Supplemental Data. Urzica et al. Plant Cell (2012) /tpc Supplemental Figure 1.

Transkript

1 Supplemental Figure 1. 1

2 Supplemental Figure 1. Common and distinct genes targeted in photoautotrophically vs. photoheterotrophically grown C. reinhardtii cells. C. reinhardtii cells were grown photoheterotrophically (acetate) or photoautotrophically (CO 2 ) under iron-replete (20 M iron), - deficient (1 M iron) and -limited (0.25 M iron) conditions. Cells were collected at a density of 3x10 6 cells ml -1. A. Illustrates growth conditions and comparisons E, F and G. Pale green color depicted for ironlimited conditions indicates that the cells are chlorotic. B. The Venn diagram shows the number of differentially expressed genes (fold change 8 for comparisons E, F and G) identified under tested conditions. The numbers in the intersections represent the overlapping genes between two analyses (see Supplemental Data Set 4 online). C. shows the number of induced (yellow) or repressed (blue) genes in each iron nutritional condition when comparing photoautotrophic versus photoheterotrophic conditions. In parentheses are indicated the comparison shown in (B) to which each set of bars correspond. D. Heatmap of the expression patterns for differentially expressed genes. Hierarchical clustering of RNA-Seq count data was performed using a model-based approach with negative binomial distribution (Si et al. unpublished). The heatmap displays the log 2 fold change of each gene for each experimental condition. The first branching separates gene clusters with higher expression in photoautotrophically-grown cells (on the left) from those showing the opposite trend (clusters on the right), in agreement with the principal component results shown in Figure 3. Changes in the colormap inside each cluster reveal second-order responses to different iron levels. Si, Y., Liu, P., Li, P., and Brutnell, T.P. Model-based clustering for RNA-seq data. Unpublished manuscript, preprint available at 2

3 Supplemental Figure 2. Fold changes estimated from RNA-Seq vs. real-time PCR experiments are well correlated. Comparison of fold changes in mrna abundance between iron-replete vs. iron-deficient (A, C) and iron-replete vs. iron-limited (B, D) cells estimated from RNA-Seq vs. real-time PCR experiments is shown. Each data point represents the change in abundance of one mrna (average of two independent experiments). The correlation coefficient was calculated from the regression line derived with a 95% confidence interval. The slope for each curve is

4 Supplemental Figure 3. * 20 * 40 * 60 * 80 * 100 Cre_NRAMP4 : MGGPGARLGPS TVIRIQSDVALDPDAYKFSFKRLLQFMGPGILMSIAYVDPGNLESDLQVGAQA : 64 Vca_56724 : MPSIAEECRNGAAP SVVTVEDSDWEESSQRHFSFRRLLKFMGPGILMSIAYVDPGNLESDLQVGAQA : 67 Coc_C169_36686 : MLEGGLSFRQIGAEDEFVDDAHDRRVTTSSPR------TLFDDDRYSGDDLPKNWFSWRKLWLFMGPGFLMSIAYLDPGNLESQLQAGANS : 85 Chl_NC64A_7420 : MLYLPCRPGALMCIAFVDPGNLEADLQTGATT : 31 MicpuN3_67467 : MDDDDSFSLRKFAAFVGPGFLMCIAYVDPGNFESNLQAGCQF : 42 OstRCC809_12332 : MKTDVGASGDAR EGDGDVQILGLDDEDHRFNLKTLWRFMGPGFLMCIAYVDPGNFESDLQAGTLF : 65 Olu_NRAMP1 : MGPGFLMCIAYVDPGNFESDLQAGTLF : 27 Ostta_26923 : MGPGFLMCIAYVDPGNFESDLQAGVLF : 27 Hsa_SLC11A1 : MTGDKGPQRLSGSSYGSISSPTSPTSPGPQQAPPR--ETYLSEKIPIPDTKPGTFSLRKLWAFTGPGFLMSIAFLDPGNIESDLQAGAVA : 88 Mmu_DCT1 : MVLDPKEKMPDDGASGDHGDSASLGAINPAYSNSSLPHSTGDSEEPFTTYFDEKIPIPEEEYSCFSFRKLWAFTGPGFLMSIAYLDPGNIESDLQSGAVA : 100 Ath_NRAMP3 : MPQLENNEPLLINEEEEEETAYDETEKVHIVRN-----EEEDDLEHGVGCGGAPPFSWKKLWLFTGPGFLMSIAFLDPGNLEGDLQAGAVA : 86 Ath_NRAMP4 : MSETDRERPLLASEERAYEETEKVLIVGI DEEEDADYDDDPGNSPKFSWKKLWLFTGPGFLMSIAFLDPGNLESDLQAGAIA : 82 TM1 TM2 * 120 * 140 * 160 * 180 * 200 Cre_NRAMP4 : GYVLLWLLLLSTLMGLVVQL QAAKLGVVTGRHLAQHCRRQYPRLPRLVLWVMAEVAIIGSDVQEVIGSAIALALLSGGRLPLWAGV : 150 Vca_56724 : GYVLLWLLLLSTVMGLVVQM QAAKLGVVTGQHLAQHCRWQYPPGPRIALWLMAEIAIIGSDIQEVIGSAIALSLLSGGRLPLWAGV : 153 Coc_C169_36686 : GYTLIWVLMWVIIMGYLMQM LAVKLGVATGLNLAQQCRKVYPVFPRYILWIMMEIAIIGSDIQEVVGSAIAISLLTYGAVPLWAGV : 171 Chl_NC64A_7420 : GYRLLWVLLWSTTMGYLLQARQILSPGWLCCLPSLAARLGVATGKHLAEHCREQYPPGVRHVLWVMAELAIIGSDIQEVIGTSIALLLLSRGAVPLWGGV : 131 MicpuN3_67467 : GYSLLWVLLWATLMGLYIQA LSVRLGLATGWHLARVMRDEYPTPARYSLWIVTELAIIASDVPEVIGTALALKLIFG--LPTVWGV : 126 OstRCC809_12332 : GYKLLWVLLWATLGGWYIQG LTVRLALATGWDLARCFREEYPDPVRYALWVISELAIIASDVPEVIGTALALKLIFN--IPTWVGV : 149 Olu_NRAMP1 : GYKLLWVLLWATLGGWYIQG