Relationship between relocation of phototropin to the chloroplast periphery and the initiation of chloroplast movement in Marchantia polymorpha.

Abstract The blue‐light photoreceptor kinase phototropin (phot) mediates chloroplast movement in response to light and temperature. Phot predominantly localizes at the plasma membrane, but also resides in the cytosol and the chloroplast periphery. Although the phot localized to the chloroplast periphery is thought to mediate chloroplast movement, the localization mechanism is unknown. In this study, we found that chloroplast movement does not occur in 0‐day‐old gemma cells of the liverwort Marchantia polymorpha but that the movement is induced in 1‐day‐old gemmaling cells. Along with this physiological change, the subcellular localization of phot also changed: In 0‐day‐old gemma cells, phot localized at the plasma membrane and the cytosol, but in 1‐day‐old gemmaling cells, the phot disappeared from the cytosol and appeared at the chloroplast periphery. When the relocalization was tracked using a photoconvertible fluorescent protein, the cytosolic phot relocated to the plasma membrane, and the plasma membrane‐resident phot relocated to the chloroplast periphery. The blue‐light‐dependent activation of phot kinase activity enhanced this relocalization. Mutated phot deficient in blue‐light reception or kinase activity had a severely reduced ability to localize at the chloroplast periphery. These findings suggest that photoactivated phot localizes at the chloroplast periphery to initiate chloroplast movement.

tion, and cold-avoidance responses are considered to share signaling pathways mediated by phot (Fujii et al., 2017;Kodama et al., 2008).
Phot is a blue-light (BL) photoreceptor kinase, which mediates various physiological responses such as phototropism, stomatal opening, and chloroplast movement (Huala et al., 1997;Jarillo et al., 2001;Kagawa et al., 2001;Kinoshita et al., 2001;Sakai et al., 2001). For example, A. thaliana has two phot proteins, Atphot1 and Atphot2, which redundantly mediate the accumulation response; in addition, Atphot2 mediates the avoidance response and the coldavoidance response (Fujii et al., 2017;Jarillo et al., 2001;Sakai et al., 2001). In M. polymorpha, phot is encoded by a single-copy gene (Mpphot). Because Mpphot mediates both the accumulation and avoidance responses, the function of Mpphot is similar to that of A. thaliana Atphot2, but not Atphot1 (Komatsu et al., 2014). In M. polymorpha, Mpphot was discovered to have thermosensory functions in the cold-avoidance response (Fujii et al., 2017). Phot has two light-oxygen-voltage (LOV) domains at the Nterminal region, and the LOV domains perceive BL and temperature changes (Christie, 2007;Fujii et al., 2017). After perceiving BL and low temperature, the LOV domains stimulate activity of the C-terminal serine/threonine kinase domain. The kinase domain induces autophosphorylation, which mediates the intracellular positioning of the chloroplasts. The phot kinase activity is essential for inducing chloroplast movement in A. thaliana (Inoue et al., 2011;Kong et al., 2007). However, the intracellular process underlying the role of phot after BL-dependent kinase activation remains to be determined.
In this study, we observed behaviors of chloroplasts and Mpphot fused with a fluorescent protein in gemma cells of M. polymorpha and found relocalization of Mpphot to the chloroplast periphery for initiation of chloroplast movement. This study provides new insight into a phot-mediated intracellular process in plants.
Gemmae were obtained from gemma cups of approximately 1-month-old Tak-1 or transgenic thalli (G 1 generation) and used as the 0-day-old sample. After gemmae culture on agar for 24 hr, the gemmalings (immature thalli grown from gemmae) were used as the 1-day-old sample. Cytosolic localization of Mpphot-Citrine (or Mpphot-Dendra2) in the 0-day-old cells was observed in almost all gemmae, and the cytosolic localization was completely gone after a 1-day culture. However, in our preliminary experiments, we found that Mpphot-Citrine was slightly localized at the chloroplast periphery in some 0-day-old gemmae ( Figure S1). This variation was expected to be dependent on the position of the gemmae in the gemma cup on the thallus. In M. polymorpha, gemmae originate from a single cell at the bottom of the gemma cup and develop in the gemma cup (Barnes & Land, 1908;Ishizaki, Nonomura, Kato, Yamato, & Kohchi, 2012). The bottom of the gemma cup accumulates hormones, such as auxin, which may stimulate gemma dormancy (Ishizaki et al., 2012). However, during development, gemmae are detached from the bottom of the gemma cup and older gemmae are pushed up into the upper position by newly forming gemmae. The gemmae in the upper position can retain dormancy. However, because the gemmae in the upper position are exposed to light, this could explain why some Mpphot-Citrine is slightly localized at the chloroplast periphery in some 0-day-old gemmae. Based on this expectation, in all experiments of this study, we obtained gemmae from the bottom of the gemma cup when possible. When we took gemmae from this position, we did not observe chloroplast-peripheral localization of Mpphot-Citrine in the cells from 0-day-old gemmae.

| Temperature-regulated microscope with a blue-light microbeam
For observation of chloroplast movement, gemmae were cultured on agar under 70 µmol photons/m 2 s -1 of continuous white fluorescent light (FL40SW, NEC Corporation) at 22°C and then observed using a temperature-regulated microscope, which is based on an inverted light microscope (Leica DM IL LED), with a BL microbeam (Fujii et al., 2017;Tanaka et al., 2017). To avoid the motion of the gemmae under the microscope, we used a fast-forming hydrogel with an ultra-low polymeric component (Hayashi et al., 2017;Sakai et al., 2008). A white LED of the inverted light microscope with a red filter (No. 21, Tokyo Butai Showmei Co Ltd) was used as a red light source for observation light. To induce the avoidance response, we used a BL microbeam of 10 W/m 2 (approximately 430 µmol photons/m 2 s -1 ) at 22°C. For induction of the accumulation and cold-avoidance responses, we used a BL microbeam of 1 W/m 2 (approximately 30 µmol photons/m 2 s -1 ) at 22°C and 5°C, respectively. ImageJ software was used to measure the distances that the chloroplasts moved.
Mpphot-Dendra2 was designed as a Gateway entry clone by us and synthesized by Fasmac Co Ltd. The resulting plasmid (pUCFa vector backbone: Fasmac Co Ltd) was mixed with the binary vector pMpGWB302 (Ishizaki et al., 2015), and the LR reaction (Gateway cloning technology; Invitrogen) was performed. The sequence of Mpphot-Dendra2 in the pUCFa vector is shown in Figure S2.

| Genetic transformation of M. polymorpha
Agrobacterium-mediated genetic transformation of M. polymorpha was performed by the G-AgarTrap method as described in our previous studies Tsuboyama, Nonaka, Ezura, & Kodama, 2018;Tsuboyama-Tanaka & Kodama, 2015). In this study, the Mpphot KO mutant line (Komatsu et al., 2014) was used as the genetic background to produce all transformants. Transgenic G2 gemmae were used for all experiments.

| Confocal microscopy
To observe Mpphot-Citrine, Mpphot-Dendra2, calcofluor white, and chlorophyll autofluorescence in gemma cells, we used a SP8X Fluorescence intensity of DenR at the plasma membrane (n = 10) and the chloroplast periphery (n = 10) was measured using ImageJ.
For statistical analysis, Tukey's test was performed. The time-gated method was used to block chlorophyll fluorescence in the imaging of Citrine and Dendra2 with a detection time of 0.5 to 12.0 ns (Kodama, 2016). To visualize cell walls, gemmae were stained with 1 µg/ml calcofluor white (Sigma-Aldrich). A 405-nm laser was used for excitation, and a range of 456 to 508 nm was detected by a hybrid detector. For observation of chlorophyll autofluorescence, a wavelength range of 650 to 700 nm (when Citrine observation) or 679 to 755 nm (when calcofluor white observation) was detected by a photomultiplier tube.

