Red Light-Induced Phytochrome Relocation into the Nucleus in Adiantum capillus-veneris

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1 RESEARCH ARTICLE Red Light-Induced Phytochrome Relocation into the Nucleus in Adiantum capillus-veneris Hidenori Tsuboi a, Sachihiko Nakamura b, Eberhard Schäfer c and Masamitsu Wada a,b,c,d,1 a Kyushu University, Hakozaki 6 1 1, Fukuoka , Japan b Tokyo Metropolitan University, Minami-Osawa 1 1, Hachioji, Tokyo , Japan c Biologie II/Institut für Botanik, University of Freiburg, D-7914 Freiburg, Germany d Freiburg Institute for Advanced Studies, School of Life Sciences, Freiburg, Germany ABSTRACT Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. Intracellular distribution of PHY GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphy1, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination. Key words: Adiantum capillus-veneris; far-red light; microbeam; phytochrome; red light; spore germination. INTRODUCTION It has long been known that seed germination is promoted by red light (R) and inhibited by far-red light (FR) since Borthwick and his coworkers (1952) research with Grand Rapids lettuce seeds. Almost 1% of the seeds that received a short R pulse germinated, but the R effect was fully reversed by a following short FR treatment. The R and FR effects were repeatedly reversible, as a hallmark of phytochrome involvement. The light dependency of fern spore germination was also well documented. R in the nm region most effectively induced spore germination, whereas subsequent irradiation with FR at nm, far ultra violet light at 26 nm, near ultra violet light (UV) at 38 nm, and blue (B) at 44 nm inhibited the R- induced germination in Pteris vittata (Sugai and Furuya, 1967; Sugai, 1971; Sugai et al., 1984; Sugai and Furuya, 199). The inhibition of R-induced spore germination by UV and B was also shown in Adiantum capillus-veneris L. (Sugai and Furuya, 1985). In Arabidopsis thaliana, PHYA and PHYB among five phytochrome genes (PHYA E) play the main roles in seed germination (Sharrock and Quail, 1989; Clack et al., 1994). PhyA mediates seed germination at a very low fluence of monochromatic light ranging from 3 to 78 nm, and this phya-dependent response is not reversible by FR. Seed germination regulated by phyb is photoreversible by alternate irradiations with either or nm under the fluence rates of.1 1 lmol m 2 (Shinomura et al., 1996). Interestingly, phye is required for germination under continuous FR irradiation (Hennig et al., 22). Phytochromes are mainly localized in the cytosol but partially diffused in a nucleus under dark conditions, and transported into nucleus under light and formed speckles (Yamaguchi et al., 1999; Gil et al., 2; Hisada et al., 2; Kim et al., 2; Kircher et al., 22; Oka et al., 28). A single, brief (;5 min) FR, R, or B pulse, or white light induced nuclear import of a phya::gfp fusion protein within a few minutes after the inductive light pulse in etiolated seedlings of A. thaliana (Kim et al., 2; Kircher et al., 22). Nuclear import of a phyb::gfp fusion protein in etiolated seedlings was, however, insensitive to single red, blue, and far-red pulse, although it was induced by continuous R irradiation (Gil et al., 2; Kircher et al., 22). Although light induced phyb translocation into the nucleus, the phyb import was at least one order of magnitude slower than that of phya and reached its maximum level after 6 h (Kircher et al., 22). Considering the difference of 1 To whom correspondence should be addressed at address a. wadascb@kyushu-u.org, tel , fax ª The Author 212. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: 1.193/mp/ssr119 Received 2 October 211; accepted 11 December 211

2 612 Tsuboi et al. transport speed into the nuclei between the phya and phyb, very fast accumulation of phya in nuclei should require an efficient active transport system for the molecule to be moved into the nucleus. It was reported that the nuclear accumulation of phya required at least two small, plant-specific proteins: FAR RED ELONGATED HYPOCOTYL 1 (FHY1) and its homologue FHY1 LIKE (FHL) (Hiltbrunner et al., 25; Zhou et al., 25). AtFHY1 was sufficient for AtphyA transport into nucleus in isolated nuclei of Acetabularia acetabulum (Pfeiffer et al., 29). FHY1 and FHL were shown to interact with phya and to be positive regulators of phya signaling during deetiolation (Desnos et al., 21; Zhou et al., 25). Recently, it was shown in fhy1/fhl double mutant plants that the light-induced nuclear accumulation of phya was completely abolished, suggesting an important role of these two proteins in the nuclear-transport mechanism for phya (Hiltbrunner et al., 26). Other components of physical interaction of phytochrome are a small subset of constitutively nuclear, basic helix loop helix (bhlh) transcription factors, named Phytochrome- Interacting Factors (PIFs) (Castillon et al., 27). The PIF proteins was initially identified by a yeast two-hybrid screen as phyb-interacting proteins, and subsequently shown to bind phytochrome, in a photoreversible fashion, to the Pfr form of both phya and phyb (Ni et al., 1999; Shimizu-Sato et al., 22). Thus, the phytochrome property of translocation from cytoplasm to nuclei in Arabidopsis has been well established. Moreover, the functions of phytochrome-interacting factors such as FHY1 and FHL or PIFs were resolved at the molecular level. However, the precise experiments showing translocation of functional phytochrome from cytoplasm to nuclei has not yet been performed at cellular level. Here, we applied the partial cell irradiation technique (Wada and Furuya, 1978; Wada, 28; Tsuboi and Wada, 211a, 211b, 211c) on the spore germination of A. capillus-veneris to determine whether physiologically functional phytochromes migrate towards the nuclear region and move into nuclei. Whether Adiantum phy1, phy2, or both mediates the spore germination was also investigated. RESULTS It has been known that R-induced fern spore germination obeys the reciprocity law (Sugai and Furuya, 1967, 1985). Hence, to know the fluence for the maximum germination rate in A. capillus-veneris spores, the fluence response relationship was obtained with the constant fluence rate of 4.2 W m 2 of R and different irradiation periods for 3 24 s by whole spore irradiation with a microbeam ( lm). Germination rate was determined at 5 d after the light irradiation (Figure 1A). Regardless of the length of the irradiation up to 6 s, the germination rate was proportional to the total fluence. As the spore germination rate showed the saturation at 6% with 252 J m 2 (4.2 W m 2 for 6 s: Figure 1A, arrow), this fluence was used for the following experiments. A fluence response curve for FR reversibility on the R-induced spore germination was obtained (Figure 1B). Immediately after A Germination rate (%) B Germination rate (%) C Germination rate (%) (days) R Fluence (J m ) Fluence (J m ) 5 R FR X hour Time (hour) (days) R FR (days) Figure 1. Fluence Response Relationship for Spore Germination. (A) Spores imbibed for 5 d under darkness were irradiated with a R microbeam ( lm) with 4.2 W m 2 for various periods of time (from 3 to 24 s). Arrow indicates the fluence (252 J m 2 ) used for the following experiments. (B) Fluence response relationship for FR reversibility on the R- induced spore germination. Whole spore was irradiated with 4.2 W m 2 of R microbeam ( lm) followed by an FR microbeam ( lm) irradiation for various periods of time (from 3 to 48 s). Arrow indicates the fluence (144 J m 2 ) used for the following experiments. (C) Escape response from FR reversibility in R-induced spore germination. Spores were irradiated with an R microbeam ( lm; 4.2 W m 2 for 6 s) and then incubated in the dark for various periods of time from to 12 h. Then, the spores were irradiated with an FR microbeam ( lm; 6. W m 2 for 24 s) and incubated in the dark for 5 d. Each point represents the mean 6 SE determined from two or three experiments. More than 1 spores were counted in each experiment. 12

3 Tsuboi et al. 613 whole-spore irradiation with a R microbeam ( lm) of 4.2 W m 2 for 6 s, the spores were irradiated with a FR microbeam ( lm) of 6. W m 2 for 3 48 s. The R-induced germination was almost completely eliminated by 144 J m 2 (6. W m 2 for 24 s: Figure 1B, arrow) of FR, so that this fluence was used for subsequent experiments. To investigate an escape response from FR reversibility, spore germination was induced by 4.2 W m 2 R for 6 s and then the whole spore area was treated with 6. W m 2 FR for 24 s after different dark-incubation periods( 12 h). As showninfigure1c, FRirradiation completely reversed the R effect on the induction of spore germination if given immediately after the R irradiation. If FR was given later than 4 h after the R treatment, the FR reversible effect was completely lost. Next, the effect of partial spore irradiation on spore germination was examined with an R microbeam. Either a nuclear region or a part of cytoplasmic area of a spore imbibed for 5 d under darkness (Figure 2A, inset) was irradiated with an R microbeam (circle, 12 lm in diameter) of 4.2 W m 2 for 6 s (Figure 2A). Chlorophyll auto-fluorescence of chloroplasts was defined as spore germination. About 6% of spores germinated following both treatments, although nearly 1% spores germinated if whole spore was irradiation with the microbeam of the same fluence, suggesting that the phytochrome that mediated spore germination existed equally in the nuclear and cytoplasmic regions under dark conditions. Next, the effect of partial spore irradiation with FR on the FR reversibility of R-induced spore germination was tested (Figure 2B). Whole spores were pre-irradiated with R of 4.2 W m 2 for 6 s, and then FR microbeam (circle, 12 lm in diameter) of 6. W m 2 was immediately given for 24 s at either the nuclear region or a part of the cytoplasmic region (Figure 2B, white bars) or 2-h darkness (Figure 2B, gray bars) after the R treatment. The FR irradiation at the nuclear region was much more effective for the inhibition of spore germination even immediately after the whole R irradiation than the irradiation on the cytoplasmic area, where the FR had almost no effect, suggesting that the phytochrome for spore germination works in the nuclear region. Furthermore, the FR microbeam given 2 h after R was more effective than that given immediately after R, indicating the migration of phytochrome molecules to the nuclear region during the period (Figure 2B). To apply another test for phytochrome accumulation in the nuclear region as Pfr, either a nucleus or a part of the cytoplasmic area was irradiated with an R microbeam (circle, 12 lm in diameter) of 4.2 W m 2 for 6 s after the whole spore had first been irradiated with 4.2 W m 2 R for 6 s, incubated for 2 h in the dark, and then irradiated with 6. W m 2 FR for 24 s (Figure 2C). The irradiation on the nuclear region was more effective than that on a cytoplasmic region, indicating that the phytochrome in the nuclear region is involved in the spore germination. To determine which phytochrome species is involved in the spore germination, transcriptional levels of AcPHY1 and AcPHY2 were analyzed using reverse transcription PCR (RT PCR) in dry spores (Dry), spores imbibed for 5 d under darkness (5D), spores imbibed for 5 d under darkness and then incubated under R for 3 d(3r), and protonemata incubated for 8 d under R after sowing (8R) (Figure 3). The transcript levels of AcPHY1 and AcPHY2 are shown as the amounts of PCR products derived from AcPHY1 and AcPHY2 relative to that of AcCRY2 (A. capillus-veneris CRYP- TOCHROME2). AcCRY2 was used as an internal standard because its transcript level is almost identical in various developmental stages and under different light treatments (Imaizumi et al., 2). AcPHY1 and AcPHY2 showed very low expression in dry spores but were highly expressed in the spores imbibed for 5 d under darkness. The expression levels of AcPHY1 and AcPHY2 were similarly high in the spores incubated under R. Phytochrome localization in germinated spores was investigated by transient expression of a phytochrome and glucuronidase (GUS) fusion gene by particle bombardment followed by staining with DAPI. Because fern spore germination was promoted by R and inhibited by B irradiations, the transfected cells were incubated under either R or in the dark to examine the effects of light on the intracellular distribution of phytochrome proteins. Representative patterns of intracellular staining of phy GUS fusion proteins are shown in Figure 4A. phy2 GUS proteins were found in the nuclear region under R (white arrow heads). However, phy1 GUS proteins under both R and dark conditions and phy2 GUS proteins under darkness tended to localize throughout cells. Nuclear staining with DAPI could not be detected in most cases because of the interference of spore coat fluorescence and blue color of GUS. A good example of phy2 GUS in the nucleus with DAPI fluorescence is shown in Figure 4B left panels, confirming the phy2 GUS protein localization under R in the nucleus (white arrow heads) or surrounding regions. Similar localization of phy2 GUS proteins in the nucleus under R was found in GUS NLS protein under darkness (Figure 4B, right panels). The intracellular localization of GUS activity varied among cells, probably because of the levels of damage by particle bombardment or the difference in expression levels of the GUS. Hence, we show in Figure 4C only the representative tendency of the intracellular distribution pattern of each phy GUS fusion protein. The patterns of GUS staining were classified into three categories (Figure 4C): stained evenly all over the cell (C), stained all over the cell with a well stained spot (C+N), and stained at one spot (N). The levels of GUS staining in each case were shown with a color scale from black to white attached to the right side of the panel. Intracellular distribution of phy1 GUS and phy2 GUS proteins is shown in Figure 4D and 4E, respectively. In more than 8% spores, phy1 GUS proteins were in the C category under both darkness (black bars, n = 31) and R (white bars, n = 44) (Figure 4D). On the contrary, phy2 GUS proteins were found either in the C or C+N categories under darkness (black bars, n = 56), while most phy2 GUS proteins were either in the C+N or N categories under R (white bars, n = 71) (Figure 4E), suggesting that phy2 GUS migrated from the cytoplasmic region to the nucleus during the period of R irradiation.

