Functional characterization of Ostreococcus tauri phototropin

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1 Research Functional characterization of Ostreococcus tauri phototropin Stuart Sullivan*, Jan Petersen*, Lisa Blackwood, Maria Papanatsiou and John M. Christie Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bower Building, Glasgow, G12 8QQ, UK Author for correspondence: John M. Christie Tel: Received: 12 June 215 Accepted: 9 August 215 doi: /nph.1364 Key words: Arabidopsis thaliana, blue light, flavin, Ostreococcus tauri, Light, Oxygen and Voltage (LOV) domain, phosphorylation, phototropin. Summary Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought. Introduction Light is important for directing the lifecycles of plants and algae. The effect of light on regulating their development and physiology is mediated by specific photoreceptor proteins that span a wide range of the electromagnetic spectrum, from the UV-B wavelengths ( nm) to the red/far red region (6 75 nm). In particular, UV-A/blue light (32 5 nm) is detected by several different flavoprotein photoreceptors. In land plants, these include cryptochrome (cry), phototropin (phot) and members of the Zeitlupe (ztl) family (Christie et al., 215). Although crys were first identified in the model flowering plant Arabidopsis, they have since been identified in bacteria, fungi, algae and animals (Chaves et al., 211). Ztl-related photoreceptors, in contrast, are found exclusively in land plants, whereas phots are restricted to the green plant lineage, from photosynthetic algae to flowering plants (Suetsugu & Wada, 213). Phots are plasma membrane-associated serine/threonine kinases that undergo light-dependent autophosphorylation in response to UV/blue-light irradiation (Christie et al., 1998). The C-terminal kinase domain of phots belongs to the AGCVIII (protein kinase A, cyclic GMP-dependent protein kinase and protein kinase C) subfamily (Barbosa & Schwechheimer, 214), whereas their N-terminal region comprises the photosensory *These authors contributed equally to this work. region containing two structurally similar Light, Oxygen and Voltage sensing (LOV) domains (Christie et al., 215). Both the LOV1 and LOV2 domains bind one molecule of flavin mononucleotide (FMN) noncovalently as a UV/blue light-absorbing chromophore (Christie et al., 1999). Blue-light irradiation induces the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain (Salomon et al., 2). This light-driven photoadduct formation acts as a trigger to propagate conformational changes within the protein that alleviates the inhibitory action of the photosensory region on the kinase domain to promote receptor autophosphorylation and photoreceptor activation (Christie et al., 215). Arabidopsis contains two phots (phot1 and phot2), which overlap in function to regulate a variety of processes that optimize photosynthetic efficiency and promote growth (Takemiya et al., 25; de Carbonnel et al., 21). These include phototropism, leaf positioning and expansion, chloroplast relocation movements and stomatal opening (Christie, 27). Phot1 is the primary photoreceptor for phototropism in Arabidopsis operating over a wide range of fluence rates, whereas phot2 functions predominantly at high light intensities (Sakai et al., 21). Phot1 is also involved in regulating hypocotyl growth inhibition to light (Folta & Spalding, 21), the suppression of lateral root growth (Moni et al., 215) and the destabilization of a small number of nuclear- and chloroplast-encoded transcripts at high light intensities (Folta & 612

2 New Phytologist Research 613 Kaufman, 23; Ohgishi et al., 24), whereas phot2 is responsible for chloroplast and nuclear avoidance movements (Kagawa et al., 21; Tsuboi et al., 27). Arabidopsis plants lacking both phot1 and phot2 are deficient in all these responses and thus exhibit impaired growth (Takemiya et al., 25). Two phots have been identified in Mougeotia scalaris, both of which function to mediate chloroplast relocation movements in this green alga (Suetsugu et al., 25). By contrast, only one PHOT gene is present in the unicellular green algae Chlamydomonas reinhardtii and its multicellular relative Volvox carteri (Huang et al., 22; Prochnik et al., 21). Chlamydomonas reinhardtii phot (Crphot) is localized to the plasma membrane (Huang et al., 22) but is smaller than its vascular plant counterparts (c. 8 kda versus c. 12 kda), sharing 3 4% overall amino acid identity with phots from Arabidopsis (Kianianmomeni & Hallmann, 214). Several functions have been ascribed to Crphot in Chlamydomonas, including light regulation of various aspects of the sexual lifecycle (Huang et al., 22; Huang & Beck, 23; Ermilova et al., 24), the control of eyespot development and phototactic behaviour (Trippens et al., 212) and mediating changes in the abundance of transcripts encoding enzymes associated with chlorophyll and carotenoid biosynthesis (Im et al., 26). Despite its divergent function in Chlamydomonas, expression of Crphot can restore phot-mediated responses when expressed in the phot1 phot2 double mutant of Arabidopsis (Onodera et al., 25), implying that the basic mechanism of action between plant and algal phots is highly conserved. However, this conclusion is derived solely from studies on Crphot. Additional algal phots have recently been identified, including one from the marine picoalga Ostreococcus tauri (Kianianmomeni & Hallmann, 214). The LOV1 domain of O. tauri phototropin (Otphot) exhibits photochemical properties that are consistent with those of phots from vascular plants (Veetil et al., 211), but further characterization of this protein is still lacking. In this study, we analysed the photochemical and biochemical properties of Otphot and examined whether this algal photoreceptor, like Crphot, can replace the function of Arabidopsis phots. Our results indicate that, while Otphot exhibits similar photochemical and biochemical properties to those of vascular plants, including light-dependent receptor autophosphorylation, plasma membrane association and partial, albeit