| Cyt/CP ratio
To quantify the subcellular localization of Mpphot-Citrine, images of Mpphot-Citrine expressed in gemmae were captured with a SP8X microscope system (Leica Microsystems). Fluorescence intensities in the cytosol (Cyt) and at the chloroplast periphery (CP) were measured using ImageJ. For the CP signal, a line (35 pixels) was drawn Cyt. The average of the Cyt signals was divided by the average of the CP signals to obtain a Cyt/CP ratio ( Figure S3d). The procedures were repeated three times, and the average and standard deviation were calculated. For statistical analysis, Tukey's test was performed.
When Mpphot-Citrine is located in the cytosol, the Cyt/CP ratio is close to 1; when Mpphot-Citrine is located at the chloroplast periphery, the ratio is close to 0 ( Figure S3d).

| Chloroplasts anchor to the plasma membrane during gemma culture
In a previous study with M. polymorpha, we used 1-day-old gemmalings (thallus developed from gemma) to analyze chloroplast movement (Ogasawara et al., 2013). Chloroplasts were dispersed throughout the cells in 0-day-old gemmae immediately after detaching from the gemma cup of the thalli (Ogasawara et al., 2013). After a 1-day culture (24 hr) under continuous weak white light at 22°C, the chloroplasts clearly accumulated in the periclinal position (Ogasawara et al., 2013). To further explore this phenomenon, we observed chloroplasts in cells of 0-day-old gemmae and 1-day-old gemmalings by confocal microscopy. To visualize cell shape, calcofluor white was used to stain the cell walls. Chlorophyll autofluorescence in the cells of 0-day-old gemmae showed that chloroplasts localized in an irregular pattern along the periclinal wall; scanning in the X-Z direction revealed that chloroplast distribution was random, suggesting that they were not attached to the plasma membrane (Figure 1a,b). By contrast, in the cells of 1-day-old gemmalings, the chloroplasts localized in a regular pattern along the periclinal wall, likely anchoring to the plasma membrane ( Figure 1a,b). These results suggested that chloroplasts approach the plasma membrane during the first day of gemma culture.

| Chloroplasts relocated during gemma culture
Chloroplasts must anchor to the plasma membrane in order to relocate in the cell (Oikawa et al., 2008). We analyzed the three types of chloroplast movement (accumulation, avoidance, and cold-avoidance responses) in cells of 0-day-old gemmae and 1-day-old gemmalings by time-lapse video recording using temperature-regulated microscopy with BL microbeam irradiation (Fujii et al., 2017;Tanaka et al., 2017).
To avoid undesired movement of the gemmae/gemmalings under the microscope, we used a fast-forming hydrogel with an ultra-low polymeric component, as described previously (Hayashi et al., 2017;Sakai et al., 2008). To quantify chloroplast movement, we determined how long it takes for three chloroplasts to move away from the microbeam

