highintensity blue light (100 Wm22 ) for the avoidance response (right). Bar = 25 mm. Plant Physiol. Vol. 166, 2014

other land plants. Because the red-light-induced chloroplast photorelocation movement has been observed only in P. patens among the bryophytes examined so far, it is suggested that chloroplast movement is not induced by red light in most bryophytes. In addition to the blue-light-induced chloroplast movement, we showed the specific distribution pattern of 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 the anticlinal walls with neighboring cells but not to the peripheral walls that lack neighboring cells after dark treatment. This dark positioning differed from that of Arabidopsis, in which chloroplasts accumulate at the bottom of the cell in darkness (Suetsugu et al., 2005a). In MpphotKO, the chloroplasts were distributed randomly in cells and did not accumulate along specific cell walls after dark treatment. The defects in dark positioning of phot2 mutants were previously reported for Arabidopsis (Suetsugu et al., 2005a) and A. capillus-veneris (Tsuboi et al., 2007). Thus, these results indicate that regulation of dark positioning by phototropins is conserved in land plants. Intriguingly, in M. polymorpha, only one phototropin (Mpphot) mediates all three types of chloroplast photorelocation movement (i.e. dark positioning, the low-light-induced accumulation response, and the high-light-induced avoidance response; Figs. 1 and 6). In most land plant species, two or more PHOT genes mediate chloroplast photorelocation movement. Although multiple phototropins show some degree of functional redundancy to mediate chloroplast photorelocation movement, they also exhibit functional divergence. For example, in Arabidopsis, although phot1 and phot2 redundantly mediate the accumulation response, phot1 contributes to the accumulation response at a much weaker blue-light intensity compared with phot2 (Sakai et al., 2001). The phot2 protein primarily regulates the avoidance response and dark positioning, whereas the contribution of phot1 to these responses is negligible (Suetsugu et al., 2005a; Luesse et al., 2010). Similar to Arabidopsis, phot2 specifically mediates the avoidance response and dark positioning in the fern A. capillus-veneris (Kagawa et al., 2004; Tsuboi et al., 2007). The moss P. patens has seven PHOT genes categorized into two groups, namely four PpPHOTAs and three PpPHOTBs (Rensing et al., 2008), of which PpphotAs contribute more to the avoidance response than PpphotBs do (Kasahara et al., 2004). Thus, during evolution, land plants acquired multiple functionally differentiated phototropins with which to fine-tune chloroplast movements 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 a single PHOT gene. Thus, liverworts may occupy an ancestral position in the evolution of the photoreceptor system for chloroplast photorelocation movement. 421

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Molecular Mechanisms of Phototropin-Mediated Chloroplast Photorelocation Movement

Our physiological analyses of MpphotKO indicate that Mpphot regulates three types of chloroplast movement. How does Mpphot mediate these different responses? The kinase activity of phototropins is essential for the regulation of chloroplast photorelocation movement (Kagawa et al., 2004; Kong et al., 2006, 2007; Sullivan et al., 2008; Suetsugu et al., 2013). In particular, autophosphorylation in the activation loop of the kinase domain is necessary for induction of chloroplast photorelocation movement (Inoue et al., 2008, 2011; Suetsugu et al., 2013). As shown in Supplemental Figure S2, the kinase domain is highly conserved in phot of M. polymorpha. Two Ser residues in the activation loop that were identified as the essential autophosphorylation sites in Arabidopsis (Inoue et al., 2008, 2011) are conserved in Mpphot. The kinase activity and autophosphorylation of Mpphot is likely to be required for chloroplast photorelocation movement in M. polymorpha as shown in other plants. In this study, we demonstrated the blue-lightinduced autophosphorylation activity of Mpphot in vitro and in vivo (Fig. 8). Because the activity was nullified in MpphotD922N, which carried a kinasenegative amino acid substitution, the kinase activity of Mpphot is necessary for the autophosphorylation. Thus, Mpphot is a blue-light-regulated kinase, similar to other phototropins. Mpphot was slightly autophosphorylated in darkness in vitro, as reported for other phototropins (Matsuoka and Tokutomi, 2005; Jones et al., 2007; Jones and Christie, 2008; Aihara et al., 2012). Autophosphorylation activity may be involved in chloroplast dark positioning, which is mediated by Acphot2 (Tsuboi et al., 2007), Atphot2 (Suetsugu et al., 2005a), and Mpphot. After blue-light-irradiated plants were returned to the dark, autophosphorylated Mpphot was gradually dephosphorylated. Dephosphorylation of Arabidopsis phot2 is implicated in the finetuning of phot2 activity during blue-light-mediated responses (Tseng et al., 2012). The A1 subunit of Ser/Thr protein phosphatase2A (PP2AA1) interacts with phot2 and mediates phot2 dephosphorylation (Tseng et al., 2012). A PP2AA1 homolog is present in the M. polymorpha genome sequences and thus protein phosphatase2A might mediate Mpphot dephosphorylation. Furthermore, the kinase domains of phot1 and phot2 in Arabidopsis are responsible for localization of the proteins on the plasma membrane and specific lightinduced internalization from the plasma membrane (Kong et al., 2006, 2013b; Kaiserli et al., 2009). Similar to other phototropins, Citrine-fusion proteins of Mpphot localized on the plasma membrane (Fig. 7), indicating that Mpphot functions on the plasma membrane. Phototropins of many plant species, including Mpphot, have no transmembrane regions. Thus, it is assumed that phototropins may be anchored to the plasma membrane by interacting with another factor localized in the plasma 422

