In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

Molecular Plant

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Volume 1

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Number 1

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Pages 178–194

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January 2008

In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1 Stuart Sullivana,*, Catriona E. Thomsona,*, Douglas J. Lamontb, Matthew A. Jonesa and John M. Christiea,1 a Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow, Scotland, UK b FingerPrints Proteomics Facility, Post-Genomics and Molecular Interactions Centre, School of Life Sciences, MSI/WTB/CIR Complex, University of Dundee, Dundee, Scotland, UK

ABSTRACT Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser58, Ser85, Ser350, and Ser410), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

INTRODUCTION Plants are dependent on the surrounding light environment to direct their growth. Genetic analysis using Arabidopsis thaliana has shown that the effects of UV-A/blue light (320– 500 nm) on plant development are mediated by three distinct classes of photoreceptors: cryptochromes, phototropins and a new class of blue-light receptors known as the ZTL/ADO family (Briggs, 2006). Cryptochromes play a key role in promoting plant photomorphogenesis (Batschauer, 2005) whereas phototropins operate to control processes that serve to optimize photosynthetic efficiency and promote growth (Christie, 2007). Members of the ZTL/ADO family include proteins that mediate targeted degradation of components associated with circadian clock function (Kiba et al., 2007; Kim et al., 2007) and flowering (Sawa et al., 2007). Phototropins are ubiquitous in higher plants and have been identified in several plant species, including the biflagellate unicellular green alga Chlamydomonas reinhardtti (Briggs et al., 2001). Arabidopsis contains two phototropins (phot1 and phot2) that share partially overlapping functions but exhibit different photosensitivities (Christie, 2007). Phototropins were first identified as photoreceptors for phototropism in Ara-

bidopsis (Huala et al., 1997; Sakai et al., 2001) but have been shown to regulate additional photoresponses including chloroplast relocation movements (Kagawa et al., 2001), light-induced stomatal opening (Kinoshita et al., 2001), cotyledon and leaf expansion (Sakamoto and Briggs, 2002; Ohgishi et al., 2004; Takemiya et al., 2005), and leaf positioning (Inoue et al., 2008). Protein sequences of plant phototropins can be separated into two segments: a photosensory domain at the N-terminus linked to a canonical serine/threonine kinase domain at the Cterminus (Christie, 2007). The N-terminal photosensory region contains two motifs of ;110 amino acids designated LOV1 and LOV2. LOV (light, oxygen, and voltage-sensing) domains are members of the large and diverse superfamily of PAS (Per, ARNT, Sim) domains associated with cofactor binding and mediating protein–protein interactions (Taylor and Zhulin, 1999).

1 To whom correspondence should be addressed. E-mail [email protected]. ac.uk, fax +44 141 330 4447.

* These authors contributed equally to the work. ª The Author 2007. Published by Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssm017, Advance Access publication 4 December 2007

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Both LOV1 and LOV2 function as blue-light sensors for the protein (Salomon et al., 2000) and bind flavin mononucleotide (FMN) as chromophore when expressed heterologously (Christie et al., 1998, 1999), but exhibit different photochemical properties and functional roles. LOV2 plays a major role in regulating phototropin kinase activity and function in Arabidopsis (Christie et al., 2002; Cho et al., 2007; Jones et al., 2007), whereas LOV1 is proposed to mediate receptor dimerization (Salomon et al., 2004) and modulates the photoreactivity of LOV2 (Matsuoka and Tokutomi, 2005). Phototropins belong to the large AGC family of protein kinases (cAMP-dependent protein kinase, cGMP-dependent protein kinase G, and phospholipid-dependent protein kinase C) and are members of the AGC-VIIIb subfamily (Bogre et al., 2003) which includes PINOID —a key regulator of polar auxin transport and signaling (Christensen et al., 2000; Benjamins et al., 2001). Phototropin activation appears to operate through a mechanism by which LOV2 functions as a repressor of the Cterminal kinase domain in the dark (Matsuoka et al., 2007). This repression is alleviated upon photoexcitation, resulting in receptor autophosphorylation that can be monitored both in vivo and in vitro (Christie, 2007). However, information as to the biological consequences of phototropin receptor phosphorylation is lacking. Whether the C-terminal kinase domain phosphorylates additional proteins involved in phototropin signaling is also not known, as only casein has been identified as a substrate for phot2 kinase activity in vitro (Matsuoka and Tokutomi, 2005). To date, the only phosphorylation sites associated with the phototropins have been assigned either by in-vitro phosphorylation of a bacterially expressed N-terminal portion of oat phot1 with bovine protein kinase A (PKA) (Salomon et al., 2003) or through in-vitro binding of a 14–3–3 protein to phot1 from stomatal guard cells of Vicia faba (Kinoshita et al., 2003). To understand the mechanisms underlying the early events associated with phototropin receptor activation and signaling, we have purified the native photoreceptor from Arabidopsis and identified the sites of in-vivo phosphorylation under dark and light conditions using a mass spectrometry approach. Our findings confirm that plant-derived phototropins bind flavin mononucleotide as chromophore and provide direct information as to the sites of receptor phosphorylation in vivo. In addition, structurefunction analysis of Arabidopsis phot1 suggests that the LOV2kinase region of the receptor protein, which lacks the main sites of receptor phosphorylation, is sufficient to elicit a range of phot1-mediated responses. The implications of these findings with respect to the mode of phototropin action are discussed.

RESULTS Phot1 Purified from Arabidopsis Binds FMN as Chromophore A transgenic Arabidopsis line expressing full-length phot1 fused to green fluorescent protein (phot1-GFP) under the control of the native PHOT1 promoter has been shown previously to complement the null phot1-5 allele (Sakamoto and

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Briggs, 2002). Native phot1 from several plant species, including Arabidopsis, is plasma membrane associated and exhibits reduced electrophoretic mobility after blue-light irradiation in vivo, consistent with autophosphorylation on multiple sites (Knieb et al., 2005). Phot1-GFP from the aforementioned transgenic line exhibits a similar reduction in electrophoretic mobility upon blue-light treatment (Figure 1A), demonstrating that the in-vivo phosphorylation status of the GFP-fusion reflects that of the wild-type protein. In contrast, no change in electrophoretic mobility was observed for the soluble enzyme UDP-glucose pyrophosphorylase (UGPase) in response to blue light (Figure 1A) that was used as a control for protein loading (Kaiserli and Jenkins, 2007). To purify phot1 from Arabidopsis, we used the transgenic line expressing phot1-GFP. Total membrane proteins were extracted from dark-grown seedlings that had been exposed briefly to blue light. Membrane proteins extracted from non-irradiated seedlings were included as a control. In each case, membrane proteins were solubilized and immunoprecipitated with anti-GFP antibody linked to magnetic microbeads. Immunoprecipitation resulted in purification of phot1-GFP that was clearly visible upon SDS-PAGE followed by Coomassie blue staining (Figure 1B). Purification was specifically due to the presence of the GFP tag, as no phot1 was detected upon immunoprecipitation of the plasma membrane-marker fusion GFP-LTI6b (Figure 1C). Moreover, the identity of the protein purified was confirmed to be phot1 by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) following tryptic digestion of the protein elution (Supplemental Figure 1). Autophosphorylation of phot1-GFP was clearly evident in preparations purified from light-treated seedlings, as indicated by the reduced electrophoretic mobility of the fusion protein relative to the dark control (Figure 1B). Phototropin LOV domains bind the blue-light-absorbing cofactor flavin mononucleotide (FMN) when expressed heterologously in E. coli or insect cells (Christie et al., 1998, 1999; Kasahara et al., 2002). Whether plant-derived phot1 also binds FMN has not been reported. To assess the in-planta chromophore status of phot1, phot1-GFP immunoprecipitated from Arabidopsis was heat denatured to release any cofactor that may be bound noncovalently to the apoprotein. Cleared fractions were monitored for the presence of flavin by fluorescence spectroscopy. Fluorescence excitation and emission spectra showed that flavin was released upon denaturation of phot1-GFP (Figure 1D), the identity of which was subsequently confirmed as FMN by thin-layer chromatography according to its mobility relative to known flavin standards (Figure 1E). As a control, no flavin fluorescence was detected upon immunoprecipitation of the plasma membrane–marker fusion GFP-LTI6b (Figure 1D).

In-Vivo Phosphorylation of phot1 Occurs Upstream of LOV2 To identify the in-vivo phosphorylation sites of Arabidopsis phot1, transgenic seedlings expressing phot1-GFP were grown in the dark and subjected to a brief irradiation (5 min) with

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Figure 1. Immunopurification of phot1-GFP from Arabidopsis and Identification of FMN as its Chromophore. (A) Western blot analysis of phot1 protein levels in wild-type (gl1) and phot1-GFP transgenic plants. Total protein extracts (10 lg) were prepared from 3 d old etiolated seedlings maintained in the dark (D) or irradiated with 100 lmol m 2 s 1 blue light for 5 min (L) and probed with anti-phot1 antibody. Dashed lines indicate lowest mobility edge of phot1 and phot1-GFP. As a control for equal protein loading, blots were probed with anti-UGPase antibody. (B) Coomassie blue-stained SDS-PAGE gel of elutions from anti-GFP immunoprecipitations. Immunoprecipitations from Triton X-100 solubilized microsomal membranes prepared from phot1-GFP seedlings treated as described in (A). Dashed line indicates lowest mobility edge of phot1-GFP. (C) Western blot analysis of anti-GFP immunoprecipitations from microsomal membranes prepared from 3 d old etiolated phot1-GFP and GFP-LTI6b expressing seedlings. Immunoprecipitations were probed with anti-GFP antibody. Positions of the respective fusion proteins are indicated with an asterisk (*). (D) Fluorescence excitation and emission spectra of the chromophore released from anti-GFP immunoprecipitations from phot1-GFP (solid line) and GFP-LTI6b (dashed line) expressing seedlings isolated as described in (C). (E) Identification of the chromophore bound to phot1-GFP as FMN. The chromophore bound to immunopurified phot1-GFP was released by boiling and used for thin-layer chromatography. Retardation factor (Rf) values for the phot1-GFP chromophore and flavin standards are shown.

high-intensity blue light (100 lmol m 2 s 1) to ensure maximal autophosphorylation (Salomon et al., 2003). Non-irradiated samples were included in the analysis so that the in-vivo phosphorylation status of phot1 under dark and light conditions could be assessed by quadrapole-linear ion trap (QTRAP) nLC-MS/MS analysis (Williamson et al., 2006). Specifically, a directed approach known as precursor ion scanning (Laugesen et al., 2004) was used which exploits the property that phosphopeptides undergo facile loss of phosphate during collisioninduced dissociation (CID). Consequently, release of the diagnostic ion PO3– at mass to charge ratio (m/z) 79 can be monitored to identify the phosphopeptides present. Precursor ion scans for tryptic digests of dark- and lighttreated phot1-GFP are shown in Figure 2. Four peaks corresponding to doubly charged peptides were detected for

light-treated phot1-GFP (Figure 2A). Two peaks (2 and 3) at m/z 639.4 and 835.5, respectively, were particularly prominent in comparison to those identified at m/z 587.3 and 1059.6 (1 and 4). In contrast, only one phosphopeptide was detected for dark-treated phot1-GFP (Figure 2B), which corresponded to peak 4 found in the precursor ion scan for light-treated phot1-GFP (Figure 2A). For both dark- and light-treated samples, four additional peaks were detected at m/z 829.5, 974.5, 992.5, and 1039.6, respectively. However, these were later assigned as missed cleavage products from a contaminant, bovine a-casein that had arisen during SDS-PAGE sample preparation (data not shown) and were therefore eliminated from our analysis. Extracted ion chromatographs were generated for each of the precursor ions identified to determine their relative

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Sequence identities of the phosphopeptides were obtained from the tandem MS fragmentation spectra by database searching (Table 1). Based on their calculated mass and charge state, a single phosphorylation site was assigned to each phosphopeptide and the identity of the phosphorylated residue verified by de-novo interpretation. This is illustrated in Figure 4 for the phosphopeptide corresponding to peak 1, where the site of phosphorylation was determined as KSSLSFMGIK. Phosphorylation sites in the remaining peptides were confirmed in a similar manner (Supplemental Figure 2) and are summarized in Table 1. All together, four phosphoserine residues were identified within Arabidopsis phot1 (Figure 5)—two residing upstream of LOV1 (Ser58, Ser185) and two within the linker region between LOV1 and LOV2 (Ser350, Ser410). Phosphopeptide ALSESESTNLHPFMTK containing Ser350 was found predominantly as a methionine sulphoxide ion as a result of methionine oxidation.

LOV2-Kinase Region of phot1 Is Membrane Localized

Figure 2. Automated nLC-MS/MS Analysis Using Precursor Ion Scan of m/z –79 on Tryptic Digests of phot1-GFP Immunoprecipitated from (A) Blue Light-Treated and (B) Dark-Treated Etiolated ArabidopsisSeedlings. Mass and charge states for the precursor ions detected over the nLC separation is shown. Peptides ions 1–4 were subsequently identified as phosphorylated peptides derived from phot1-GFP. Peptide ions denoted with an asterisk (*) were found to correspond to a phosphopeptide originating from a contaminant derived from bovine a-casein. m/z, mass to charge ratio in atomic units (amu); cps, counts per second.

abundance in light- and dark-treated samples (Figure 3). Phosphopeptides corresponding to peaks 1, 2, and 3 were only detected following illumination. Signal intensities for peaks 2 and 3 were over one order of magnitude higher than that observed for peak 1, indicating that these precursor ions were of higher abundance. By comparison, peak 4 appeared equally abundant in both dark- and light-treated samples (Figure 3D), implying that phosphopeptides corresponding to peaks 1 and 4 are in lower abundance relative to those represented by peaks 2 and 3.

To investigate the potential physiological consequences of receptor phosphorylation, we used the phot1-5phot2-1 double mutant to examine the functionality of a truncated version of phot1 lacking the phosphorylation sites mapped in this study. Stable transformation of this mutant with PHOT1 cDNA from Arabidopsis driven by the constitutively expressed cauliflower mosaic virus 35S promoter has been shown to restore wild-type levels of phot1-responsiveness (Christie et al., 2002; Cho et al., 2007). We transformed the phot1phot2 double mutant with the PHOT1 cDNA region encoding a truncated LOV2-kinase version of the photoreceptor under the control of the 35S promoter (Figure 6A). Based on the segregation of kanamycin resistance, four independent homozygous lines, each with a single transgene, were obtained. Data from two LOV2-kinase lines (L2K) are presented, but are representative of the results attained for all lines. Western analysis using a peptide antibody that was raised against the extreme C-terminal region of Arabidopsis phot1 showed that a protein of the expected size (;69 kDa) was detectable in membrane extracts isolated from the L2K transgenic lines, demonstrating that this region is sufficient to mediate plasma membrane association (Figure 6B). Lowermolecular-weight proteins in addition to phot1 were also detectable in extracts from wild-type seedlings (gl1 background), but most likely represented proteolytic cleavage products of the native photoreceptor, as these were not detected in extracts from phot1phot2 double mutant. In contrast to full-length phot1 (Figure 1A), phot1 LOV2-kinase showed no change in electrophoretic mobility in response to in-vivo blue-light treatment (Figure 6C), consistent with an absence of the receptor phosphorylation sites detected by nLC-MS/MS (Figure 5). Similarly, in-vitro phosphorylation analysis showed that no light-induced autophosphorylation activity could be detected for the LOV2-kinase protein in membrane fractions isolated from dark-grown seedlings (Figure 6D).

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Figure 3. Combined Extracted Ion Chromatographs for Phosphorylated Peptides Identified in Tryptic Digests from Light- and Dark-Treated Arabidopsis phot1-GFP. Doubly charged precursor ions were selected for comparison of their relative abundance in light- (blue) and dark-treated (red) phot1-GFP. (A) Precursor ion m/z 587.3 (peak 1; Figure 2). (B) Precursor ion m/z 639.4 (peak 2; Figure 2). (C) Precursor ion m/z 835.5 (peak 3; Figure 2). (D) Precursor ion m/z 1059.6 (peak 4; Figure 2).

Previous studies have reported that a fraction of phot1 is rapidly internalized (within minutes) from the plasma membrane upon blue-light irradiation (Sakamoto and Briggs, 2002; Knieb et al., 2004). However, no relocalization to the soluble fraction was detected for the LOV2-kinase protein by Western analysis (Figure 6C), whereas partial relocalization of phot1 to the soluble fraction was routinely observed for wild-type seedlings using the same conditions (data not shown).

Phot1 LOV2-Kinase Mediates Phototropism and Leaf Positioning under Moderate Light Intensities Phot1 is the primary phototropic receptor in Arabidopsis mediating phototropism from low to high fluence rates (0.1–100 lmol m 2 s 1) of blue light (Sakai et al., 2001). To determine the functionality of the L2K lines generated, we first examined the ability of the truncated LOV2-kinase protein to restore phototropic responsiveness in the phot1phot2 double mutant. Phototropism was examined over a range of fluence rates. As reported previously (Sakai et al., 2001; Inada et al., 2004; Cho et al., 2007), wild-type seedlings showed phototropic curvature in response to a broad range of fluence rates of blue light (Figure 7A). Minimal curvature was observed for the phot1phot2 double mutant over the light conditions tested.

Table 1. Identification of In-Vivo Phosphorylation Sites in phot1GFP Immunoprecipitated from Dark or Blue-Light-Treated Arabidopsis by nLC–MS/MS. Pre 79 peaka

Peptide sequenceb

Positionc

Dark 4

GTSPQPRPQQEPAPSNPVR

56–74

Light 56–74

4

GTSPQPRPQQEPAPSNPVR

2

SGIPRVSEDLK

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ALSESTNLHPFmTK

348–361

1

KSSLSFMGIK

408–417

179–189

a

Peaks assigned as depicted in Figure 2. Sequence of peptide containing phosphorylation sites. Residues in bold underlined represent definitively identified phosphorylation sites. Met sulfoxide is denoted as m. c Position of peptide shown in protein sequence of Arabidopsis phot1. b

Similarly, no curvature was detected in lines expressing phot1 LOV2-kinase at fluence rates below 1 lmol m 2 s 1. Yet, strong curvature was observed in the L2K transgenic lines at higher fluence rates (.1 lmol m 2 s 1) and was particularly

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the higher fluence-rate dependency detected for the phototropic responsiveness of the L2K transgenic lines (Figure 7A).

Phot1 LOV2-Kinase Mediates Leaf Expansion and Chloroplast Accumulation Movement

Figure 4. In-Vivo Phosphorylation Site Identification of Phosphopeptide KSSLSFMGIK. Tandem mass spectrum obtained for precursor ion at m/z 589.4/2+ (peak 1; Figure 2A) was used in conjunction with MS-Product (ProteinProspector v3.4.1) to annotate C-terminal (y) fragment ions generated by collision-induced dissociation. Only ions relevant to the analysis are shown, to reduce complexity. Subscript denotes the ion position within the identified peptide (inset). Superscripts + and 2+ indicate singly and doubly protonated ions, respectively. The phosphoserine residue within the peptide denoted S was determined by monitoring for the presence of a modified serine residue (dehydroalanine) as indicated by loss of 69 Da between y8H3PO4 and y7. This is to be expected for a phosphoserine residue that has undergone loss of phosphoric acid by b-elimination. Presence of unmodified y6 and y9 ions exclude other serine residues within the peptide as potential sites of phosphorylation. Presence of the originating, doubly charged precursor ion [M+2H]2+ and its H3PO4 loss are also indicated. m/z, mass to charge ratio in atomic units (amu); cps, counts per second.

evident at 10 lmol m 2 s 1, implying that the LOV2-kinase region of phot1 is sufficient to elicit phototropic curvature at moderate light intensities. Phototropins also regulate leaf positioning in Arabidopsis to blue light as a result of differential petiole growth (Inoue et al., 2008). We also observed that leaf positioning is dependent on phototropin receptor activity under white-light conditions. One-week-old seedlings grown under moderate white light (50 lmol m 2 s 1) were transferred to either low white light (10 lmol m 2 s 1) or kept under moderate white light for a further 5 d. Petioles of wild-type seedlings transferred to both low and moderate light intensities grow obliquely upward so that the first leaflets faced the light source from above (Figure 7B). Petioles of the phot1phot2 double mutant, however, grew horizontally under these light conditions, with their leaves slanted downwards. Expression of phot1 LOV2-kinase restored the leaf positioning response in the phot1phot2 double mutant, but more effectively under moderate light intensities (Figure 7B), in agreement with

Genetic analysis has shown that either phot1 or phot2 is sufficient to support normal leaf expansion in Arabidopsis (Sakamoto and Briggs, 2002; Takemiya et al., 2005). Rosette leaves of the phot1phot2 double mutant, grown under 70 lmol m 2 s 1 white light for 3 weeks, are epinastic and curl downward at the sides in comparison to leaves from mature wild-type plants (Figure 8A). Leaf expansion was fully restored in the L2K transgenic lines as measured by monitoring the leaf expansion index relative to those of wild-type and phot1phot2 plants (Figure 8B), demonstrating that phot1 LOV2-kinase is sufficient to bring about this response. Given the restoration of leaf expansion observed in the L2K transgenic lines, we also investigated whether phot1 LOV2kinase complemented chloroplast accumulation movement in detached leaves of mature plants (Onodera et al., 2005). Under low light conditions, phot1 and phot2 act redundantly to induce chloroplast accumulation movement to the upper cell surface to promote light capture for photosynthesis (Kagawa et al., 2001). Detached leaves were transferred to low-intensity blue light (1.5 lmol m 2 s 1) to induce chloroplast accumulation movement or kept in darkness as a control. Chloroplasts accumulated at the upper cell surface under weak blue light relative to dark controls in leaves from both wild-type and the L2K transgenic lines (Figure 9), showing that phot1 LOV2-kinase is also biologically active for this response. The level of response obtained for the L2K transgenic lines was slightly lower compared to that observed for wild type under the light conditions used. Irradiation with higher blue-light intensities (10 lmol m 2 s 1) resulted in a modest enhancement of the chloroplast accumulation response in the L2K transgenic lines, whereas the chloroplasts of wild type underwent avoidance movement owing to the presence of functional phot2 (Figure 9). Correspondingly, the localization of chloroplasts in darkness was less sparse in wild type relative to that detected in leaves of the phot1phot2 mutant and in the L2K transgenic lines. These findings are in accordance with known role of phot2 in the positioning of chloroplasts to the anticlinal cell walls in darkness (Suetsugu et al., 2005; Tsuboi et al., 2007).

Arabidopsis Lacking Functional Phototropins Exhibit Enhanced Drought Tolerance In addition to the known physiological responses mediated by the phototropins (Christie, 2007), we have found from our plant growth experiments that the phot1phot2 double mutant is drought tolerant compared to wild-type plants. Individual plants, grown in soil, were regularly watered for 3 weeks prior to drought stress by complete termination of irrigation. For plants that had not been irrigated for 8 d, wild-type plants

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Figure 5. Amino Acid Alignment of the N-terminal and LOV-linker Regions of Phototropins from A. thaliana (AtPhot1 and AtPhot2), Vicia faba (VfPhot1a and VfPhot1b), Oryza sativa (OsPhot1 and OsPhot2) and Avena sativa (AsPhot1a). Phosphorylated serine residues in AtPhot1 are indicated above the sequence and conserved serine residues in other phototropin sequences are boxed. Phosphorylated serine residues identified previously in AsPhot1a by in-vitro phosphorylation using PKA (Salomon et al., 2003), and 14–3–3 binding in vitro to VfPhot1a and VfPhot1b are grey-shaded (Kinoshita et al., 2003). Vertical dashed lines indicate the boundaries of the LOV1 domain.

wilted, whereas the phot1phot2 double mutant remained green and turgid (Figure 10A). Drought tolerance of the phot1phot2 double mutant was further investigated by determining the relative water content (RWC) as a measurement of whole-plant transpiration under water stress conditions. RWC is a reliable indicator of the plant water status, as it closely reflects the balance between water supply and transpiration rate (Cominelli et al., 2005). Transpirational water loss, as determined by RWC after 8 d, was greatly reduced (approxi-

mately 50%) in the phot1phot2 double mutant relative to wild-type plants (Figure 10B). Nevertheless, no difference in transpirational water loss was detected between the phot1phot2 double mutant and wild-type plants after prolonged drought stress (12 d; Figure 10B) such that plants appeared equally wilted (data not shown). We therefore examined whether the L2K transgenic lines showed a drought-sensitive phenotype similar to that of wild type as an indicator of functional activity. L2K transgenics

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Figure 6. Expression and Subcellular Localization of phot1 LOV2-kinase in Transgenic Arabidopsis. (A) Schematic diagram showing the generation of the LOV2-kinase expression vector. In-vivo phosphorylation sites of full-length phot1 are indicated upstream of the LOV2 domain. The LOV domains are shown with bound FMN as chromophore. The positions of the Ja-helix (grey) and the kinase domain (black) are indicated. (B) Western blot analysis of membrane proteins extracted from 3 d old etiolated wild-type (gl1), phot1phot2 double mutant (p1p2), and two independent transgenic lines expressing the LOV2-kinase construct (L2K8 and L2K9). Protein extracts (20 lg) were probed with antiphot1 antibody. (C) Western blot analysis of phot1 LOV2-kinase localization from 3 d old etiolated transgenic seedlings expressing the LOV2-kinase construct given a dark (D) or 20 lmol m 2 s 1 blue-light treatment for 60 min (L). Total protein extract (T) was fractionated into soluble (S) and membrane (M) fractions by ultra-centrifugation. Equal volumes of each fraction were probed with anti-phot1 antibody. The dashed line indicates lowest mobility edge of LOV2-kinase. Blots were also probed with anti-UGPase antibody, which recognizes the soluble UGPase protein. (D) Autoradiograph showing in-vitro phosphorylation activity in membranes isolated from 3 d old etiolated wild-type (gl1), phot1phot2 double mutant (p1p2), and a transgenic line expressing the LOV2-kinase construct (L2K8). Protein samples (20 lg) were given a mock irradiation (D) or irradiated with white light (L) at a total fluence of 30 000 lmol 2 prior to the addition of radiolabelled ATP.

showed an intermediate phenotype when compared to wildtype and phot1phot2 mutant plants following 8 d of drought treatment. Although the L2K transgenic lines appeared wilted and less turgid than the phot1phot2 double mutant, the phenotype was not as severe as that observed for wildtype plants (Figure 10A). RWC measurements following 8 d without irrigation confirmed that transpirational water loss was reduced in L2K transgenic lines relative to wild type (Figure 10B), but was not as pronounced as that measured for the phot1phot2 double mutant, implying that expression of phot1 LOV2-kinase can only partially restore drought sensitivity.

DISCUSSION Purification and Localization of phot1 from Arabidopsis Considerable progress has been made in characterizing the mode of phototropin activation by light through the expression of full-length or truncated forms of the receptor in heterologous systems (Christie, 2007). To date, very little work has been done using the photoreceptor protein obtained directly

from a plant source owing to difficulties in purifying suitable quantities for biochemical and photochemical characterization. Previous attempts to purify phot1 from oat employed the use of the reactive dye Cibachron Blue 3GA, which has a high affinity for the nucleotide binding site of protein kinases (Knieb et al., 2004). Our present findings demonstrate the feasibility of isolating microgram quantities of phot1 from transgenic plants expressing the protein as a C-terminal GFP fusion (Figure 1B). A similar epitope-tagging approach was used to purify the blue-light-sensing White Collar Complex (WCC) from the filamentous fungus Neurospora crassa (He et al., 2002). Whereas WCC binds flavin adenine dinucleotide (FAD), presumably via the LOV domain of WC-1 (Froehlich et al., 2002; He et al., 2002), we conclusively show that Arabidopsis phot1 utilizes FMN as chromophore (Figure 1) analogous to its heterologously expressed counterparts (Christie et al., 1998, 1999). Further modification of the purification protocol described may yield sufficient quantities of photoreceptor holoprotein to facilitate biophysical and structural analyses of plant-derived phototropins.

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Figure 7. Hypocotyl Phototropism and Leaf Positioning in phot1 LOV2-kinase Expressing Seedlings. (A) Phototropism fluence rate response curves in 3 d old etiolated wild-type (gl1), phot1phot2 double mutant (p1p2) and two independent transgenic lines expressing the LOV2-kinase construct (L2K8 and L2K9). Curvatures were measured after 24-h exposure to unilateral blue light at the indicated fluence rate. Each value is the mean 6SE of at least 15 seedlings. (B) Leaf positioning of wild-type (gl1), phot1phot2double mutant (p1p2) and two independent transgenic lines expressing the LOV2kinase construct (L2K8 and L2K9) in response to 10 (LW) or 50 lmol m 2 s 1(MW) continuous white light. White solid arrowheads show the first true leaves. The white bar represents 1 cm.

The mechanism by which phototropins associate with the plasma membrane is currently not known. Since the LOV2kinase region of phot1 is effectively membrane localized (Figure 6C), peptide region(s) involved in this process must be contained within this part of the protein. Correspondingly, the C-terminal region of phot2, including the kinase domain, has been shown to confer plasma membrane association (Kong et al., 2006, 2007). Members of the AGC-VIII family of protein kinases, including phot1 and phot2, contain an amino acid insertion between the conserved subdomains VII and VIII of the catalytic subunit, which typically contains a stretch of

Figure 8. Leaf Expansion Measurements of Wild-Type (gl1), phot1phot2 Double Mutant (p1p2) and Two Independent Transgenic Lines Expressing the LOV2-kinase Construct (L2K8 and L2K9). (A) Leaf expansion phenotypes of 3 week old plants. The fifth rosette leaf from each plant is shown. The leaf from the phot1phot2 double mutant is curled at the edges and is therefore lying on its side. The white bar represents 1 cm. (B) The leaf-expansion index of the fifth rosette leaves from the plants shown in (A). The leaf-expansion index was expressed as the ratio of the leaf area before and after artificial uncurling. Each value is the mean 6SE of five leaves.

basic residues (Bogre et al., 2003). Recent data suggest a role for this insertion in the subcellular localization (Zegzouti et al., 2006b). In addition, a peptide region C-terminal to the kinase domain of phot2 is essential for mediating chloroplast avoidance movement in the fern Adiantum capillus-veneris (Kagawa et al., 2004), but whether this region is required for phototropin plasma membrane association has yet to be investigated. No partial relocalization of phot1 LOV2-kinase from the membrane to the soluble fraction was detectable by Western blotting (Figure 6C), while this is well documented for native phot1 in wild-type seedlings under similar conditions (Sakamoto and Briggs, 2002; Knieb et al., 2004). Impairment of phot2 kinase activity has been shown to prevent

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Figure 10. Increased Tolerance of phot1phot2 Mutants to Desiccation.

Figure 9. Chloroplast Relocation in Wild-Type (gl1), phot1phot2 Double Mutant Plants (p1p2) and Two Independent Transgenic Lines Expressing the LOV2-kinase Construct (L2K8 and L2K9). (A) Rosette leaves were detached from 4 week old plants. The leaves were treated with 1.5 (low blue, LB) or 10 (medium blue, MB) lmol m 2 s 1 blue light on agar plates, or kept in darkness (D) before observation. Chlorophyll autofluorescence was detected with a confocal laser scanning microscope. The white bar represents 20 lm. (B) Numbers of chloroplasts at the front face per mesophyll cell after light treatment. Each value represents the mean 6SE of the numbers observed in 25 cells. Light treatments were as described in (A).

light-stimulated relocalization from the plasma membrane to the Golgi apparatus (Kong et al., 2006). Therefore, one possible consequence of receptor phosphorylation upstream of LOV2 may be to promote changes in subcellular localization. However, we cannot exclude the possibility that a partial relocalization of phot1 LOV2-kinase was beyond the level of detection in our biochemical fractionation analysis. Further work to examine the localization of phot1 LOV2-kinase as a translational fusion to GFP will now be necessary to confirm whether regions upstream of LOV2 are required to promote lightinduced changes in phot1 subcellular localization.

(A) Representative wild-type (gl1), phot1phot2 double mutant plants (p1p2) and two independent transgenic lines expressing the LOV2-kinase construct (L2K8 and L2K9) 8 d after the cessation of irrigation. The white bar represents 1 cm. (B) Changes in the RWC during drought. Plants grown under normal watering conditions for 24 d were drought-stressed by complete termination of irrigation. Each value represents the mean 6SE of at least eight plants.

In-Vivo Phosphorylation of Arabidopsis phot1 Purification of phot1-GFP from Arabidopsis enabled identification of in-vivo sites of receptor phosphorylation. From our analysis, phot1 is phosphorylated on multiple serine residues consistent with previous reports (Palmer et al., 1993; Short et al., 1994; Salomon et al., 2003). Four abundant phosphorylation sites were identified in the present study (Table 1), namely Ser58, Ser185, Ser350, and Ser410, the latter three being induced by light. Although the general location of these sites (Figure 5) corresponds to those identified in an earlier study (Salomon et al., 2003), the residues that are in effect phosphorylated do not. This discrepancy most likely results from the difference in techniques employed; in-vitro phosphorylation of a bacterially expressed N-terminal portion of oat phot1 by bovine PKA in combination with site-directed mutagenesis was used previously (Salomon et al., 2003) in contrast to the in-vivo mass spectrometry approach adopted here. The in-vivo phot1 phosphorylation sites identified in this study conform to PKA-like consensus sequences RxS or RxxS (Ubersax and Ferrell, 2007), strongly suggesting that phototropins are, as had been proposed previously (Salomon et al., 2003), light-activated PKA-like kinases. However, whether

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the phosphorylation status detected results exclusively from receptor autophosphorylation requires further analysis, since we cannot exclude at this point that these phosphorylation events are mediated by a kinase(s) other than phot1 itself. Ser410 within Arabidopsis phot1 corresponds closely to one of the residues (Ser349) assigned to oat phot1 by in-vitro phosphorylation by PKA (Salomon et al., 2003). Relative to Ser185 and Ser350, phosphorylation of Ser410 was detected in lower abundance (Figure 3), suggesting that a smaller percentage of immunoprecipitated phot1 is phosphorylated at this residue. Ser410 is not conserved between phot1 and phot2 protein sequences from different plant species, although equivalent PKA-like RxS or RxxS motifs are located near to this site (Figure 5). Contrary to the other phosphorylation sites identified, Ser58 appears to be equally phosphorylated in both dark- and lighttreated phot1-GFP samples (Figure 3D) and concurs with the basal dark levels of kinase activity detected for phot1 both in vivo and in vitro (Christie, 2007). Nevertheless, this site occurs within an amino acid region that is highly variable between different phototropins (Figure 5). One consequence of phototropin autophosphorylation is to mediate 14–3–3 binding (Shimazaki et al., 2007). Specifically, 14–3–3 binding to phot1a and phot1b from Vicia faba requires phosphorylation of Ser358 and Ser344, respectively, situated in the linker region between LOV1 and LOV2 (Kinoshita et al., 2003). Amino acid sequence identity between phototropins is less conserved within this peptide region, but there are two serine residues situated nearby that adopt a PKA-like consensus in Arabidopsis phot1: Ser376 and Ser387 (Figure 5). Our preliminary analysis indicates that phosphorylation of Ser387 is not required for 14–3–3 binding to Arabidopsis phot1 (C.E. Thomson and J.M. Christie, unpublished), where Ser376 was not identified as a potential phosphorylation site in the present study. Further analysis is now required to identify the phosphoserine motif necessary for 14–3–3 binding to Arabidopsis phot1 in vivo. A possible candidate is Ser350, which was the most abundant light-induced phosphorylation site detected in our analysis (Figure 3). This residue is highly conserved among phot1 protein sequences, but not in phot2 sequences from rice and Arabidopsis (Figure 5). Whether phot2 exhibits 14–3–3 binding remains to be assessed, as does the biological significance of 14–3–3 binding to phot1. Ser185, like Ser350, is abundantly phosphorylated in response to blue light (Figure 3). Ser185 is located within a highly conserved amino acid region just before the LOV1 domain of phot1 and phot2 (Figure 5). Light-induced structural changes are well documented for LOV2, and involve unfolding of a neighbouring C-terminal a-helix (named the Ja-helix; Figure 6A) from the surface of the LOV2-core (Harper et al., 2003) which is required for kinase activation (Harper et al., 2004; Jones et al., 2007). The fungal photoreceptor VIVID from Neurospora crassa has recently been shown to operate through a similar mechanism whereby photoactivation of the LOV-domain results in displacement of residues N-terminal to the LOVcore (Zoltowski et al., 2007). So far, no information has been

attributed to sequences in the N-terminal to LOV1 domain of plant phototropins. Regulation of protein kinases typically involves phosphorylation of residues within the activation loop (Ubersax and Ferrell, 2007). For instance, phosphoinositol-dependent protein kinase 1 (PDK1) is a potent enhancer of PINOID kinase activity in Arabidopsis and acts by phosphorylating one or more serine residues within the activation loop (Zegzouti et al., 2006a). By contrast, phototropins lack the hydrophobic docking site that facilitates PDK1 binding (Bogre et al., 2003). However, no in-vivo phosphorylation sites were detected downstream of LOV2 in our analysis. Likewise, no in-vitro autophosphorylation activity was detected for phot1 LOV2kinase in microsomal membrane fractions (Figure 6D). Nonetheless, it should be noted that complete determination of phosphorylation sites by LC-MS/MS carries some difficulties owing to insufficient sequence coverage and peptides phosphorylated at substoichiometric levels (Steen et al., 2006). Therefore, phosphorylation sites in addition to those identified here (Figure 5) may be present within Arabidopsis phot1 that were not detected in our analysis.

Implications Regarding the Functional Activity of phot1 LOV2-Kinase Our previous studies have shown that the photochemical reactivity of LOV2 is necessary to bring about both phot1- and phot2-mediated phototropism and leaf expansion in Arabidopsis, whereas the photoactivity of LOV1 is not (Christie et al., 2002; Cho et al., 2007). At least for the physiological responses examined in this study, truncation of phot1 to the LOV2-kinase region does not seem to abolish protein functionality. These data are in accordance with an earlier report demonstrating that an equivalent truncation of phot2 is active in mediating chloroplast avoidance movement in Adiantum (Kagawa et al., 2004). Together, these findings would argue against the importance of LOV1 and receptor phosphorylation in phot1-mediated signaling. Interpretation of the biological activity of phot1 LOV2kinase is somewhat difficult because the phot2-1 allele used in our analysis is not a complete null and has been shown to express a low level of phot2 protein (Cho et al., 2007). In fact, the level of residual phot2 resulting from the leaky phot2-1 allele appears to be extremely low such that it is not readily detectable by Western blotting of total protein extracts from mature plants (Supplemental Figure 3). Our physiological analyses on young seedlings show that expression of phot1 LOV2-kinase in the phot1-5phot2-1 mutant restores both phototropism and leaf positioning under moderate light intensities (Figure 7). Interestingly, such properties are more representative of phot2 fluence-rate dependencies rather than phot1 (Christie, 2007). Thus, expression of phot1 LOV2-kinase could somehow potentiate the activity of residual phot2 present in the phot1-5phot2-1 mutant to bring about the biological activity observed. While this possibility is difficult to exclude, it is worth noting that chloroplast avoidance

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movement, which is solely mediated by phot2, was not detected in the L2K transgenic lines (Figure 9), even following exposure to 100 lmol m 2 s 1 blue light (data not shown). In addition, phot1 LOV2-kinase was able to induce chloroplast accumulation movement effectively at blue-light intensities below 2 lmol m 2 s 1 (Figure 9), which is not consistent with the fluence-rate dependency of phot2 for this response (Kagawa and Wada, 2000; Sakai et al., 2001). An alternative explanation for the phenotypes observed in the L2K transgenic lines could be that residual phot2 interacts with and elicits activation of phot1 LOV2-kinase that would otherwise be non-functional. Indeed, a similar heterointeraction mechanism has been proposed to account for the phototropic responsiveness detected under moderate intensities for a LOV-inactivated version of full-length phot1 expressed in the phot1-5phot2-1 mutant, and is hypothesized to involve cross-phosphorylation between functional phot2 and inactive phot1 (Cho et al., 2007). If this mechanism holds for the L2K transgenic lines, cross-phosphorylation between residual phot2 and phot1 LOV2-kinase would have to occur at sites other than those identified in this study (Table 1). It is also interesting to note that expression of a LOV-inactivated version of full-length phot1 is unable to restore leaf expansion in the phot1-5phot2-1 mutant (Cho et al., 2007), whereas phot1 LOV2-kinase is fully functional in this regard (Figure 8). This discrepancy would therefore imply that residual phot2 present in the phot1-5phot2-1 mutant has a greater capacity to activate phot1 LOV2-kinase. A more plausible and simple explanation for the phenotypes observed in the L2K transgenic lines is that phot1 LOV2-kinase itself is biologically active. The presence of LOV1 has been reported to extend the lifetime signal of phototropin activity by slowing its dark recovery process (Christie et al., 2002; Kagawa et al., 2004). Consequently, deletion of the N-terminal region of phot1 including the LOV1 domain would be expected to enhance dark recovery and account for the reduced photosensitivity observed for both phototropism and leaf positioning in the L2K transgenic lines (Figure 7). Ascribing the biological activity observed for the L2K transgenic lines solely to phot1 LOV2-kinase would, as mentioned above, imply that regions upstream of LOV2, including the sites of receptor phosphorylation, are not important for receptor signaling and raise questions as to how phot1 initiates signalling. One possibility is that the kinase domain of phot1 mediates signaling through the phosphorylation of, as yet unidentified, protein substrates that are not detectable by in-vitro phosphorylation analysis of crude microsomal membrane fractions (Figure 6D). Support for this conclusion comes from a recent study showing that ectopic expression of the C-terminal kinase region of phot2 leads to constitutive activation of phototropin responsiveness even in dark conditions (Kong et al., 2007). The enhanced drought tolerance phenotype observed for the phot1phot2 double mutant is reminiscent of that reported for Arabidopsis mutants deficient in cryptochrome blue-light receptors correlating with a reduction in stomatal perfor-

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mance (Mao et al., 2005). As cryptochromes function additively with phototropins in mediating blue-light-induced stomatal opening (Kinoshita et al., 2001; Mao et al., 2005), the drought tolerance phenotype of the phot1phot2 double mutant reported here may also correlate with a reduction in stomatal opening. Drought sensitivity was only partially restored in the L2K transgenic lines (Figure 10), suggesting that phot1 LOV2kinase is partly active with respect to stomatal function. 14–3–3 binding could be important in this regard, given that activity of the guard cell H+-ATPase and stomatal opening are regulated by 14–3–3 proteins (Kinoshita and Shimazaki, 2002). With knowledge of the in-vivo phosphorylation sites of phot1 at hand, it will now be possible to perform a more detailed functional characterization of the roles of receptor autophosphorylation and 14–3–3 binding to phot1 in Arabidopsis.