weak, internalization upon blue-light stimulation, it is unable to fully restore phot responsiveness in the phot1 phot2 mutant of Arabidopsis, particularly for phototropism and chloroplast avoidance. Together, these findings highlight key differences between the modes of action of plant and algal phots that have so far gone unrecognized. Materials and Methods Plant material and growth Wild-type (WT) Arabidopsis thaliana L. Heynh. (gl-1, ecotype Columbia) and the phot1-5, phot2-1 and phot1-5 phot2-1 mutants have been described previously (Liscum & Briggs, 1995; Kagawa et al., 21). Kanamycin-resistant transgenic seedlings were identified by rapid selection (Harrison et al., 26) of seedlings grown on silicon dioxide (Davis et al., 29). Seeds were planted on silicon dioxide and watered with quarterstrength Murashige and Skoog (MS) salts medium (supplemented with kanamycin for transgenic lines) or surface-sterilized and planted on half-strength MS salts medium with.8% agar (w/v). After cold treatment (4 C) for 2 4 d, seeds were exposed to 8 lmol m 2 s 1 white light for 4 6 h to induce germination before incubation in the dark for 64 7 h. Seedlings on silicon dioxide were exposed to 8 lmol m 2 s 1 white light for 16 h to induce selection before seedlings were transferred to soil and grown in a controlled environment room 66 (Fitotron; Weiss- Gallenkamp, Loughborough, UK) under 16 h 22 C: 8 h 18 C, light : dark cycles (8 lmol m 2 s 1 white light). The fluence rate for all light sources was measured with a Li-25A and quantum sensor (Li-Cor). Protein expression in Escherichia coli and purification For the expression of N-terminal tagged 7His-Strep II-SUMO Otphot LOV1+2, the coding sequence (amino acids ) was cloned into the expression vector phs (Christie et al., 212) via NcoI and NotI. Protein expression of the fusion protein was performed in the Escherichia coli expression strain Rosetta BL21 (DE3)pLysS (Merck, Darmstadt, Germany). Cultures were grown in LB medium at 37 C to an optical density at 6 nm of.6, then induced with.5 lm isopropyl b-d-1-thiogalactopyranoside (IPTG) (final concentration). Induction was carried out for a total of 18 h at 24 C. Afterwards the culture was harvested, lysed and processed for tandem affinity purification, including cleavage of the tag, as previously described (Christie et al., 212). Amino acid mutations of the active-site cysteine residues C66 and C241 to alanine were introduced by PCR as described previously (Christie et al., 22). Spectroscopic analysis Absorption spectra were collected at room temperature using a Shimadzu MultiSpec-151 diode array spectrophotometer (Shimadzu, Milton Keynes, UK). Light-induced absorption changes and dark recovery kinetics were recorded using a bluelight emitting diode (k 455 nm) with 8 lmol m 2 s 1. Halflives (t 1/2 ) were calculated by fitting the photoadduct decay at 45 nm to a double exponential curve using SIGMA PLOT (Systat Software, San Jose, CA, USA). Insect cell expression The coding sequence of OtPHOT was cloned into the baculovirus transfer vector pachlt-a (BD Biosciences, San Jose, CA, USA) via the restriction sites NcoI and NotI. Recombinant baculovirus was generated using the Bacmagic TM transfection kit (Merck) in accordance with the supplier s instructions. Expression of recombinant phot was performed as previously described (Christie et al., 1998; Sakai et al., 21). For the separation of the soluble and membrane fractions, the insect cell extract was

3 614 Research New Phytologist centrifuged at 1 g for 75 min at 4 C. The supernatant was recovered, and the pellet was washed in kinase buffer and applied to a second round of centrifugation. The membrane pellet was resuspended in kinase buffer. Immunoblot analysis was performed as described below using monoclonal anti-his-tag antibody (Merck) for the immunodetection of phot, anti-gp64 antibody (Abcam, Cambridge, UK) as a membrane marker and anti-a-tubulin antibody (Abcam) as a marker for the soluble fraction. In vitro phosphorylation analysis Kinase assays were performed as previously described (Sakai et al., 21). Protein extract (1 lg) was either mock irradiated under red light or treated for 1 s with white light at a total fluence of 3 lmol m 2. Reactions were performed for 2 min at room temperature and stopped by the addition of sodium dodecyl sulfate (SDS) sample buffer. Immunoblot analysis Total proteins were extracted from 3-d-old etiolated Arabidopsis seedlings by grinding 5 seedlings in 1 ll of29 SDS sample buffer. For fractionation, total proteins were extracted as described previously (Sullivan et al., 28) under a dim red safe light and separated into soluble and membrane proteins by centrifugation at 1 g at 4 C for 75 min. After separation by SDS-polyacrylamide gel electrophoresis (PAGE), proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ, USA) by electroblotting and detected with anti--hfp monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany), anti-ugpase antibody (Agrisera, V ann as, Sweden), anti-phot1 polyclonal antibody (Cho et al., 27) and anti-non-phototropic Hypocotyl 3 (NPH3) polyclonal antibody (Tsuchida-Mayama et al., 28). Blots were developed with horseradish peroxidase (HRP)-linked secondary antibodies (Promega) and Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). promoter region to generate pezr-patphot1- (Preuten et al., 215). The full-length coding sequence of AtPHOT1 was amplified from cdna and inserted into pezr-patphot1- using the restriction sites HindIII and BamHI. Constructs were transformed into the phot1-5 phot2-1 double mutant with Agrobacterium tumefaciens as described previously (Christie et al., 22). Based on the segregation of kanamycin resistance, independent T2 lines were selected for analysis. Phototropism Blue-light sources for phototropism were as described previously (Kaiserli et al., 29). Etiolated seedlings were grown on silicon dioxide as described above and irradiated with 1 lmol m 2 s 1 unilateral blue light for 8 h before representative seedlings were photographed. expression for in seedlings was confirmed by