membrane. The mechanism by which phototropin localizes on the plasma membrane may be common to land plants. Evolution of Phototropin Genes

In our phylogenetic analysis (Fig. 3B), Mpphot was sister to the clade of land plant phototropins. Recent extensive cloning and phylogenetic analysis revealed that duplications of PHOT genes occurred independently in different lineages (i.e. in seed plants as well as in ferns and mosses), and that many liverwort species have a single PHOT (Li et al., 2014), as is the case for M. polymorpha. Mpphot may have retained the ancestral functions of phototropin that were gained before the evolutionary diversification of PHOT genes in land plants. If the ancestral PHOT originated as a single-copy gene, it should have possessed the ability to mediate both the accumulation and avoidance responses. Indeed, the transient expression of Mpphot in the A. capillusveneris phot2 mutant rescued the defects in the avoidance response (Fig. 9), and the expression of Mpphot in the Arabidopsis phot1 phot2 double mutant rescued the defects in both accumulation and avoidance responses (Fig. 10). Importantly, the green alga C. reinhardtii has a single PHOT gene and, similar to Mpphot, the expression of Crphot in the Arabidopsis phot1 phot2 double mutant rescued the defects in both accumulation and avoidance responses (Onodera et al., 2005). Thus, Crphot and Mpphot are phot2-like in that they can mediate both the accumulation and avoidance responses. The chloroplast avoidance response is essential for plant survival under sunlight (Kasahara et al., 2002). During early land plant evolution, the chloroplast avoidance response may have made a greater contribution to plant survival than the accumulation response, because there was no dense canopy to intercept light and the ancestral land plants were directly exposed to sunlight. However, after the explosive evolution and diversification of trees, many plants had to live in shade and thus needed to use the weak light levels under the dense canopy. Duplication of PHOT genes and subsequent acquisition of a weak-lightspecific phototropin, such as the seed plant phot1, is one strategy for adaptation to weak light. Another strategy would be integration of phototropin signaling with the phytochrome system; the acquisition of chimeric photoreceptor neochromes, which consist of a phytochrome chromophore-binding domain and phototropin in ferns and 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 phytochrome and phototropin are implicated in the phytochromesignaling pathway (Kasahara et al., 2004; Jaedicke et al., 2012). We observed that in M. polymorpha chloroplast movement is not induced by red light at any fluence rate examined (Table I) and no neochrome has been identified in the M. polymorpha genome. Thus, the liverwort M. polymorpha is indicated to have the most simple photoreceptor system for chloroplast photorelocation Plant Physiol. Vol. 166, 2014

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

amplified by PCR. The membranes were hybridized in Church hybridization buffer (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 described (Chiyoda et al., 2008).

Phylogenetic Analysis of Phototropins MATERIALS AND METHODS Culture and Growth Conditions of Marchantia polymorpha Male and female accessions of M. polymorpha, Takaragaike (Tak)-1 and Tak-2, respectively, were asexually maintained as previously described (Ishizaki et al., 2008). Plants were incubated on one-half-strength Gamborg’s B5 agar medium (Gamborg et al., 1968) at 22°C under continuous white light (approximately 20 Wm22).