LTIRLALATGWDLARCMREEYPDPTRYALWAMTELAIIASDVPEVIGTALALKLIFG--IPTWVGV : 111 Ostta_26923 : GYKLLWVLLWATAGGWYIQG LTIRLALATGWDLARCLREEYPDPVRYALWVISELGIIASDVPEVIGTALALKLIFS--IPTWVGV : 111 Hsa_SLC11A1 : GFKLLWVLLWATVLGLLCQR LAARLGVVTGKDLGEVCHLYYPKVPRTVLWLTIELAIVGSDMQEVIGTAIAFNLLSAGRIPLWGGV : 174 Mmu_DCT1 : GFKLLWVLLLATIVGLLLQR LAARLGVVTGLHLAEVCHRQYPKVPRIILWLMVELAIIGSDMQEVIGSAIAINLLSAGRVPLWGGV : 186 Ath_NRAMP3 : GYSLLWLLMWATAMGLLVQL LSARLGVATGRHLAELCRDEYPTWARMVLWVMAELALIGSDIQEVIGSAIAIKILSNGILPLWAGV : 172 Ath_NRAMP4 : GYSLIWLLMWATAIGLLIQL LSARLGVATGRHLAELCREEYPTWARMVLWIMAEIALIGADIQEVIGSAIAIKILSNGLVPLWAGV : 168 TM3 TM4 TM5 * 220 * 240 * 260 * 280 * 300 Cre_NRAMP4 : LCTAVVSFSLLFIERLGIRVLEGFFGAMV-GVMVVAFGVMYARAD-VPAGEVLRGFTVPSLP---KKDLPVAVALMGSLIMPHNIYLHSALVQT---RKL : 242 Vca_56724 : LLTAADSFALLFIERLGIRVLEGFFAGMV-GVMVGTFGVMYGRAG-VPLEQVARGFVVPQLP---RKDIPVAVALLGSLIMPHNIYLHSALVQT---RRL : 245 Coc_C169_36686 : LITAVASFMLLLLERWGVRNLEALFAVLI-GVMAVSFGVMYILAG----GTGVAGLLVPRLP---RSALKMAVAIVGAGIMPANFYLHSALVHS---RKI : 260 Chl_NC64A_7420 : LLAAAAAYTLLFLERLGVRYLEVVFELLIAGARVLCSWLAARLAT---DRGCRAGLALPSLP---RAAIPTACGLLGAIIMPHNLFLHSALVHSSRQRPR : 225 MicpuN3_67467 : LVTSASTLLFLALQQFGVRKLEAFMGSLV-GVMSVCFVAQMGMLEGGDGGEVIKGIVVPIFQN--GKALFIGISIIGAVVMPHNLFLHSALVLS---RSF : 220 OstRCC809_12332 : VLTSMSTMVFLGLQSFGVRKLEAFMASLV-GVMSLCFLAEVTFVD-APASSVVAGVVLPRLPS--TKALYIAISLVGAVVMPHNLFLHSALVLS---RGF : 242 Olu_NRAMP1 : VLTSMSTMVFLGLQSFGVRKLEAFMAALV-GVMSLCFLAEVTFVD-AKASSVVAGIVLPRLPG--SKALYIAVSLVGAVVMPHNLFLHSALVLS---RGF : 204 Ostta_2692 : VLTSMSTMVFLGLQSFGVRKLEAFMASLV-GVMSLCFLAECTYVD-AAVSSVTAGIIIPRLPG--STALYIAISLVGAVVMPHNLFLHSALVLS---RGF : 204 Hsa_SLC11A1 : LITIVDTFFFLFLDNYGLRKLEAFFGLLI-TIMALTFGYEYVVAR-PEQGALLRGLFLPSCPGCGHPELLQAVGIVGAIIMPHNIYLHSALVKS---REI : 269 Mmu_DCT1 : LITIADTFVFLFLDKYGLRKLEAFFGFLI-TIMALTFGYEYITVK-PSQSQVLRGMFVPSCPGCRTPQVEQAVGIVGAVIMPHNMYLHSALVKS---RQV : 281 Ath_NRAMP3 : VITALDCFVFLFLENYGIRKLEAVFAVLI-ATMGVSFAWMFGQAK-PSGSELLIGILVPKLS---SRTIQKAVGVVGCIIMPHNVFLHSALVQS---REV : 264 Ath_NRAMP4 : VITALDCFIFLFLENYGIRKLEAVFAILI-ATMALAFAWMFGQTK-PSGTELLVGALVPKLS---SRTIKQAVGIVGCIIMPHNVFLHSALVQS---REV : 260 4