METHODS Plant Material and Growth Conditions The wild-type (gl-1, ecotype Columbia) and phot-deficient mutants phot1-5 (nph1-5) and phot1-5phot2-1 (nph1-5cav11) have been described previously (Liscum and Briggs, 1995; Kagawa et al., 2001), as have transgenic Arabidopsis expressing phot1-GFP (Sakamoto and Briggs, 2002) and GFP-LTI6b (Cutler et al., 2000). Seeds were planted on soil or surface sterilized and planted on half-strength Murashige and Skoog (MS) salts medium with 0.8% agar (w/v). After cold treatment (4
C) for 3 d, seeds were grown in a controlled environment room (Fitotron, Weiss-Gallenkamp, Loughborough, UK) under 16/ 8 h 22/18
C light–dark cycle (70 lmol m 2 s 1) unless otherwise stated. The fluence rate for all light sources was measured with a Li-250A and quantum sensor (LI-COR, Lincoln, NE).

Plasmid Construction and Transformation of Arabidopsis The 35S::LOV2-KINASE transformation vector was constructed using the modified binary expression vector pEZR(K)-LC containing an N-terminal 6XHis epitope tag (Christie et al., 2002). A DNA fragment encoding the LOV2-kinase region of Arabidopsis phot1 (residues 448–996) was PCR amplified using the Arabidopsis PHOT1 cDNA and cloned into the transformation vector using EcoRI and SalI and confirmed by DNA sequencing. The resulting 35S::LOV2-KINASE construct was transformed into the phot1-5phot2-1 double mutant with Agrobacterium tumefaciens as described previously (Christie et al., 2002). Based on the segregation of kanamycin resistance, T2 lines that contained a single transgene locus were selected and homozygous T3 seeds were harvested for further studies.

Protein Extraction Unless otherwise indicated, all procedures were carried out under a dim red safe light at 4
C. Plant tissue was ground in a mortar and pestle in extraction buffer containing 50 mM Tris-MES pH 7.5, 300 mM sucrose, 150 mM NaCl, 10 mM potassium

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acetate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor mixture (Complete EDTA-free; Roche Diagnostics, Mannheim, Germany) and clarified by centrifugation at 10 000 g, 4
C for 10 min. The supernatant was used as the total protein fraction. For separation of soluble and membrane proteins, total protein extract was centrifuged at 100 000 g, 4
C for 75 min. The resulting supernatant was used as the soluble fraction while the pellet was resuspended in extraction buffer and used as the membrane fraction. Protein concentrations were determined by the Bradford colorimetric method (Bio-Rad, Hercules, CA). All samples were mixed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% b-mercaptoethanol, 0.004% bromophenol blue), boiled for 5 min and subjected to 7.5% SDS-PAGE.

Western Blot Analysis Plant proteins were detected by Western blot analysis on nitrocellulose membrane (Bio-Rad, Hercules, CA) with anti-phot1 and anti-phot2 purified polyclonal antibodies raised against a peptide fragment at the extreme C-terminal region of Arabidopsis phot1 or phot2 (Cho et al., 2007), anti-GFP monoclonal antibody (BD Biosciences, Palo Alto, CA), and antiUGPase antibody (AS05086, AgriSera, Va¨nna¨s, Sweden). Blots were developed with either horseradish peroxidase (HRP)linked secondary antibodies and Immobilon Western chemiluminescence HRP substrate (Millipore, Billerica, MA) or alkaline phosphatase-linked secondary antibodies and 5-bromo4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) solution (Sigma-Aldrich, St Louis, MO) for colorimetric development.

In-Vitro Phosphorylation Analysis In-vitro phosphorylation analysis of microsomal membrane proteins was carried out under dim red light in the presence of 1% Triton X-100 as described previously (Christie et al., 2002).

Immunoprecipitation of GFP-Tagged Proteins All procedures were carried out under a dim red safe light. Microsomal membranes were prepared from 3 d old, dark-grown seedlings as detailed above and irradiated with a blue-light source as described (Wade et al., 2001) at 100 lmol m 2 s 1 for 5 min or mock-irradiated. For the generation of samples for phosphorylation site mapping, the extraction buffer was supplemented with both half-strength phosphatase inhibitor cocktail 1 and half-strength phosphatase inhibitor cocktail 2 (Sigma-Aldrich, St Louis, MO). The microsomal membrane pellet was resuspended in extraction buffer containing 1% Triton X-100, incubated on ice for 30 min and centrifuged at 100 000 g, 4
C for 30 min. The resulting supernatant containing solubilized microsomal membrane proteins was used for immunoprecipitation using the lMACS GFP isolation kit (Miltenyi Biotech, Surry, UK). Immunoprecipitated proteins were eluted with 0.1 M Triethylamine pH 11.8/0.1% Triton X-100 and neutralized with elution buffer containing 1 M MES pH 3.

Spectral Analysis and Thin-Layer Chromatography Microsomal membranes were immediately used for immunoprecipitation upon resuspension to reduce the loss of chromophore due to prolonged detergent exposure. After elution, samples in elution buffer were boiled for 5 min to release bound chromophore, chilled on ice and centrifuged at 100 000 g for 5 min. Fluorescence spectra of the supernatants were obtained with a LS-55 luminescence spectrometer (PerkinElmer Life Sciences). Fluorescence excitation spectra were obtained by monitoring the emission at 520 nm. Fluorescence emission spectra were measured by using an excitation wavelength of 380 nm. Flavin standards (FAD, FMN, and riboflavin) were diluted in elution buffer and the supernatants from anti-GFP immunoprecipitations, lyophilized to dryness and resuspended in 70% ethanol. Thin-layer chromatography was performed using silica gel powder plates (Merck KGaA, Darmstadt, Germany) with n-butanol/acetic acid/water, 3:1:1 (v/v) as solvent.

