Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the Liverwort Marchantia polymorpha

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 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, 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, 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-light-dependent 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-light-dependent 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 chromophore-binding domain of phytochrome and complete phototropin domains (Nozue et al., 1998). Neochrome is the 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-light-induced 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 phototropin-mediated 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 analyzed the molecular mechanism underlying the photoreceptor system through molecular genetic analysis of M. polymorpha phototropin.