confocal microscopy. Petiole positioning and leaf expansion For petiole positioning, Arabidopsis seedlings were grown on soil for 1 d before representative plants were photographed. Measurement of leaf expansion was carried out as described previously (Takemiya et al., 25) from 4-wk-old plants grown on soil. Leaf areas were measured before and after uncurling and the ratio of the curled to uncurled area designated as the leaf expansion index. Leaf area was measured using IMAGEJ software ( rsb.info.nih.gov/ij/). Chloroplast relocation Measurements of chloroplast positioning were performed as described previously (Inoue et al., 211). Rosette leaves detached from 3-wk-old plants grown on soil were placed on agar plates and irradiated with 1.5 or 1 lmol m 2 s 1 blue light through a 1-mm slit for 1 h. The plates were placed on a white light transilluminator and photographed. Band intensities were quantified using IMAGEJ software and the relative band intensities expressed as the ratio of the irradiated to the nonirradiated areas. Immunoprecipitation of -tagged proteins Microsomal membrane proteins were extracted and green fluorescent protein () immunoprecipitations were performed as described previously (Sullivan et al., 28) using the lmacs isolation kit (Miltenyi Biotech). Transformation of Arabidopsis Transformation vectors for PHOT1::AtPHOT1- and 35S:: OtPHOT- were constructed using the modified binary expression vector pezr(k)-ln (Kaiserli et al., 29). The fulllength OtPHOT genomic sequence was cloned into pezr(k)- LN using the restriction sites EcoRI and BamHI. For PHOT1:: AtPHOT1-, the 35S promoter was removed using restriction sites SacIandHindIII and replaced with the native AtPHOT1 Stomatal opening Stomatal apertures were measured as in Kinoshita et al. (21) with modifications. Epidermal peels obtained from the abaxial side of rosette leaves from dark-adapted 3-wk-old plants were placed in standard buffer comprising 1 mm Na + -MES, ph 6.1 (1 mm MES titrated to ph 6.1 with NaOH), and 5 mm KCl and kept in the dark for 1 h. Epidermal peels were subsequently irradiated with 1 lmol m 2 s 1 blue light superimposed on background of 5 lmol m 2 s 1 red light using a Red, Green and Blue (RGB) light source (Li-Cor, 64-18) for 2 h. Stomata were imaged before and after light treatment using a laser scanning confocal microscope (Zeiss LSM 51) with a 514-nm laser at 4-fold magnification. Apertures were tracked for individual stomata and quantified using IMAGEJ. All measurements were carried out between 8: and 11: h.

4 New Phytologist Research 615 Confocal microscopy Subcellular localization of -tagged proteins in Arabidopsis seedlings was visualized using a laser scanning confocal microscope (Zeiss LSM 51) as described previously (Kaiserli et al., 29). Maximum projection images were constructed from z-stacks using IMAGEJ. Results Photochemical properties of Otphot LOV1+2 The O. tauri genome sequence contains a single putative PHOT gene (Ot16g29) (Derelle et al., 26). The Otphot protein sequence shares an overall 55% homology with the phot sequence from Crphot and a domain structure that is conserved compared with all previously identified phot sequences. Analysis of the spectral properties of the isolated LOV1 domain of Otphot expressed in E. coli showed that it functions as an FMN-bound blue-light sensor (Veetil et al., 211). However, further photochemical characterization of Otphot has not been reported. The LOV1+2 region of Arabidopsis phots (Christie et al., 22) and Crphot (Guo et al., 25) can be readily expressed and purified from E. coli in suitable quantities for absorbance spectroscopy. In the case of the Arabidopsis phots, the photochemical properties obtained for the LOV1+2 proteins are similar to those recorded for the full-length photoreceptors (Kasahara et al., 22). We therefore expressed the corresponding region of Otphot (amino acids ) in E. coli to examine the primary photochemical responses following blue-light excitation (Supporting Information Fig. S1a). The absorption spectrum for Otphot LOV1+2 showed spectral characteristics typically observed for heterologously expressed LOV domains with maximal absorbance at 449 nm (Fig. 1a). Vibronic signatures were also observed in this spectral region, as well as an absorption peak at 378 nm in the UV-A region of the spectrum. Irradiation with blue light resulted in a loss of absorbance in the blue region of the spectrum with the appearance of three isosbestic points that correspond to FMN-cysteinyl adduct formation (Fig. 1a). These photochemical changes were reversible in darkness and could be resolved into exponential components with half-lives of 115 and 1155 s, respectively (Fig. 1b). These kinetic properties of adduct decay are c. 1-fold slower compared with those reportedly previously for the LOV1+2 region of Crphot (Kasahara et al., 22). Side-chain replacement of the conserved cysteine required for LOV domain photochemistry with alanine in both LOV1 (C66) and LOV2 (C241) abolished these light-induced absorption changes (Fig. S2) and produced the appearance of two absorption peaks in the UV-A region of the spectrum at 36 and 371 nm (Fig. S1b). A similar absorption profile was observed when only the LOV1 domain was mutated (C66A), while Otphot LOV1+2 harbouring the C-A mutation in LOV2 (C241A) showed absorption properties similar to those of the wild-type protein (Figs 1, S1b). Both single C-A mutants, particularly C66A, exhibited light-induced absorption changes that were diminished compared (a) WT (b).16 WT Wavelength (nm) Fig. 1 Photochemical properties of Ostreococcus tauri phototropin (Otphot) Light, Oxygen and Voltage sensing 1+2 (LOV1+2). (a) Blue light-induced absorption changes for wild type (WT) Otphot LOV1+2, C66A and C241A. Consecutive spectra were recorded at, 2, 4, 6, 8 and 1 s after the start of irradiation (8 lmol m 2 s 1 ). The arrows indicate absorbance peaks present at the initial time-point. (b) Darkrecovery kinetics for WT Otphot LOV1+2, C66A and C241A. The main panel presents absorption changes at 45 nm recorded every 3 s after blue-light excitation (8 lmol m 2 s 1 ) for 2 min. Light-minusdark absorption difference spectra showing the recovery to the dark state after photoexcitation are shown in the inset, with spectra recorded every 4 min. Absorbance C66A C241A Wavelength (nm) Δ Absorbance (45 nm) C66A C241A Wavelength (nm) Wavelength (nm) Time (s)

5 616 Research New Phytologist with WT (Fig. 1a). More pronounced light-induced absorption changes were observed for C241A compared with C66A, suggesting that LOV1 may be more photochemically active than LOV2. Photoadduct decay kinetics recorded for C241 (t 1/2 = 92 and 99 s) were similar to those of WT, indicating that photochemically active LOV1 alone is sufficient to mediate these slow dark recovery properties (Fig. 1b). C66A, in contrast, showed moderately faster photoadduct decay kinetics by comparison (t 1/2 = 17 and 693 s). Otphot exhibits light-dependent autophosphorylation In order to determine whether Otphot showed light-dependent autophosphorylation, the full-length protein was expressed in Sf9 insect cells as done previously for Arabidopsis phots (Christie et al., 22) and Crphot (Onodera et al., 25). In vitro kinase assays on insect cell extracts showed that Otphot undergoes lightdependent autophosphorylation (Fig. 2a), although the overall signal was reduced compared with insect cells expressing phot1 from Arabidopsis (Atphot1). Fractionation of the protein extract from insect cells showed that Otphot has the ability to associate with the membrane fraction, although much of the protein is soluble (Fig. 2b). A similar fractionation profile was observed for Atphot1 (Fig. S3), indicating that, despite being hydrophilic in nature, both plant and algal phots possess the necessary properties to facilitate membrane association when expressed in insect cells. Antibodies recognizing glycoprotein-64 and a-tubulin were used to confirm the purity of membrane and soluble fractions, respectively. C-A mutations were next incorporated into full-length Otphot to probe the role of LOV1 and LOV2 in regulating light-dependent autophosphorylation activity. Kinase activity in response to saturating light was still observed when LOV1 was photochemically inactivated (C66A), albeit at a somewhat reduced level compared with wild-type Otphot (Fig. 2c). By contrast, light-induced kinase activity was considerably reduced in comparison to WT when LOV2 was mutated (C241A). However, a residual level of light-induced activity could still be observed, whereas no increase in phosphorylation was observed in response to irradiation when both LOV domains were inactivated (C66A C241A). These results indicate that both LOV1 and LOV2 of Otphot are capable of eliciting light-dependent phosphorylation at saturating light conditions, with LOV2 playing a prominent role, as noted previously for Arabidopsis phot2 (Christie et al., 22). (a) (c) Atphot1 Atphot1 Otphot D L D L Otphot (b) C66A WT C66A C241A C241A D L D L D L D L WT C66A C241A C66A C241A T S M Otphot gp64 α-tubulin Fig. 2 Autophosphorylation activity and membrane association of Ostreococcus tauri phototropin (Otphot) expressed in insect cells. (a) Autoradiograph showing light-dependent autophosphorylation of Arabidopsis phot1 (Atphot1) and Otphot expressed in insect cells. Insect cell extracts were given a mock irradiation (D) or irradiated with white light (L) at a total fluence of 3 lmol m 2 before the addition of radiolabelled ATP. Immunoblot analysis of protein levels with anti-his antibody is shown in the lower panel. (b) Membrane association of Otphot expressed in insect cells. The total protein extract (T) was fractionated into soluble (S) and membrane (M) fractions by ultracentrifugation. Fractions were analysed by immunoblotting using anti-his antibody to detect Otphot. Anti-a-tubulin antibody was used as a soluble marker and antigp64 antibody as a marker for the membrane fraction. (c) Autoradiograph showing light-dependent autophosphorylation of wild type (WT) Otphot and C66A, C241A and C66A C241A mutants expressed in insect cells. Immunoblot analysis of protein levels with anti-his antibody is shown in the lower panel. Expression of Otphot in Arabidopsis To assess the functionality of Otphot in Arabidopsis, Otphot fused to was expressed under the control of the cauliflower mosaic virus 35S promoter () in the phot1 phot2 double mutant (Kagawa et al., 21) and three independent T2 lines were isolated (4, 14 and 16). Immunoblot analysis of total protein extracts from 3-d-old etiolated seedlings maintained in darkness or irradiated with blue light showed that lines 4 and 14 expressed to levels comparable to those of a transgenic line expressing Atphot1- under the control of its native PHOT1 promoter, while line 16 had a lower level of expression (Fig. 3a). Atphot1- is expressed to levels comparable to those of endogenous phot1 in WT seedlings (Fig. S4). While Atphot1- showed reduced electrophoretic mobility upon blue-light irradiation, consistent with autophosphorylation on multiple residues, no change in the mobility of was detected (Fig. 3a). Fractionation of total protein extracts confirmed that, like Atphot1-, localized to the membrane in Arabidopsis (Fig. 3b). Blue light-induced changes in Otphot subcellular localization In Arabidopsis, blue-light irradiation leads to a rapid relocalization of a portion of phot1 and phot2 from the plasma membrane to the cytosol and Golgi, respectively (Sakamoto & Briggs, 22; Kong et al., 26). Phot kinase activity has been shown to be required for this response (Kong et al., 26; Kaiserli et al., 29). Confocal observation of etiolated seedlings showed that localized to the plasma membrane in darkness, like Atphot1- (Fig. 4a). Scanning with the blue light laser used to excite led to rapid internalization of Atphot1-, but

6 New Phytologist Research 617 (a) Atphot p1p2 D L D L D L D L D L (a) Atphot1 - scan 1 scan 2 scan 3 UGPase 4 (b) Atphot1-4 p1p2 T S M T S M T S M 14 UGPase 16 Fig. 3 Expression and subcellular localization of Ostreococcus tauri phototropin (phot)- in transgenic Arabidopsis. (a) Immunoblot analysis of total protein extracts from 3-d-old etiolated seedlings expressing Atphot1- or three independent lines expressing Otphot- (lines 4, 14 and 16) in the phot1 phot2 double mutant (p1p2). Seedlings were either maintained in darkness (D) or irradiated with 2 lmol m 2 s 1 of blue light (L) for 15 min. Protein extracts were probed with anti- antibody (upper panel) and antibody raised against UDPglucose pyrophosphorylase (UGPase; lower panel) as a loading control. (b) Immunoblot analysis of total protein extract (T) separated into soluble (S) and membrane (M) fractions by ultracentrifugation. Equal volumes of each fraction were probed with anti- antibody (upper panel) and anti- UGPase (lower panel) as a soluble protein marker. this response was attenuated in the expressing lines. To determine whether the reduced movement of was attributable to reduced sensitivity, etiolated seedlings of the two highest expressing lines (4 and 14) and Atphot1- were irradiated with high-intensity blue light (5 lmol m 2 s 1 for 15 min) before confocal imaging. Whereas Atphot1- showed extensive internalization following highintensity blue-light irradiation, only a small proportion of was observed to move away from the plasma membrane (Fig. 4b). Otphot mediates leaf positioning but not phototropism in Arabidopsis Crphot was able to complement several phot-mediated responses when expressed in the Arabidopsis phot1 phot2 double mutant (Onodera et al., 25). To assess the functionality of the Otphot- lines, we first examined the ability of to restore phototropism in the phot1 phot2 double mutant. Phototropism was examined over a range of fluence rates (data not shown) but no phototropic response was observed in any of the three Otphot1- expressing lines. Fig. 5(a) shows representative images of 3-d-old etiolated seedlings after exposure to 1 lmol m 2 s 1 of unilateral blue light for 8 h. WT and the phot1-5 and phot2-1 single mutant seedlings all displayed a robust (b) Atphot Fig. 4 Blue light-induced changes in the subcellular localization of Arabidopsis phototropin 1 (Atphot1) - and Ostreococcus tauri phototropin (Otphot)- in hypocotyl epidermal cells. (a) Composite maximum projection images of 3-d-old etiolated seedlings expressing Atphot1- or three independent lines expressing (4, 14 and 16). Seedlings were scanned immediately (scan 1) and twice more at 1-min intervals (scan 2 and scan 3) with darkness between each scan. Each scan took 12 s to complete. is shown in green and autofluorescence in red. White arrows indicate internalization. Bars, 2 lm. (b) Composite maximum projection images of 3-d-old etiolated seedlings expressing Atphot1- or two independent lines expressing (4 and 14) after irradiation with 5 lmol m 2 s 1 of blue light for 15 min. is shown in green and autofluorescence in red. White arrows indicate internalization. Bar, 2 lm. phototropic response. The expressing lines did not show any reorientation of growth towards the blue light and were indistinguishable from the phot1 phot2 double mutant. Therefore, was unable to mediate phototropic curvature in Arabidopsis. Phots are also required to mediate the optimal positioning of leaves to maximize light capture (Inoue et al., 28). The petioles of WT, phot1-5 and phot2-1 seedlings grew obliquely upwards so that the first true leaves faced the light source from above (Fig. 5b). By contrast, petioles of the phot1 phot2 double mutant seedlings grew horizontally with their leaves slanted downwards. Expression of restored the leaf positioning response in the phot1 phot2 double mutant in all three transgenic lines (Fig. 5b), demonstrating that Otphot was able to replace the function of Arabidopsis phots for this response.

7 618 Research New Phytologist (a) WT p1-5 p2-1 p1p2 (a) Leaf expansion index WT p1-5 p2-1 p1p (b) WT p1-5 (b) Light Dark p2-1 p1p Stomatal aperture (µm) WT p1-5 p2-1 p1p Fig. 5 Functional analysis of Ostreococcus tauri phototropin (Otphot)- in the phot1 phot2 double mutant of Arabidopsis. (a) Phototropism response in 3-d-old etiolated wild type (WT) and phot1-5 (p1-5), phot2-1 (p2-1) and phot1 phot2 (p1p2) mutants and three independent lines expressing (4, 14 and 16). Seedlings were irradiated with 1 lmol m 2 s 1 of unidirectional blue light for 8 h. (b) Leaf positioning of WT and phot1-5 (p1-5), phot2-1 (p2-1) and phot1 phot2 (p1p2) mutants and three independent lines expressing (4, 14 and 16). Plants were grown under 8 lmol m 2 s 1 of white light (16 h : 8 h, light : dark cycles) for 1 d. Otphot partially complements leaf expansion, stomatal opening and plant growth Rosette leaves of the phot1 phot2 double mutant grown under 8 lmol m 2 s 1 of white light for 4 wk were epinastic and curled downwards at the side in comparison to leaves from WT plants (Fig. 6a). Under our growth conditions, phot1 and phot2 acted redundantly to mediate leaf expansion; leaves of the phot1-5 and phot2-1 single mutants were identical to those of WT plants. Expression of led to a partial complementation of the leaf expansion phenotype in the phot1 phot2 double mutant background for all three expressing lines (Fig. 6a). A similar trend was also observed for plant growth under the same growth conditions (Fig. S5). Fresh weight measurements of total rosette leaves showed that expression of was able to partially complement the reduced growth phenotype seen in the phot1 phot2 double mutant (Fig. S5). Fig. 6 Leaf expansion and stomatal opening in Ostreococcus tauri phototropin (Otphot)- expressing Arabidopsis plants. (a) Leaf expansion index of wild type (WT) and phot1-5 (p1-5), phot2-1 (p2-1) and phot1 phot2 (p1p2) mutants and three independent lines expressing (4, 14 and 16). The leaf expansion index was expressed as the ratio of the leaf area before and after artificial uncurling. Each value is the mean SE of 1 leaves. Images of leaf sections illustrate the leaf expansion phenotype. (b) Stomatal movement in WT and phot1-5 (p1-5), phot2-1 (p2-1) and phot1 phot2 (p1p2) mutants and three independent lines expressing (4, 14 and 16). Epidermal peels from rosette leaves of 3-wk-old plants were placed in darkness (dark) or irradiated with 5 lmol m 2 s 1 red light and 1 lmol m 2 s 1 blue light (light) for 2 h. Each value is the mean SE of 46 stomata. Images of stomata after irradiation illustrate the stomatal opening phenotype. Bar, 1 lm. Both phot1 and phot2 are known to overlap in function to regulate stomatal opening in response to blue light (Kinoshita et al., 21). Consequently, phot1-5 and phot2-1 single mutants still displayed blue light-induced stomatal opening whereas this response was lost in the phot1 phot2 double mutant (Fig. 6b). The reduced sensitivity of stomatal opening in phot1-5 is consistent with earlier studies using 1 lmol m 2 s 1 or less of blue light (Kinoshita et al., 21). was able to restore stomatal opening in all three of the transgenic lines to a level comparable to the activity observed for phot2 in the phot1 single mutant background

8 New Phytologist Research 619 (Fig. 6a). These findings indicate that Otphot, like Crphot (Onodera et al., 25), can complement stomatal function when expressed in Arabidopsis. (a) WT p1-5 p2-1 p1p Otphot mediates chloroplast accumulation but not chloroplast avoidance Chloroplast accumulation is mediated by both phot1 and phot2 in Arabidopsis in response to low-fluence blue-light irradiation, whereas only phot2 mediates chloroplast avoidance movement in response to high-fluence blue-light irradiation. Chloroplast movements were assessed in the Otphot- expressing lines via the slit band assay (Kagawa et al., 21; Suetsugu et al., 25). When leaves of WT, phot1-5 and phot2-1 plants were irradiated with 1.5 lmol m 2 s 1 of blue light through a 1-mm slit, a dark band was visible on the leaf as a result of chloroplast accumulation (Fig. 7a upper panel). When leaves were irradiated with 1 lmol m 2 s 1 of blue light, a white band was visible on the leaves of WT and phot1-5 plants as a result of chloroplast avoidance (Fig. 7a lower panel). As a consequence of the lack of phot2, the leaves of the phot2-1 mutant showed a weak accumulation response under 1 lmol m 2 s 1 of blue light, whereas the leaves of the phot1 phot2 double mutant showed no response under both light treatments. The expressing lines 4 and 14 showed chloroplast accumulation but not chloroplast avoidance, whereas line 16 did not show any chloroplast relocation movement (Fig. 7a). Quantification of the slit band assays showed that the accumulation response of expressing lines 4 and 14 was reduced compared with that of the WT and phot2-1 mutant, but was more similar to the level of response in the phot1-5 mutant (Fig. 7b). The reduced sensitivity of chloroplast accumulation in phot1-5 is in agreement with previous reports (Kagawa et al., 21; Suetsugu et al., 25; Inoue et al., 211). While a weak chloroplast accumulation response was still observed in the phot2-1 single mutant at high blue-light intensities, this was not the case for the transgenic lines expressing Otphot (Fig. 7b). The lack of a response in the expressing line 16 could be attributable to the reduced level of expression in this line compared with lines 4 and 14 (Fig. 3a). Otphot is unable to complex with NPH3 The inability of Otphot to restore phototropism and chloroplast avoidance movement in Arabidopsis may stem from impaired signalling for these responses. NPH3 is required for both phot1- and phot2-mediated phototropism and is known to physically interact with phot1 (Motchoulski & Liscum, 1999) and phot2 (de Carbonnel et al., 21). NPH3 is rapidly dephosphorylated upon phot1 activation (Tsuchida- Mayama et al., 28) and acts early in the phototropic signalling pathway upstream of auxin redistribution (Haga et al., 25; Pedmale & Liscum, 27). We therefore investigated the phosphorylation status of NPH3 in the expressing lines in response to blue-light irradiation (b) Band Intensity WT p1-5 p2-1 p1p Fig. 7 Light-dependent chloroplast positioning of Ostreococcus tauri phototropin (Otphot)- expressing Arabidopsis plants. (a) Slit band assays of chloroplast movement in wild type (WT) and phot1-5 (p1-5), phot2-1 (p2-1) and phot1 phot2 (p1p2) mutants and three independent lines expressing (4, 14 and 16). Detached leaves were irradiated with 1.5 lmol m 2 s 1 of blue light for chloroplast accumulation (upper panel) or 1 lmol m 2 s 1 of blue light for chloroplast avoidance (lower panel) through a 1-mm slit for 6 min. Arrowheads indicate the irradiated areas. (b) Quantification of the slit band assays. The slit band intensity was quantified using IMAGEJ and the relative band intensities expressed as the ratio of the irradiated to the nonirradiated areas. Ratios > 1 indicate accumulation, while those < 1 indicate avoidance. The dashed line indicates a ratio of 1. Each value is the mean SE of five leaves. Immunoblot analysis of total protein extracts from 3-d-old etiolated seedlings maintained in darkness or irradiated with blue light showed an enhanced electrophoretic mobility of NPH3 in blue light-irradiated samples of seedlings expressing Atphot1-, but not in the phot1 phot2 double mutant (Fig. 8a), consistent with protein dephosphorylation. No change in the electrophoretic mobility of NPH3 in response to blue-light irradiation was observed in seedlings expressing, demonstrating that it is unable to mediate the dephosphorylation of NPH3 in Arabidopsis. Co-immunoprecipitation analysis was performed to determine whether Otphot still has the ability to form a complex with NPH3 in Arabidopsis. -tagged phot proteins were immunopurified using anti- antibodies (Sullivan et al., 28) from 3-d-old etiolated seedlings either kept in darkness or exposed to a brief irradiation (15 min) with blue light (2 lmol m 2 s 1 ). NPH3 was found to co-purify with Atphot1- under both nonirradiated and irradiated conditions (Fig. 8b). However, 1.5 1