Observation of Chloroplast Photorelocation Movement in M. polymorpha For observation of chloroplast distribution, the marginal region of 3-d-old gemmalings was observed. A single or a few cell layers were present on the marginal region. Plants were cultured on one-half-strength Gamborg’s B5 agar medium at 22°C for 3 d under continuous white light, and then incubated under the different light conditions indicated in the text. A blue LED illuminator (MIL-B18; SANYO Electric), red LED illuminator (MIL-R18; SANYO Electric), and neutral density filter (Smoke 20; Sumitomo 3M) were used. For observation of chloroplast dark positioning, plants were incubated in darkness for 3 d before observation. For microbeam irradiation, gemmalings were transferred to a custom-made cuvette (25 mm in diameter and 5 mm in height) that consisted of two sets of a steel ring and a round glass with a silicone-rubber ring spacer in the dark (Wada et al., 1983). The cuvette containing the gemmaling was placed on the sample stage of a microbeam irradiator (Yatsuhashi and Wada, 1990; Tsuboi et al., 2006; Wada, 2007). Microbeam irradiation was performed as previously described (Yatsuhashi and Wada, 1990; Tsuboi et al., 2006; Wada, 2007) with some modifications. The gemmalings were irradiated with a microbeam at different intensities of red or blue light. Chloroplast movement was observed using infrared light. Samples were prepared under a dim green safe light. The paths of chloroplast movement in response to a red or blue microbeam were traced by taking photographs at 1-min intervals under infrared light. The size of the light-irradiated area was 50 mm 3 10 mm. The resulting images were processed and analyzed with ImageJ version 1.45s software (http://rsbweb.nih.gov/ij/). The fluence of the red- and bluelight microbeams at 1 Wm22 used in these experiments was approximately 5.5 and 3.8 mmol photons m22 s21, respectively.

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

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

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 blotted onto 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, and 1000 bp, respectively) for gene-targeting analysis (Supplemental Fig. S4) were Plant Physiol. Vol. 166, 2014

A multiple alignment of amino acid sequences of phototropins was constructed using the MUSCLE program (Edgar, 2004) implemented in Geneious software (version 6.1.8; Biomatters; http://www.geneious.com/) with default parameters. Unaligned gaps were first removed from the resulting alignment using Gblocks (http://molevol.cmima.csic.es/castresana/Gblocks_server. html), and then the conserved region covering the Ja helix and the C-terminal Ser/Thr kinase domain was extracted before the phylogenetic tree construction, which was performed using Markov chain Monte Carlo simulations by MrBayes 2.0.9 (Huelsenbeck and Ronquist, 2001) implemented in the Geneious software. The parameters used were as follows: rate matrix, blosum; rate variation, 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. The accession numbers of analyzed proteins are as follows: Arabidopsis (Arabidopsis thaliana), 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 capillusveneris, Ac_PHOT1 (BAA95669), Ac_PHOT2 (BAD16730); Selaginella moellendorffii, Sm_PHOT1-1 (EFJ32904), Sm_PHOT1-2 (EFJ15768), Sm_PHOT2-1 (EFJ27458), and Sm_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 is shown in Supplemental Figure S3.

Targeted Gene Knockout of MpPHOT To generate the MpPHOT-targeting vector, pJHY-TMp1 was used (Ishizaki et al., 2013a). The 59 and 39 homology arms (3492 and 3482 bp, respectively) were amplified from genomic DNA by PCR amplification using KOD FX Neo (Toyobo) with the following primer pairs: PHOT-5IF-L/PHOT-5IF-R (59-CTAAGGTAGCGATTAAGTGGTGGCAAACGAGGTAG-39/59-CCGGGCAAGCTTTTACTGGAAAGAAGCGAGAGCAT-39) for the 59 homology arm, and PHOT-3IF-L/PHOT-3IF-R (59-AACACTAGTGGCGCGTCATCATCTACGTCGCTTCG-39/59-TTATCCCTAGGCGCGCGATGCTCTGCGAGACATTA-39) for the 39 homology arm. The PCR products of the 59 and 39 homology arms were cloned into the PacI and AscI sites of pJHY-TMp1, respectively, using the In-Fusion HD Cloning Kit (Clontech). Introduction of the targeting construct into M. polymorpha was performed with Agrobacterium tumefaciens C58C1 GV2260 as previously described (Ishizaki et al., 2008, 2013a). F1 spores generated by crossing Tak-1 and Tak-2 were used for transformation. Isogenic lines (designated as G1 lines) were obtained by isolating gemmae, which develop from single cells, from independent T1 lines (Ishizaki et al., 2012) and were screened for gene-targeted lines (designated as MpphotKO) by genotyping using the method previously described (Ishizaki et al., 2013a) with minor modifications. The PCR program was 94°C for 2 min, followed by 40 cycles of 98°C for 10 s, and 68°C for 5 min. The following primer pairs were used: GT-L2/GT-R3, GT_L0/P1R, and HIF/GT_R5 (59-ATGGGGAGTGCTGATGAAGA-39/59-TCCCTGGAGAAATCGACTGT-39, 59-GAATCTGGCAAGGAGTTCCA-39/59-GAAGGCTTCTGATTGAAGTTTCCTTTTCTG-39 and 59-GTATAATGTATGCTATACGAAGTTATGTTT-39/59-GGCCTAGGAAAGACAACACG-39, respectively). After PCR screening, two independent MpPHOT knockout lines were identified. Plants grown from gemmae of G 1 lines (the G 2 generation) were used for phenotypic analysis and protein blotting.