4 614 Tsuboi et al. DISCUSSION In Adiantum capillus-veneris, two conventional, full-length phytochrome genes (AcPHY1 and AcPHY2) have been cloned from a genomic library and have also been sequenced as cdnas (Okamoto et al., 1993; Wada et al., 1997). Other phytochromerelated sequences are AcPHY3 and AcPHY4. The former is a chimera photoreceptor between a phytochrome chromophore binding domain and a full-length phototropin (Nozue et al., 1998), renamed as neochrome1 (neo1) (Suetsugu et al., 25), which mediates R-induced chloroplast movement and phototropism (Kawai et al., 23). Acneo1 is localized along the plasma membrane and the Acneo1-mediated chloroplast movement is induced in enucleated cells (Wada, 1988, 27), indicating that Acneo1 plays its roles without involvement of the nucleus. AcNEO1 is a partial phytochrome that contains phytochrome exon 1, and has only been cloned from a genomic library (Nozue et al., 1997). These results indicate that the phytochrome-mediating spore germination could be either Acphy1 or Acphy2. The AcPHY1 amino acid sequence had about 55% similarity to those of AtPHYA and AtPHYB and 68% similarity to that of Selaginella PHY, which resembles the PHYB of seed plants (Okamoto et al., 1993), while the AcPHY2 amino acid sequence has about 59% similarity to those of AtPHYA and AtPHYB and approximately 75% to that of Selaginella PHY (Tsuboi, unpublished data). From amino-acid sequencedata,bothacphy1andacphy2morecloselyresemble AtPHYB than AtPHYA. Adiantum capillus-veneris spore germination shows complete R FR reversibility, while UV and B inhibit R-induced spore germination (Sugai and Furuya, 1985). Previous experiments using a microbeam irradiator demonstrated that the photoreceptive site of B was in the nucleus in both centrifuged or noncentrifuged spores (Furuya et al., 1997). However, these experiments could not distinguish any specific region by R microbeam for germination induction. Here, we showed why they could not detect any localized R effect in spore germination. We showed, for the first time, that the induction and reversal effects of microbeam light treatments were altered by pre-irradiations in a time-dependent manner, indicating a slow light-induced migration of phytochrome from the cytosol into the nucleus, leading to the depletion of cytosolic phytochrome (compare Figure 2A and 2C) and an increase in nuclear phytochrome (Figure 2B). Figure 2. Translocation of Phytochrome Response from Cytoplasm to a Nuclear Region. (A) Whole or either a nucleus or a part of cytoplasmic area of a spore (inset) imbibed for 5 d under darkness was irradiated with R microbeam (mr: circle, 12 lm in diamater; 4.2 W m 2 for 6 s). Germination rate was counted 5 d after the light treatments. Germination rate without light treatment was also shown. (B) Either nucleus or a part of cytoplasmic area was irradiated with FR microbeam (mfr in the upper panel: circle, 12 lm in diameter; 6. W m 2 for 24 s) just after (white bars) or 2 h (gray bars) after R irradiation (wr: 4.2 W m 2, 6 s). Others are the same as in (A). (C) Whole spore was irradiated with R (wr: 4.2 W m 2, 6 s), incubated for 2 h in darkness, and then irradiated whole spore with FR (wfr: 6. W m 2, 24 s). Finally, either nucleus or a part of cytoplasmic area was irradiated with an R microbeam (mr: circle, 12 lm in diameter; 4.2 W m 2 for 6 s). Each point represents the mean 6 SE determined from two or three experiments. More than 2 spores were counted in each experiment. Other details are the same as in Figure 1.

5 Tsuboi et al. 615 Figure 3. Phytochrome Transcription Levels under Different Light Conditions. The dry spores (Dry), the spores imbibed under darkness for 5 d (5D), the spores imbibed under darkness for 5 d and then cultivated under R for 3 d (3R), and protonemata incubated under R for 8 d after sowing (8R) were tested. RT PCR analyses were performed using primers specific for AcPHY1, AcPHY2, and AcCRY2. As the very low fluence rate response, which is mediated by phya in seed plants, has not yet been found in fern photomorophogenic responses, the phytochrome-mediating spore germination is likely related to phyb. AtphyA nuclear import is dependent on FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like) (Genoud et al., 28). However, AtphyB accumulates independently in the nucleus. As we found a FHY1- like sequence in our A. capillus-veneris cdna library (Yamauchi et al., 25), it will be interesting to determine whether the fern FHY1-like protein is involved in phytochrome nuclear import during spore germination. Because of the close phylogenic relationship between ferns and seed plants, functional analyses of fern FHY1-like protein may become a tool to investigate when and how seed plants invented phya. To elucidate which phytochrome (Acphy1 or Acphy2) accumulates in the nucleus and mediates the spore germination, we applied the GUS staining method to already germinated spores, not only because stable transformation is not available in this fern, but also because particle bombardment through spore coat is not possible. We found that Acphy2 GUS protein was preferentially localized in the nucleus only under R light and that Acphy1 GUS protein was equally localized in cytoplasm and nucleus in darkness and very much reduced and preferentially localized to the cytoplasm (Figure 4D and 4E). Considering, however, that most phytochromes function in the nucleus in seed plants, both fern phytochromes (phy1 and phy2) could also function in the nucleus. In the previous studies of phytochrome localization in A. thaliana, both AtphyA and AtphyBeventuallymigratedintothenucleus(FranklinandQuail, 21). The different localizations of Acphy1 and Acphy2 described in this paper might be simply a difference in nucleus migration speed, with Acphy2 moving rapidly into nuclei under R light but Acphy1 migrating slowly. In physiological experiments using microbeam irradiation in Figure 2, we showed that the phytochrome for spore germination accumulated at the nuclear region within 2 h. Together with the timing of escape from FR shown in Figure 1C, that is 4 h, we suggest that the Figure 4. Intracellular Distribution of phy GUS Fusion Proteins in Germinating Cells. (A) Representative distribution patterns of phy1 GUS and phy2 GUS fusion proteins under R or darkness were taken by Nomarski optics. (B) Co-localization of GUS activity (upper panels) and DAPI staining (lower panels) were shown in phy2 GUS fusion proteins (left panels) and GUS NLS fusion proteins (right panels). Note that the phy2 GUS under R and GUS NLS proteins under darkness localized in the nucleus (white arrow heads). (C) Three different categories of GUS staining taken by Nomarski optics (upper panels) and staining levels expressed by color scale (lower panels): stained only one restricted area (N), stained whole area with one well-stained area (C+N), stained evenly around whole area (C). (D, E) Histogram of intracellular distribution of phy1 GUS (D) and phy2 GUS (E) proteins under the dark (black bars) and R (white bars) conditions. Bars: 1 lm. phytochrome for spore germination moves to the proper place and functions within 4 h of the onset of R. In conclusion, the phytochrome mediating the fern spore germination is likely to be phy2.

6 616 Tsuboi et al. METHODS Plant Materials and Culture Conditions Spores of the fern Adiantum capillus-veneris L. were collected in a greenhouse at Koishikawa Botanical Gardens in 21 and stored at about 4 C until use. For microbeam irradiation, spores were sterilized for 3 s with.5% (v/v) Antiformin (Wako Pure Chemical Industries, Osaka, Japan), and were sown on the surface of solidified agar medium (.5% INA agar, Funakoshi, Tokyo, Japan), containing White s components (Tsuboi et al., 27), in a 3-cm Petri dish and covered with a coverslip (Tsuboi and Wada, 21) and incubated in the dark for 5 d. After the various light treatments mentioned below, the spores were incubated for 5 d in the dark and the germination was identified by auto-fluorescence of chloroplasts. For particle bombardment or mrna isolation, sterilized spores of A. capillus-veneris were sown on a sheet of cellophane spread on White s medium solidified with.5% INA agar in a Petri dish (6 cm in diameter), and then cultured under light conditions appropriate for the experiments described below. All cultures and experimental procedures were conducted at 25 C. Light Sources Fluorescent lamps (FL4SD Toshiba Lighting and Technology, Tokyo, Japan) were used as the source of white light. Red light was obtained from the same lamps filtered through a red plastic filter (Shinkolite A no. 12, Mitsubishi Rayon, Tokyo, Japan). Microbeam Irradiation Dark-imbibed spores were placed on a sample stage of a microbeam irradiator (Tsuboi et al., 26, 29) under dark conditions. A part of a spore was irradiated with a microbeam with various light intensities of either R with a transmissionpeak at 66 nm obtained through an interference filter (half band-width, 34 nm; IF-BPF-66, Vacuum Optics Co. of Japan, Tokyo, Japan) or FR with a transmission peak at 74 nm obtained through an FR filter (half band-width, 36 nm; IF- BPF-74, Vacuum Optics Co. of Japan, Tokyo, Japan). Spores were observed using infrared light obtained from a halogen lamp (Focusline 12V-1W HAL, Philips Lighting, Eindhoven, Netherland) through an infrared filter (IR85, Hoya Corp., Tokyo, Japan) on a monitor screen connected to a camera (ARTCAM-13MI-NIR, Artray Co., Ltd, Tokyo, Japan) that is sensitive to infrared light. All procedures for the microbeam irradiations were performed under a dim green safe light. DNA and RNA Isolation Total RNA derived from either spores or protonemata was isolated by using the cetyl trimethyl ammonium bromide (CTAB) method (Imaizumi et al., 2). When tissue was ground with a mortar and pestle, an amount of sterilized quartz sand equal to 1 times the volume (w/w) was added to each sample to help break the cell walls in liquid nitrogen. Frozen tissue was incubated with an amount of 2 X CTAB solution (2 X CTAB solution is 2% (w/v) CTAB,.1 M Tris-HCl, ph 8., 2 mm EDTA, and 1.4 M NaCl) equal to 1 times the volume (w/v) containing 5% (v/v) 2-mercaptoethanol at 6 C. The solution was treated with chloroform three times to remove the proteins. RNA was precipitated with LiCl. The precipitate was dissolved in diethyl pyrocarbonate-treated water. RNA were then reverse-transcribed from an oligo(dt) primer using the SUPER SCRIPT III preamplification system for first-strand cdna synthesis (Invitrogen, Carlsbad, CA, USA). An aliquot of first-strand cdna was used for PCR amplification with Ex Taq polymerase (Takara Bio, Otsu, Japan) and transcript-specific primers. Equal volumes of amplified products were electrophoresed on an agarose gel and stained with ethidium bromide. The amount of PCR products obtained using the AcPHY1- and AcPHY2-specific primers was normalized to that obtained using the AcCRY2- specific primers. Analysis of GUS Intracellular Localization in Germinated Spores Gold particles (1.6 lm in diameter) coated with PHY GUS plasmids were introduced into germinated spores precultured for 5 d in the dark and then for 3 d under unilateral red light, by the Biolistic PDS-1/He Particle Delivery System (Bio-Rad, Hercules, CA, USA). To bombard gold particles, 11-psi rupture discs were used according to the manufacturer s procedure. The transfected cells were incubated under red light of 1.2 W m 2 or in the dark for 24 h at 25 C and then stained with 1 mm sodium phosphate (ph 7.), 1 mm EDTA,.3% (v/v) Triton X-1,.5 mm potassium ferricyanide,.5 mm potassium ferrocyanide,.1 lg ml 1 4,6-diamidino- 2-phenylindole (DAPI), 2% methanol and 1 mm X-gluc (Imaizumi et al., 2) at 37 C overnight under the same light conditions. Nomarski and fluorescence images were taken under a fluorescence microscope (Axioskop, Carl Zeiss, Jena, Germany) with a color digital camera (DP2, Olympus Co., Tokyo, Japan). FUNDING M.W. would like to thank the Humboldt Foundation and the Freiburg Institute for Advanced Studies (FRIAS) for supporting his stay in Freiburg, Germany. This work was partially supported by the Japanese Ministry of Education, Sports, Science and Technology (MEXT and to M.W.) and the Japan Society for the Promotion of Science (JSPS , and to M.W.). ACKNOWLEDGMENTS We thank Dr Winslow R. Briggs, Carnegie Institution for Science, for his critical reading and editing of this manuscript. We thank Dr Tetsuo Tohma for his kind effort in collecting Adiantum capillus-veneris spores at Koishikawa Botanical Gardens. No conflict of interest declared.

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