Identification of Phosphorylation Sites by nLC-MS/MS Phot1-GFP samples were separated by SDS-PAGE, stained with Coomassie blue and the gel bands excised and digested in 20 mM ammonium bicarbonate with 12.5 lg ml 1 trypsin (Roche, Sequencing Grade) at 30
C for 18 h. Multiple phot1GFP immunoprecipitates were pooled such that approximately 10 lg of phot1-GFP was used for phosphorylation site mapping. The supernatant was removed and the gel pieces extracted with 2.5% (v/v) formic acid/50% (v/v) acetonitrile. The combined extracts were dried under vacuum. Digests were reconstituted in 60 ll of 1% formic acid in water and 20 ll was analyzed by LC-MS on an LC-Packings Ultimate HPLC system interfaced to an Applied Biosystems 4000 QTRAP system. Peptides were separated on a 150 3 0.075 mm PepMapC18 column equilibrated in 0.1% formic acid in water at a flow rate of 300 nl min 1 and eluted with a discontinuous acetonitrile gradient at the same flow rate. The column eluate was mixed with a sheath liquid of propan-2-ol/water (4:1 v/v) at 100 nl min 1 using a capillary Mixing Tee (Upchurch Scientific) and the combined flow plumbed into the microionspray head of the 4000 QTRAP system mass spectrometer fitted with a New Objectives Picotip emitter (FS-360 75 15 N). Electrospray MS was carried out in an automated precursor of 79 duty cycle (6 s total) in negative ion mode (–2300 V), with Q1 masses scanned between 500 and 2000 m/z (3 s), collided with a variable collision energy of –65 to –110 V and daughter ions detected in Q3 after trapping and expelling from the linear ion trap (50 ms fill time). If a daughter ion of PO3– at (m/z) 79 was detected then an enhanced resolution scan was performed to determine accurate mass and charge state. The polarity at the microionspray head was then automatically switched to positive ion mode (+2400 V after 700 ms dwell) and an enhanced product ion scan (tandem MS) of the precursors found in negative ion mode was performed. The polarity was then switched back to –2300 V and the duty cycle

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repeated. All tandem MS spectra were searched against local databases using the Mascot search engine (Matrix Science) run on a local server, and sites of phosphorylation were manually assigned from individual MS spectra viewed using Analyst software (Applied Biosystems). Phosphopeptide mapping analysis was performed with two biological replicas, giving consistent results. To determine protein coverage, 5 ll of phot1-GFP digest was analyzed using a LTQ Orbitrap (Thermo Scientific) and the coverage assigned using Mascot.

Measurement of Phototropic Curvature Hypocotyl curvatures were assayed as described (Lasceve et al., 1999) on square Petri dishes containing half-strength MS/0.8% agar (w/v). Blue light (20 lmol m 2 s 1 and below) was provided by a white fluorescent lamp (18 W/26–835, Osram) filtered through two (for 0.1 lmol m 2 s 1) or one layer (for above 0.1 lmol m 2 s 1) of blue Plexiglas. Images of the seedlings were captured using an Umax PowerLook 1100 scanner. Hypocotyl curvature was measured using ImageJ software (http://rsb.info.nih.gov/ij/).

Observation of Leaf Positioning Arabidopsis seeds were grown on soil under white light at 50 lmol m 2 s 1 for 7 d in a 16/8 h light–dark cycle. Plants were then transferred to 10 or 50 lmol m 2 s 1 continuous white light for 5 d before representative plants were photographed.

Chloroplast Relocation Chloroplast positions were determined as described (Onodera et al., 2005). Rosette leaves detached from 4 week old plants grown on soil were placed on agar plates and irradiated with 1.5 or 10 lmol m 2 s 1 blue light, or placed into darkness for 3 h. Chloroplasts were observed in the mesophyll cells from the adaxial side of the leaves by confocal scanning microscopy.

Measurement of Leaf Expansion Measurement of leaf expansion was carried out as described (Cho et al., 2007). Plants were grown on soil under white light at 70 lmol m 2 s 1 for 3 weeks. The fifth rosette leaves were detached and scanned using an Umax PowerLook 1100 scanner. They were then uncurled manually and scanned again. 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.

Drought Stress and Determination of Relative Water Content Drought stress and plant water status was evaluated by estimating relative water content (RWC) as described (Cominelli et al., 2005). Plants grown individually on soil under white light at 70 lmol m 2 s 1 were regularly watered for 24 d

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and then subjected to drought stress by complete termination of irrigation. Before irrigation was terminated, pots were covered with Saran wrap to reduce soil evaporation. RWC was measured as described (Ascenzi and Gantt, 1999).

ACKNOWLEDGMENTS We thank Winslow Briggs for supplying the phot1-5phot2-1 double mutant. We also thank Eirini Kaiserli for help with confocal microscopy, Margaret Ennis for technical support and Gareth Jenkins for critical reading of the manuscript. This work was supported by UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/C000366/1 (to J.M.C.) and PhD studentships from the BBSRC (to C.E.T.) and the Gatsby Charitable Foundation (to M.A.J.). J.M.C. is grateful to the Royal Society for the award of a University Research Fellowship.

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Supplemental Figure 1. Tandem MS Coverage of phot1 Immunoprecipitated from Etiolated Arabidopsis Seedlings. Amino acid sequence of Arabidopsis phot1 showing the peptides sequenced by tandem MS (underlined). Phosphorylated peptides are indicated in bold. An overall coverage of 68% was attained. Supplemental Figure 2. In-Vivo Phosphorylation Site Identification of Phosphopeptides Derived from Arabidopsis phot1GFP. (A) Tandem mass spectrum obtained for precursor ion at m/z 640.9/2+ (peak 2; Figure 2A) was used in conjunction with MSProduct (ProteinProspector v3.4.1) to annotate b and y fragment ions required to identify the phosphorylation site within the peptide. Subscript denotes the ion position within the identified peptide SGIPRVSEDLK (inset). Superscripts + and 2+ indicate singly and doubly protonated ions, respectively. The identified phosphoserine residue within the peptide denoted S was determined by monitoring for the presence of a modified serine residue (dehydroalanine), as indicated by loss of 69 Da between b7-H3PO4 and b6. Presence of the originating, doubly charged precursor ion [M+2H]2+ is also indicated. m/z, mass to charge ratio in atomic units (amu); cps, counts per second. (B) Tandem mass spectrum obtained for precursor ion at m/z 836.5/2+ (peak 3; Figure 2A) was used to annotate b and y fragment ions as described in (A). Subscript denotes the ion position within the identified peptide ALSESTNLHPFMTK (inset). Superscript + indicates singly protonated ions. The identified phosphoserine residue within the peptide denoted S was determined by monitoring for the presence of a modified serine residue (dehydroalanine) as indicated by loss of 69 Da between y12-H3PO4 and y11. The methionine sulphoxide residue (oxidized methionine) in the peptide is denoted m.

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(C) Tandem mass spectrum obtained for precursor ion at m/z 1062.1/2+ (peak 4; Figure 2) was used to annotate b and y fragment ions as described in (A). Subscript denotes the ion position within the identified peptide GTSPQPRPQQEPAPSNPVR (inset). Superscripts +, 2+, and 3+ indicate singly, doubly and triply protonated ions, respectively. The identified phosphoserine residue within the peptide denoted S was determined by monitoring for the presence of a modified serine residue (dehydroalanine), as indicated by loss of

69 Da between y172+-H3PO4 and y162+. Presence of the originating, triply charged precursor ion [M+3H]3+ is also indicated. Supplemental Figure 3. Western Blot Analysis of Total Proteins Extracted from 4 Week Old Light-Grown Wild-Type (gl1) and phot1phot2 Double Mutant (p1p2) Plants. Protein extracts (30 lg) were probed with anti-phot2 antibody. As a control for equal protein loading, blots were probed with anti-UGPase antibody.