Volume 1, Issue 1, Pages 178-194 (January 2008) In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1  Sullivan Stuart , Thomson Catriona E. , Lamont Douglas J. , Jones Matthew A. , Christie John M.   Molecular Plant  Volume 1, Issue 1, Pages 178-194 (January 2008) DOI: 10.1093/mp/ssm017 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 μg) were prepared from 3 d old etiolated seedlings maintained in the dark (D) or irradiated with 100 μmol 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. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 Arabidopsis Seedlings. 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 α-casein. m/z mass to charge ratio in atomic units (amu); cps, counts per second. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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). Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 y8-H3PO4 and y7. This is to be expected for a phosphoserine residue that has undergone loss of phosphoric acid by β-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. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 Jα-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 μg) were probed with anti-phot1 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 μmol 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 μg) were given a mock irradiation (D) or irradiated with white light (L) at a total fluence of 30 000 μmol−2 prior to the addition of radiolabelled ATP. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 ±SE of at least 15 seedlings. (B) Leaf positioning of wild-type (gl1), phot1phot2 double mutant (p1p2) and two independent transgenic lines expressing the LOV2-kinase construct (L2K8 and L2K9) in response to 10 (LW) or 50 μmol m−2 s−1(MW) continuous white light. White solid arrowheads show the first true leaves. The white bar represents 1 cm. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 ±SE of five leaves. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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) μmol 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 μm. (B) Numbers of chloroplasts at the front face per mesophyll cell after light treatment. Each value represents the mean ±SE of the numbers observed in 25 cells. Light treatments were as described in (A). Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 10 Increased Tolerance of phot1phot2 Mutants to Desiccation. (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 ±SE of at least eight plants. Molecular Plant 2008 1, 178-194DOI: (10.1093/mp/ssm017) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions