SUPPORTING APPENDIX TABLE OF CONTENTS Figure S1: Compartmentalized redox proteome under steady state conditions in P. tricornutum cells. Figure S2: Redox regulation of metabolic pathways. Figure S3: Evolutionary conservation of GOGAT reactive cysteines. Figure S4: Expression levels of P. tricornutum redox sensitive nitrogen metabolism genes in EST libraries. Figure S5: Redox regulation of primary nitrate assimilation in diatoms. Figure S6: Targeting the in vivo redox sensor roGFP to different subcellular compartments in P. tricornutum cells. Figure S7: Growth of P. tricornutum cultures under low nitrogen conditions. Figure S8: GSH depletion under nitrogen starvation and oxidative stress conditions. Figure S9: In vivo quantification of the oxidation state of redox sensitive cysteine in roGFP. Table S1: Diatom redox proteome under oxidative stress. A list of P. tricornutum redox-sensitive proteins which were identified in this study. Predicted protein localization, sensitive cysteine position and degree of oxidation under steady state and H2O2 treatment are included. When the identified peptide contains more than one cysteine, the cysteine position refers to the first cysteine. P-value ranges are marked by asterisks: *** P < 0.01, ** 0.01 < P < 0.05, * P < 0.1. Table S2: Biological function enriched in P. tricornutum redox-sensitive proteome. Significantly enriched terms (p<0.05) are included. Enrichment of Gene Ontology (GO) terms was analyzed using the Ontologizer software (http://compbio.charite.de/contao/index.php/ontologizer2.html). Table S3. List of abbreviations. Supplementary methods Figure S1: Compartmentalized redox proteome under steady state conditions in P. tricornutum cells. The thiol proteome distribution under steady state condition is presented based on prediction of their subcellular localization and their level of oxidation. Chl, chloroplast; Mit, mitochondria; Nuc, nucleus; SP, signal peptide; SA, signal anchor; NT, not targeted. TCA cycle Cofactors, Prosthetic Groups, Nucleosides and Nucleotide Calvin cycle and Nitrogen Compound Electron Carrier Biosynthesis Biosynthesis photorespiration Metabolism Amino Acids Biosynthesis Secondary Metabolite Biosynthesis Pentose Phosphate Aromatic Amines and Glycolysis Compound Polyamine Fatty Acid and Lipid Pathway Biosynthesis Biosynthesis Biosynthesis Figure S2: Redox regulation of metabolic pathways. Overview of P. tricornutum peroxide-sensitive proteins that are involved in metabolic network reactions. Redox proteomics data was mapped to metabolic-map diagrams using the DiatomCyc databases at http://www.diatomcyc.org/. Each node in the diagram represents a single metabolite and each line represents a single bioreaction. Reactions catalyzed by proteins that were found as redox-sensitive (ΔOX >10%) are marked in red. Figure S3: Evolutionary conservation of GOGAT reactive cysteines. Sequence comparison of GOGAT protein from 38 organism is shown. Highly conserved cysteines, Cys120 and Cys704, which were found as redox sensetive are highlithed. Multiple sequence alignment was done and visulized using MATLAB software. 20 nitrite reductase ornithine aminotransferase glutamate synthase 1 carbamoyl-phosphate synthase 16 serine hydroxymethyltransferase isocitrate nadp-dependent serine hydroxymethyltransferase glycine dehydrogenase 12 arginase glutamate synthase 8 Frequencies of ESTs of Frequencies 4 0 OS UA AA NS NR Figure S4: Expression levels of genes encoding redox-sensitive proteins participating in nitrogen metabolism in P. tricornutum EST libraries. Expression levels in five EST libraries derived from different nitrogen conditions is presented. Level of expression is presented as frequencies of ESTs in each library. UA, Urea Adapted, AA, Ammonium Adapted, NR, Nitrate Replete, OS, Original Standard, NS, Nitrate Starved. A B 50 50 N N 15 40 15 40 30 30 20 20 Atom % excess % Atom excess % Atom 10 10 0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Time (min) Time (min) Figure S5: Redox regulation of primary nitrate assimilation in diatoms. P. 15 tricornutum cells were supplied with N-nitrate under oxidative stress (150µM H2O2) 15 and control conditions (0µM H2O2) and the enrichment of N in glutamine labeled in amide and amino groups was detected using multiple reaction monitoring (MRM) applying the following parameters: 148.1>130.1 (A) and 148.1>131.1 (B). a Chl-roGFP H4 promoter OEE tp roGFP Mit-roGFP H4 promoter GSIII tp roGFP Nuc-roGFP H4 promoter H4 sp roGFP b BF roGFP Marker Overlay roGFP mitoTracker DAPI Chl Chlorophyll autoflorescence Mit Nuc 10µm Figure S6: Targeting the in vivo redox sensor roGFP to different subcellular compartments in P. tricornutum cells (a) Schematic representation of roGFP constructs. Histone H4 (H4) promoter was used for all constructs for constitutive expression of the roGFP protein. Mit-roGFP has mitochondrial transient peptide (GSIII), nuc-roGFP has a nuclear signal peptide (Histone H4), and chl-roGFP has chloroplastic transit peptide (OEE1). (b) Verification of roGFP subcellular localization. P. tricornutum cells expressing roGFP were viewed under a fluorescence microscope. Images of expression in the chloroplast, mitochondria and nucleus are shown. For each P. tricornutum strain images of bright field (BF), roGFP fluorescence (ex: 488nm, em: 525nm) and various sub-cellular markers are presented. Chlorophyll autofluorescence (ex: 500nm, em: > 650nm), mitoTracker stain (ex: 540nm, em: 630nm), and DAPI stain (ex: 350nm, em: 460nm) were used as markers for plastid, mitochondria, and nucleus localization, respectively. Scale bar: 10µm. Mit, mitochondria; Nuc, nucleus; Chl, chloroplast. 4 + N 3 - N ) 1 - 2 Cell·ml 6 10 ( Cell abandence Cell 1 0 0 10 20 30 40 50 60 70 Time (hr) Figure S7: Growth of P. tricornutum cultures under nitrogen-starved conditions. Cells from an exponential culture (~2•105 cells/ml) were washed with nitrogen-free ¯ media and resuspended in media containing 882µM NO3 (+Nitrate) or nitrogen-free media (-Nitrate). Subsequently, cell abundance was monitored over growth phase of 72hr. 1.2 1 0.8 ++ Nitrate Nitrate 0.6 -- Nitrate Nitrate GSH (AU) ++ Nitarte, Nitrate, H H22OO2 % of control% 0.4 -- Nitrate, Nitrate, H H2022O2 0.2 0 1 2 3 4 Day Figure S8: GSH depletion under nitrogen starvation and oxidative stress conditions. Cells were exposed to nitrogen replete or depleted media and GSH level were determined with or without addition of hydrogen peroxide (150µM, 2hr) by applying the specific GSH dye monochlorobimane. Fluorescence data was analyzed by flow cytometery and presented as percentage of monochlorobimane fluorescence of control (day 1, nitrogen replete). Figure S9: In vivo quantification of the oxidation state of redox sensitive cysteine in roGFP. Exponential P. tricornutum cells were subjected to OxICAT labeling under steady state and oxidized conditions (150μM H2O2, 20 min). Mass spectra are shown for the peptide containing the Cys204 of roGFP which is responsible for the redox properties of the protein. Peptide mass signal with lower m/z value corresponds to the thiol reduced state, labeled with light 12C-ICAT reagent (blue) and higher m/z value corresponds to thiol oxidized state, which is labeled with heavy 13C-ICAT reagents (red). The difference in m/z between the ICAT labeled pairs is equal to 9.0 Da (difference in mass between light and heavy ICAT reagent) divided by the charge state of the peptide. As seen under steady state conditions, a majority of the protein is in its reduced state, while after H2O2 treatment most of the protein is in its oxidized state and the difference in degree of oxidation between these conditions is 41%. These results were comparable to the fluorescence measurement of the roGFP. Table S1 Protein Code Cystein FC intensity Protein annotation JGI code Peptide Name % oxidation rest % oxidation H2O2 deltaOxi p-value Localization NCBI position (H2O2 /rest) ROS metabolism and redox signalling alkyl hydroperoxide reductase thiol specific antioxidant mal allergen 217408144 12713 GCTLEAR 100 12.0 47.2 35.2 ** 6.64 SP peroxiredoxin 5, atypical 2-Cys peroxiredoxin 217405639 14898 VAIFGVPGAFTPGCSK 94 16.9 30.8 13.9 * 2.25 Chl thioredoxin h 217408133 56471 LAEENPDIEFVKVDVDEADDVAAHCGVR 69 35.3 45.5 10.2 ** 1.54 Cyt redoxin domain protein 217405724 22388 VVVFAIPGAFTPTCSSTHLPGYEAAYDK 98 17.8 29.9 12.1 ** 1.94 SP thioredoxin 219110721 8167 DELLDTIVGCVAK 170 22.0 37.9 15.9 ** 2.16 Chl peroxiredoxin q 217406452 21736 KPLVVYFYPADSTPGCTK 74 33.0 57.4 24.3 *** 2.77 Chl ascorbate peroxidase 219122837 47395 YGRVDASGPENCSAEGNLPDAEPGPDGK 158 12.5 34.6 22.0 * 3.77 Chl ascorbate peroxidase 219122837 47395 VDASGPENCSAEGNLPDAEPGPDGK 158 9.6 29.4 19.8 *** 3.91 Chl ascorbate peroxidase 219122832 54731 IDSNGPENCSK 125 12.3 23.0 10.7 * 2.10 Cyt dehydroascorbate reductase 219120379 36641 CSLESVLLR 167 20.5 38.5 18.1 * 2.48 Cyt cytochrome c peroxidase 219120259 13174 CPANGRLPDATQGAEHLR 127 16.6 27.3 10.7 ** 1.89 Cyt glutaredoxin 2 219123711 38124 DVLPTVYVYDHCPFCVR 73 46.2 69.5 23.3 2.64 SP Photosynthesis and Chlorophyll biosynthesis magnesium chelatase atpase subunit d 219112735 33017 DLKVAPSQMQYLCEEAIR 372 46.4 66.5 20.1 ** 2.31 Chl ferredoxin 219127706 49401 VKIPVNCQK 106 55.8 77.6 21.8 ** 2.73 Chl fucoxanthin Chlorophyll a c binding protein 219110471 54065 LAMLAAAGCMAQELANGK 186 3.2 13.4 10.2 ** 4.60 Chl pgr5-like protein 1a 217411614 42543 CYIDTGICK 192 49.8 60.4 10.6 * 1.53 Chl ferredoxin 219127706 49401 ACSTPLPTGK 126 20.9 43.9 22.9 * 2.84 Chl ferredoxin 118411008 SDCTISVHQEDELY 88 49.2 59.5 10.3