| Changes in the subcellular localization of Mpphot-Citrine
Because previous studies reported that the subcellular localization of phot is important for the regulation of chloroplast movement (Kong, Kagawa, Wada, & Nagatani, 2013;Kong et al., 2007;Kong, Suetsugu, et al., 2013), we next observed the subcellular localization of M. polymorpha phot fused with Citrine yellow fluorescent protein (Mpphot-Citrine) in the cells of 0-day-old gemmae and 1-day-old gemmalings. In our previous study with transgenic plants of M. polymorpha, Mpphot-Citrine localized at the plasma membrane and at the chloroplast periphery in cells of 1-day-old gemmalings (Kodama, 2016). This result was reproduced in this study (Figure 3). In the cells of 0-day-old gemmae, by contrast, Mpphot-Citrine was not found at the chloroplast periphery but was observed at the plasma membrane and in the cytosol (Figure 3). After the 1-day culture, Mpphot disappeared from the cytosol, but appeared at the chloroplast periphery.
To follow up on these qualitative observations, we developed a method to quantitatively evaluate the localization of Mpphot-Citrine in the cytosol or at the chloroplast periphery, based on measurements of Citrine fluorescence. To this end, we measured the fluorescence intensities in the cytosol (Cyt) and the outside edge of the chloroplast periphery (Cp) from images captured by a confocal microscope with the time-gating method (Kodama, 2016) and calculated the Cyt/CP ratio. A detailed procedure for calculating the Cyt/CP ratio is described in the EXPERIMENTAL PROCEDURES ( Figure S3).
If Mpphot-Citrine localizes in the cytosol in the 0-day-old gemma cells (0 hr) (Figure 4a), the averaged Cyt/CP ratio is close to 1 (Figure 4b). In contrast, if Mpphot-Citrine localizes at the chloroplast periphery in the 1-day-old gemmaling cells (24 hr) (Figure 4a), the averaged Cyt/CP ratio is close to 0 (Figure 4b). When the Cyt/ CP ratio was evaluated at several time points during the 1-day culture under white light (WL), a 6-hr culture was required to complete the relocalization of Mpphot from the cytosol to the chloroplast periphery (Figure 4c,d). Therefore, Mpphot does not accumulate at the chloroplast periphery in cells of 0-day-old gemmae, but it does accumulate at the chloroplast periphery after 6 hr of culture under WL.
This method represents a simple procedure to induce and quantify

| Enhancement of chloroplast-peripheral localization of Mpphot by BL
Because Mpphot is a BL photoreceptor, we investigated the light dependency of its relocalization. The relocalization of Mpphot was induced when gemmae were cultured for 6 hr under WL and when cultured in the dark (Figure 5a,b). However, the Cyt/CP ratio revealed that the amount of chloroplast periphery-localized Mpphot under WL was much higher than that in the dark condition (Figure 5a,b).
These results suggested that WL enhances the chloroplast-peripheral localization of Mpphot to a greater extent than darkness.
Next, we examined the response of Mpphot to different wavelengths of light. When gemmae were cultured for 6 hr under BL or red light (RL), the amount of chloroplast-peripheral Mpphot under BL was similar to that under WL (Figure 5a,b), whereas the amount of chloroplast-peripheral Mpphot under RL was similar to that under the dark conditions (Figure 5a,b). When we observed Mpphot-Citrine localization for 24 hr under darkness and RL, more Mpphot-Citrine eventually localized at the chloroplast periphery ( Figure S4). Therefore, BL enhances the speed of chloroplast-peripheral localization of Mpphot.

| Photoreception is involved in the chloroplastperipheral localization of Mpphot
Mpphot has two photoreceptor domains, LOV1 and LOV2, in the Nterminal region (Figure 6a), and amino acid substitutions of cysteine F I G U R E 3 Changes in the subcellular localization of Mpphot-Citrine. Representative images of the subcellular localization of Mpphot-Citrine in cells from 0-day-old gemmae and 1-day-old gemmalings. Bars = 10 µm with alanine (C328A and C628A) completely disrupt its ability to perceive BL (Fujii et al., 2017). To test whether BL-dependent en-  (Figure 6a). When the lov1-LOV2 cells were cultured for 6 hr under BL, Citrine fluorescence was still observed in the cytosol (Figure 6b). When the Cyt/CP ratio was measured, it was slightly higher than that of LOV1-LOV2 cells cultured for 6 hr under BL (Figure 6c). Similarly, when the LOV1-lov2 cells were cultured for 6 hr under BL, Citrine fluorescence was also observed in the cytosol (Figure 6b). The Cyt/CP ratio of LOV1-lov2 cells was similar to that of the lov1-LOV2 cells (Figure 6c). When the lov1-lov2 cells were cultured for 6 hr under BL, abundant Citrine fluorescence was observed in the cytosol (Figure 6b), and the Cyt/ CP ratio was higher than those of the lov1-LOV2 and LOV1-lov2 cells ( Figure 6c). When we observed lov1-LOV2, LOV1-lov2, and lov1-lov2 cells for 24 hr under BL, Mpphot C328A -Citrine, Mpphot C628A -Citrine, and Mpphot C328A/C628A -Citrine eventually localized at the chloroplast periphery ( Figure S5). These results indicate that the LOV1 and LOV2 domains are partially responsible for the BL-dependent enhancement of chloroplast-peripheral localization of Mpphot.

| The kinase activity of Mpphot enhances its chloroplast-peripheral localization
Mpphot has a serine/threonine kinase domain at the C-terminal region (Figure 6a), and the kinase activity is increased via perception of BL by the LOV domains (Fujii et al., 2017;Komatsu et al., 2014). To test whether the kinase activity promotes chloroplast-peripheral localization of Mpphot, we used a mutation (D922N) that disrupts the kinase activity of Mpphot (Komatsu et al., 2014). We  (Figure 6d). The Cyt/CP ratio was much higher than that of the lov1-lov2 cells cultured for 6 hr under BL, but slightly lower (statistically nonsignificant) than that of the 0-day-old gemma cells (Figure 6c). When we observed the Kinase-Dead cells for 24 hr under BL, Mpphot D922N -Citrine also eventually localized at the chloroplast periphery ( Figure S5). These results indicated that kinase activity is not essential for chloroplast-peripheral localization of Mpphot but strongly contributes to its BL-dependent enhancement of chloroplast-peripheral localization.
Dendra2 is a monomeric fluorescent protein that can be photoconverted from a green fluorescent form (DenG) to a red fluorescent form (DenR) upon UV or BL irradiation, for example, 405 or 490 nm, respectively (Chudakov et al., 2007). When the fusion construct

| A hypothesis for the functional role of Mpphot localization in M. polymorpha
Taking these observations together, we found that in the gemma cells within the gemma cup of M. polymorpha, Mpphot localizes at the plasma membrane and in the cytosol. After the gemma is extruded from the cup, the subcellular localization of Mpphot changes to the plasma membrane and chloroplast periphery within a period of several hours to 1 day; Mpphot relocates from the cytosol to the plasma membrane, followed by a relocation from the plasma membrane to the chloroplast periphery. This change in localization is enhanced by the BL-dependent activation of Mpphot kinase activity. When Mpphot localizes at the chloroplast periphery, chloroplast movement, such as the accumulation, avoidance, and cold-avoidance responses, are induced to optimize photosynthesis.

| D ISCUSS I ON
In this study, we found that the relocalization of Mpphot to the chloroplast periphery possibly increases with chloroplast movement in gemma cells of M. polymorpha. We observed that photoactivation of Mpphot promotes its relocalization to the chloroplast periphery, which is enhanced by the BL-dependent Mpphot kinase activity.
Furthermore, we found that Mpphot relocates from the cytosol to the plasma membrane, followed by a relocation from the plasma membrane to the chloroplast periphery. These findings reveal the intracellular changes that phot undergoes after BL-dependent kinase activation, which may function in the induction of chloroplast movement. Many studies have focused on changes in the subcellular localization of phot from the plasma membrane to cytoplasmic compartments, such as the cytosol, Golgi apparatus, and chloroplast periphery, and investigated the functional roles of this relocation (Liscum, 2016). However, the importance of the relocation of phot remains to be determined (Liscum, 2016). In A. thaliana, cytosolic Atphot1 and Atphot2 are not essential for chloroplast movement (Kong, Suetsugu, et al., 2013;Preuten, Blackwood, Christie, & Fankhauser, 2015). In this study, we found that Mpphot predominantly localizes at the plasma membrane and in the cytosol in  (Kong, Suetsugu, et al., 2013;Preuten et al., 2015).
However, cytosolic Atphot2 that has a C-terminal deletion could induce the accumulation response, but not the avoidance response, in A. thaliana (Kong, Kagawa, et al., 2013;Kong, Suetsugu, et al., 2013); therefore, further study is needed to clarify the contribution of cytosolic Mpphot in the accumulation response in M. polymorpha.
Our present data suggested that Mpphot that is localized at the chloroplast periphery mediates the induction of chloroplast movement (the accumulation, avoidance, and cold-avoidance re- from the gemma cup, expression or localization of MpNCH may change to initiate the accumulation response. Actin filaments also function in chloroplast movement. In M. polymorpha, actin filaments mediate the accumulation, avoidance, and cold-avoidance responses (Kimura & Kodama, 2016). Their organization may change during gemma growth. Further studies are needed to find the relationship between protein behavior and the initialization of chloroplast movement in M. polymorpha.
Previous studies reported the intracellular relocation of A. thaliana phot; Atphot1 and Atphot2 localize at the plasma membrane in the dark, and upon BL irradiation, Atphot1 relocates to the cytosol and Atphot2 to the Golgi apparatus (Kong et al., 2006;Sakamoto & Briggs, 2002). This study in M. polymorpha found that relocation of Mpphot from the cytosol to the chloroplast periphery involves an intermediate localization to the plasma membrane, indicating both a new direction (from the cytosol to the plasma membrane) and a new route (from the plasma membrane to the chloroplast periphery) for intracellular relocation of phot. In A. thaliana mesophyll cells, Atphot1 and Atphot2 localize at the chloroplast periphery (Kong, Suetsugu, et al., 2013). According to the present study, Atphot1 and Atphot2 seem to relocate from the plasma membrane to the chloro-  Figure S6). These punctate structures may include the Golgi apparatus; further study will be needed to determine the precise localization. In contrast to the requirement for phot2 kinase activity to localize at the Golgi apparatus observed in previous studies (Aggarwal et al., 2014;Kong et al., 2006), this study found that BL-dependent kinase activation is not essential for the localization of Mpphot at the chloroplast periphery. Although the plant species used are different, the results from these two studies suggest that the molecular mechanism for phot movement from the plasma membrane to the chloroplast periphery may differ from the mechanism for localization to the Golgi apparatus.
The BL-dependent kinase activation of Atphot2 stimulates downstream factors that induce various physiological responses, including chloroplast relocation. In a previous study with A. thaliana, the Kinase-Dead Atphot2 D720N mutant was unable to induce the avoidance and accumulation responses (Inoue et al., 2011;Kong et al., 2007). The requirement for kinase activity was similar in M. polymorpha, where the avoidance response was not induced in the cells of 1-day-old gemmalings with Kinase-Dead Mpphot ( Figure S7). In addition, we found that BL-dependent kinase activation enhanced the localization of Mpphot to the chloroplast periphery, which occurred with chloroplast movement. This is a novel insight on the intracellular process of BL-dependent kinase activation of phot2type proteins (i.e., Mpphot) in plant cells.
Finally, we hypothesized about the physiological significance of BL-dependent enhancement of the localization of Mpphot at the chloroplast periphery in M. polymorpha. In gemma cells, the localization of Mpphot at the chloroplast periphery occurred under darkness and RL, but it was promoted to a greater extent under BL. Thus, the chloroplast-peripheral localization of Mpphot is induced in a light-independent manner, but the speed of the localization is enhanced by BL. The BL-dependent enhancement of this relocalization of Mpphot may rapidly induce chloroplast movement in gemmae that have been extruded from the gemma cup during the daytime (i.e., BL). In gemmae that are extruded from the cup during the nighttime (i.e., darkness or RL), Mpphot slowly relocates to the chloroplast periphery because rapid induction of chloroplast movement is not required.

ACK N OWLED G M ENTS
We thank Dr. Takayuki Kohchi (Kyoto University, Japan) and Dr.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.