9 62 Research New Phytologist (a) NPH3 (b) NPH3 NPH3 was not detected in the immunoprecipitates, confirming that Otphot lacks the ability to complex with NPH3 in Arabidopsis. Discussion Atphot p1p2 D L D L D L D L D L Atphot1- Otphot- 4 D L D L Atphot1- Otphot- 4 D L D L Input IP Fig. 8 Ostreococcus tauri phototropin (Otphot)- does not interact with Non-Phototropic Hypocotyl 3 (NPH3) in transgenic Arabidopsis. (a) Immunoblot analysis of total protein extracts from 3-d-old etiolated seedlings expressing Atphot1- or three independent lines expressing (4, 14 and 16). Seedlings were either maintained in darkness (D) or irradiated with 2 lmol m 2 s 1 of blue light (L) for 15 min. Protein extracts were probed with anti-nph3 antibody. (b) Atphot1- and were immunoprecipitated from membranes from 3-d-old etiolated seedlings (D) or seedlings irradiated with 2 lmol m 2 s 1 of blue light (L) for 15 min. Samples were subjected to immunoblot analysis with anti- and anti-nph3 antibodies. Input (left panels) represents solubilized microsomes used for immunoprecipitation (IP; right panels). Functional characterization of algal phots has received less attention compared with those from vascular plants. To date, functional analysis of algal counterparts has been limited to phot from Chlamydomonas reinhardtii (Kianianmomeni & Hallmann, 214). Here, we chose to study the photochemical, biochemical and functional properties of phot from the smallest free-living eukaryote O. tauri. While Otphot in some aspects displays a similar mechanism of activation to that of Arabidopsis phots, the results of our analysis reveal that the mode of action is not completely conserved between plant and algal phots. LOV2 drives Otphot kinase activity Our photochemical and mutational analyses on Otphot LOV1+2 showed that both LOV domains contribute to its photochemical reactivity. More prominent light-induced absorbance changes were recorded for C241A compared with C66A (Fig. 1a). However, it is worth noting that C66A showed faster photoadduct decay kinetics, which could contribute to the reduced light-induced absorbance changes observed. Further analysis is required to determine whether the LOV1 and LOV2 domains of Otphot exhibit different quantum efficiencies. Nonetheless, photoadduct decay for Otphot LOV1+2 was measured to be approximately an order of magnitude slower than that reported previously for Crphot LOV1+2 (Kasahara et al., 22) (t 1/2 = 7 and 2 s). Photoadduct decay is important for photoreceptor activity and these properties of Otphot LOV1+2 are more comparable to those obtained previously for Atphot1 LOV1+2 (Kaiserli et al., 29) (t 1/2 = 6 and 735 s). The LOV2 domain of Arabidopsis phots plays a prominent role in regulating light-induced kinase activity (Christie et al., 22; Cho et al., 27; Jones et al., 27; Okajima et al., 212). In the present study, we have illustrated that this is also the case for Otphot. Photochemical inactivation of LOV2 severely impaired light-dependent autophosphorylation of Otphot when expressed in insect cells, whereas mutation of LOV1 photochemistry had less of an effect under the conditions examined (Fig. 2c). These findings are consistent with recent observations that LOV2 functions as the principal light sensor in regulating Crphot kinase activity (Aihara et al., 212; Okajima et al., 214). The LOV1 domain of Otphot appeared to mediate a residual level of lightinduced autophosphorylation (Fig. 2c). A similar observation has been noted for the kinase activity of Arabidopsis phot2 (Atphot2) expressed in insect cells (Christie et al., 22). Moreover, mutational analyses on Atphot2 suggest that LOV1 can still play a small functional role in light sensing when LOV2 is photochemically inactivated, at least for phototropism (Cho et al., 27; Suetsugu et al., 213). This property was not apparent in studies involving Atphot1 (Christie et al., 22; Cho et al., 27) and probably correlates with the higher quantum efficiency reported for the LOV1 domain of phot2 compared with that of phot1 (Salomon et al., 2; Kasahara et al., 22). Otphot associates with the plasma membrane Otphot showed the ability to associate with membrane fractions isolated from insect cells (Fig. 2b) and Arabidopsis hypocotyl cells (Fig. 3b), similar to that observed for Atphot1 (Figs 3b, S3). The lower level of autophosphorylation detected for Otphot compared with Atphot1 correlates well with that reported previously for Crphot expressed in insect cells (Onodera et al., 25) and probably reflects fewer phosphorylation sites within the algal proteins, which contain a smaller LOV-linker sequence compared with plant phots, as well as lacking the extreme N-terminal sequence (Kianianmomeni & Hallmann, 214). The majority of identified phosphorylation sites for plant phots reside within these two regions (Christie et al., 215). At least 21 in vivo phosphorylation sites have been identified for Atphot1 by liquid chromatography mass spectrometry (Christie et al., 215). By contrast, only one phosphorylation site has been reported so far for Otphot, which corresponds to S171 within the LOV-linker sequence (Hindle et al., 214). The reduced level of autophosphorylation in algal phots may account for the absence of an electrophoretic mobility shift for Otphot when expressed in Arabidopsis (Fig. 3a). Otphot showed changes in subcellular localization following blue-light irradiation in Arabidopsis (Fig. 4). Yet, internalization of from the plasma membrane was markedly

10 New Phytologist Research 621 reduced in comparison to Atphot1-. The reduced internalization observed for is unlikely to account for its lack of phototropic function (Fig. 5a), as recent studies indicate that phototropism is not dependent on this process (Preuten et al., 215). The functional significance of phot internalization is still not known, but our studies demonstrate that algal phots exhibit this property at least when expressed in Arabidopsis. Otphot mediates petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement in Arabidopsis Otphot was able to complement several phot-mediated processes in Arabidopsis. Petiole positioning (Fig. 5b), leaf expansion (Fig. 6a), stomatal opening (Fig. 6b) and chloroplast accumulation movement (Fig. 7) were all clearly measurable in the transgenic lines expressing. These findings coincide with those reported previously for Crphot functionality in Arabidopsis (Onodera et al., 25). The weaker responsiveness of the Otphot expressing lines for leaf expansion was not attributable to lower protein levels, at least for lines 4 and 14 (Fig. 3a). Reduced functionality of Otphot could relate to its photochemical properties. Minor absorbance changes corresponding to photoadduct formation were recorded for Otphot LOV1+2 when LOV1 was inactive (Fig. 2a), implying that the quantum efficiency of LOV2 is low in comparison to that recorded for equivalent LOV1+2 proteins derived from Arabidopsis phots under similar conditions (Christie et al., 22; Kasahara et al., 22). A lower quantum efficiency would be expected to reduce receptor photosensitivity and could account for the partial responsiveness of Otphot in mediating leaf expansion compared with WT and phot1-5 and phot2-1 single mutants (Fig. 6a). However, response measurements for Otphot-mediated stomatal opening and chloroplast accumulation were comparable to that driven by phot2 in the phot1-5 single mutant (Figs 6a, 7). These findings therefore suggest that Otphot is less effective in eliciting leaf expansion and promoting growth compared with Arabidopsis phots (Fig. S5). Otphot is unable to mediate phototropism or chloroplast avoidance movement A noteworthy finding from this study is the inability of Otphot to restore phototropic curvature and chloroplast avoidance movement when expressed in the phot1 phot2 double mutant of Arabidopsis (Figs 5a, 7). These findings are of particular interest given earlier work showing that Crphot is able to mediate these processes in addition to other phot-mediated responses (Onodera et al., 25). In contrast to Crphot, Otphot appears to lack the ability to initiate signalling events associated with phototropism and chloroplast avoidance movement, the latter being controlled solely by phot2 in Arabidopsis. Dynamic trafficking of phot2 from the plasma membrane to the Golgi apparatus is proposed to be important for this response (Kong et al., 213a,b). Unlike phot2, Otphot does not appear to exhibit blue light-dependent association with the Golgi apparatus (Fig. 4) and may therefore account for the lack of functionality observed. In the case of phototropism, this stems from the inability of Otphot to complex with NPH3 and direct early phototropic signalling events (Fig. 8). While both phot1 and phot2 have been shown to interact with NPH3, only phot1 is able to mediate NPH3 dephosphorylation. The significance of NPH3 dephosphorylation in phototropic signalling has been questioned (Tsuchida-Mayama et al., 28) and the recent work of Haga et al. (215) has proposed that the dephosphorylated form of NPH3 is inactive. The inability of Otphot to mediate phototropism is consistent with a model whereby interaction between phototropin and NPH3 is required to bring about a phototropic response. NPH3 is also involved in leaf flattening and petiole positioning (Inoue et al., 28). An inability to complex with NPH3 may account for the reduced functionality of Otphot in restoring leaf expansion and plant growth in Arabidopsis (Figs 6a, S5), yet Otphot showed robust activity in mediating petiole positioning under the conditions examined (Fig. 5b). Higher light intensities can rescue the leaf positioning phenotype in nph3 mutants (Inoue et al., 28) in a pathway that is dependent on Atphot1 (de Carbonnel et al., 21). Therefore, in this case, Otphot appears to function like Atphot1 to mediate petiole positioning in an NPH3-independent manner. Similarly, Otphot was unable to replace phot2 function in Arabidopsis as it lacked the ability to direct chloroplast avoidance movement (Fig. 7), whereas Crphot complements this response (Onodera et al., 25). Together, these findings argue against the proposal that land plant phots evolved from a phot2-like ancestor (Galvan-Ampudia & Offringa, 27) and support the more recent phototropin phylogeny of Li et al. (Li et al., 214) showing that diversification of a single phototropin ancestor into multiple isoforms occurred independently in seed plants, ferns and mosses. The function of algal phots is largely different to those found in vascular plants (Kianianmomeni & Hallmann, 214). Yet, the results from this study and previous work demonstrate that algal phots are capable of carrying out phot-mediated processes when expressed in Arabidopsis. Ostreococcus is taxonomically positioned at the base of the green plant lineage (Leliaert et al., 211). Hence, features intrinsic to Crphot, which enable it to mediate phototropism and chloroplast avoidance movement in Arabidopsis, appear to be absent from evolutionary distant algal forms such as Otphot. Further comparative structure function analyses between Otphot and Crphot will therefore shed new light in defining protein regions within phototropins that are important for initiating signalling events specifically associated with phototropism and chloroplast avoidance movement in vascular plants. Acknowledgements We thank Francois-Yves Bouget for providing Ostreococcus tauri genomic DNA as a template for PHOT coding sequence amplification and Tatsuya Sakai for providing anti-nph3 polyclonal antibody. We also thank Margaret Ennis for technical assistance.

11 622 Research New Phytologist We are grateful for funding support from the UK Biotechnology and Biological Sciences Research Council (BB/J1647/1 and BB/M2128/1 to J.M.C). L.B. was supported by a UK Biotechnology and Biological Science Research Council PhD Studentship. Author contributions J.M.C. designed and directed the research. S.S., J.P. and L.B. planned and performed experiments. M.P. conducted the stomatal opening measurements. All authors analysed data and J.M.C. wrote the manuscript. All authors commented on the manuscript. References Aihara Y, Yamamoto T, Okajima K, Yamamoto K, Suzuki T, Tokutomi S, Tanaka K, Nagatani A Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin. Journal of Biological Chemistry 287: Barbosa IC, Schwechheimer C Dynamic control of auxin transportdependent growth by AGCVIII protein kinases. 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