Complementation Lines of MpphotKO To generate complementation lines of MpphotKO, a binary vector harboring a mALS mutated acetolactate synthase gene that confers chlorosulfuron resistance was used. For construction of a plasmid containing the MpPHOT genomic fragment, the promoter and coding regions were amplified by 423

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PCR using the following primer pairs: PRO_L/PRO_R and Infusion_fw/ Infusion_rv (59-CACCATCCAGCACCATGAGAAGTA-39/59-AAGCTTGGCTCGTCCTGATTT-39 and 59-TCAGGACGAGCCAAGATGATGCCCTCCACGGATTC-39/59-CGCGCCCACCCTTCTGAATTTGACATCCTCCTAAG-39, respectively). The promoter fragment was cloned into pENTR/D-TOPO (Life Technologies). After digestion with HindIII, the amplified coding region was cloned into the HindIII site of the plasmid carrying the promoter fragment with the In-Fusion HD Cloning Kit to generate the plasmid pAI019, which contained the MpPHOT genomic fragment from 5.0-kb upstream of ATG to the 39 untranslated region (total 13 kb). The resultant MpPHOT cassette was cloned into a binary vector using LR Clonase II (Life Technologies) according to the manufacturer’s protocol. Complementation lines gMpPHOT/MpphotKO were generated by transformation of the resulting binary plasmid into regenerating thalli of MpphotKO as previously described (Kubota et al., 2013). The several transformants were obtained through selection with chlorosulfuron and used as gMpPHOT/MpphotKO lines.

Construction of Mpphot Bacterial Expression Vectors The cDNA fragments for the N-terminal region of Mpphot (amino acids 522251) as the antigen and full-length Mpphot were amplified by PCR with oligonucleotide primers for the N-terminal region of Mpphot (59-TTTGGATCCTCAGCTGCGGAAGATGCCTTGG-39/59-TTTTCTAGAGGACGCGCGGCCGGTCGA-39) and for full-length Mpphot (59-TTTACGAGTATGATGGCCCTCCAC-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 kinase activity by Asn in subdomain VII of the kinase domain (Hanks and Hunter, 1995). The amino acid substitutions were introduced by PCR with oligonucleotide primers (59-AATTTCGACCTTTCCTTCTTGAC-39/59-AGTGAGCTGCACATGCCCATCT-39). The fragments were cloned into the modified pMAL-c2 expression vector (New England Biolabs), in which the factor Xa cleavage sequence was replaced by the recognition sequence for PreScission protease (GE Healthcare) and the 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, and MBP-MpphotD922N, the Escherichia coli strain Rosetta2(DE3) was transformed with the respective expression plasmid and induced with 1 mM isopropylb-D-thiogalactopyranoside for 24 h at 18°C. Cells were collected by centrifugation 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 the cells were lysed by sonication, the recombinant proteins in the supernatants were purified by affinity chromatography using amylose resin (New England Biolabs). MBP-Mpphot and MBP-MpphotD922N were used for the in vitro phosphorylation assay. For the preparation of Mpphot antigen, the MBP moiety was removed from MBP-Mpphot(522251)-6xHis with Turbo 3C Protease (Accelagen). Mpphot (522251)-6xHis was purified by affinity chromatography using cOmplete HisTag Purification Resin (Roche) and was used for producing the rabbit polyclonal antibody (KIWA Laboratory Animals).

In Vitro Phosphorylation Assay In vitro kinase activity assay was performed as previously described (Okajima et al., 2011) with some modifications. MBP-Mpphot and MBP-MpphotD922N were incubated 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, and 200 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. Phosphorylation signals were detected with an image analyzer (FLA3000; Fujiflm). For the analysis of blue-light-induced autophosphorylation activity, MBP-Mpphot was irradiated with blue light (18 Wm22) for the first 0, 1, 5, 10, 20, or 30 min of 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 incubated for 3 d in the dark. After various light treatments, plants were frozen and 424

crushed in a mortar. For the experiment in Figure 4B, the homogenates mixed with equal volumes of a lysis buffer (1 mM EDTA, 1 mM DTT, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 100 mM NaCl, 1% (v/v) Triton X-100, and 50 mM Tris-HCl, pH 7.4) were centrifuged at 16,000g for 20 min at 4°C. For the experiments in Figure 8, C and D, the homogenates mixed with 23 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 and then centrifuged at 16,000g for 20 min at room temperature. The supernatants were subjected to immunoblot analysis with anti-Mpphot antibody as described below. Membrane fractionation was performed as previously described (Ishizaki et al., 2013b) with some modifications. Plants were grown under continuous white light for 14 d and then incubated in darkness for 3 d. Seven g of plant tissue were crushed in a mortar. The crushed tissues were mixed with 25 mL of homogenization buffer containing 500 mM Suc, 10% (v/v) glycerol, 20 mM EDTA, 20 mM EGTA, 50 mM sodium fluoride, 1% (w/v) polyvinylpyrrolidone, 10 mM ascorbic acid, 2 mM DTT, 3 mM phenylmethylsulfonyl fluoride, cOmplete Mini EDTA-Free Protease Inhibitor, and 50 mM Tris-MES, pH 8.0. The homogenates were filtered through a cell strainer (70-mm nylon; BD Biosciences). After centrifugation of the filtrate at 3,000g for 10 min at 4°C, the supernatant (total protein fraction) was further centrifuged at 100,000g for 60 min at 4°C to separate the supernatant (cytosolic fraction) and the membranecontaining precipitate. The precipitate was washed two times in homogenization buffer 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, and 62.5 mM Tris-HCl, pH 6.8) to generate the membrane fraction. Each fraction was subjected 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) for the experiment in Figure 8, C and D, and in the standard 8% (w/v) SDS-PAGE gels for other experiments. The proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories), and detected with various antibodies. 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 (GE Healthcare) was diluted to 1:10,000. Signals were detected using the ECL Plus western-blotting detection system (GE Healthcare) and the ImageQuant LAS4010 digital imaging system (GE Healthcare).

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

Analysis of Chloroplast Photorelocation Movement in MpPHOT-Transfected Cells of A. capillus-veneris phot2 Prothalli MpPHOT cDNA amplified with primers (59-CACCATGATGCCCTCCAC39/59-TTAATATTCATCAAATGAGGCGG-39) was cloned into a 35S gateway destination vector. The 35S:MpPHOT and 35S:GFP vectors were cotransfected into the phot2-deficient A. capillus-veneris prothalli by particle bombardment as previously described (Kagawa et al., 2004). GFP fluorescence was used as a marker to identify transfected cells. The MpPHOT-transfected cell (i.e. GFPpositive cells) and the adjacent nontransfected cell were irradiated with a strong blue-light microbeam. The chloroplast avoidance response induced by high-fluence blue light (15 Wm22) was recorded at 1-min intervals.

Analysis of Chloroplast Photorelocation Movement in MpPHOT-Transformed phot1 phot2 Mutant of Arabidopsis The MpPHOT cDNA was subcloned into the pGWB2 vector by the LR reaction of the Gateway system (Life Technologies). The construct was introduced into the Arabidopsis phot1 phot2 mutant (phot1-5 phot2-1) using Plant Physiol. Vol. 166, 2014

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Agrobacterium-mediated transformation. Analysis of chloroplast movement using leaf transmittance was performed as previously described (Wada and Kong, 2011). The red light (650 nm) transmittance was automatically recorded every 2 min using a microplate reader (VersaMax; Molecular Devices). The values shown 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 phototropins from M. polymorpha, Arabidopsis, and C. reinhardtii. Supplemental Figure S3. Alignment of amino acid sequences of phototropins from a variety of plant species used for the phylogenetic analysis in Figure 3B. Supplemental Figure S4. Strategy for targeted disruption of the MpPHOT locus and analysis of homologous recombination events. Supplemental Figure S5. Images of 20-d-old thalli of the wild type and MpphotKO. Supplemental Figure S6. Comparison of the ratio of the area occupied with 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, and Yukiko 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.

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