5 TM6 * 320 * 340 * 360 * 380 * 400 Cre_NRAMP4 : GSDRPAAKREAMLYFGIESALSLVVAVVINLFITAVFAAGFHGA ELPDIGLQSAGRYLGDTYGE---AVVYIWAL : 314 Vca_56724 : GSDAPGAKREALTYFGIESALSLVVAVVINLFITAVFAAGFYGA DLPDIGLQNAGRYLGETYGQ---PVVYIWAL : 317 Coc_C169_36686 : DAGQNVRKKEALAYYRIESAVALFCALLINICVVAVFARGFYGT EGVEIGLENAGAYLGETFGA---SMRIIWAI : 332 Chl_NC64A_7420 : ADHGPAVPQESVLYYNIEAGMALLVTLFINVCVISVFARGFFGR GGEEEIGLANAGEYLGATFGK---HMVVIWAV : 298 MicpuN3_67467 : ELGERS-VRRALKYNVAESALALAVTLVINFAVTIVAAQSIRDADFDDPTGEKR QGIIDRPLQNAPDMLKDVLGK---SAKGLFAA : 302 OstRCC809_12332 : SLGERS-LKMAYKYNIVESGLALAVSLFINFAVIIVGAANYAQLTDPVQM QEVRERPLQYAPEMLKQVLGS---SAKGFFAA : 320 Olu_NRAMP1 : SLGEKS-LRMAYKYNIVESGMALSVSLFINFAVVIVAASNYAHLTDPEEM RQVRERPLQYAPQMLKEVLGT---AAKGFFAA : 282 Ostta_26923 : VLGEKS-LKMAYKYNVVESGLALSVSLFINIAVVVVAAANFAHLTDPQEK NNVRDRPLQYAPQMLKEVLGP---AAKGFFAA : 282 Hsa_SLC11A1 : DRARRADIREANMYFLIEATIALSVSFIINLFVMAVFGQAFYQKTNQAAFNICANSSLHDYAKIFPMNNATVAVDIYQGGVILGCLFGP---AALYIWAI : 366 Mmu_DCT1 : NRANKQEVREANKYFFIESCIALFVSFIINVFVVSVFAEAFFEKTNKQVVEVCKNNS-SPHADLFPSDNSTLAVDIYKGGVVLGCYFGP---AALYIWAV : 377 Ath_NRAMP3 : DKRQKYRVQEALNYYTIESTIALFISFLINLFVTTVFAKGFYNT DLANSIGLVNAGQYLQEKYGGGVFPILYIWAI : 340 Ath_NRAMP4 : DPKKRFRVKEALKYYSIESTGALAVSFIINVFVTTVFAKSFYGT EIADTIGLANAGQYLQDKYGGGFFPILYIWAI : 336 TM7 TM8 TM9 * 420 * 440 * 460 * 480 * 500 Cre_NRAMP4 : GLLAAGQSSTMTGTYTGQFVMGGYLDLKVSPWARVAITRAVAIAPTLAVALLCSGSNGDGDGSALDQLNQGLNLLQSVQLPFALVPVLTFSASPAIMGRA : 414 Vca_56724 : GLLAAGQSSTMTGTYTGQFVMGGYLDLKVSPWCRVAITRAVAIAPTLLVALLC----GGGS--QLDQLNQGLNLLQSIQLPFALVPVLTFTSSPVIMG-E : 410 Coc_C169_36686 : GLLAAGQSSTITGVYTGQFVMSGFLNLRISQWKRISITRSVALVPTLLVSLLY----RQPGGTELDILNEWLNVLQSVQIPFALLPLLMLTSSQVIMGPC : 428 Chl_NC64A_7420 : GLLAAGQSSTMTGTYAGQFVMGGFLNLKISAAMRTLVTRGVAICPTLLVALSA----RSDA-TRLDTLNQWINILQSVQLPFAVIPVRAL : 383 MicpuN3_67467 : ALLASGQSSTITGTYAGQFVMEGFLEIKINPVLRAFLTRTCAILPSLLVTIIA----GDEY---AEFLIVISSVFLSFQLPFALIPLVKFCGSEKIVG-A : 394 OstRCC809_12332 : ALLASGQSSTITGTYAGQFVMDGFLELRVNPVLRAAVTRMCAILPSLAVVLIA----GDSY---SESLIVISSTVLAIQLPYALIPLIKFTASPNMMG-P : 412 Olu_NRAMP1 : ALLASGQSSTITGTYAGQFVMDGFLELRVNPVLRAFVTRMCAIVPSLIVVLAA----GDKY---SESLIVISSTILAIQLPYALIPLIKFTASERIMG-P : 374 Ostta_26923 : ALLASGQSSTITGTYAGQFVMDGFLELRVNPVLRAFVTRMCAIVPSLAVVLIA----GDQY---SESLIVISSTILAIQLPFALIPLIKFTASSNIVG-P : 374 Hsa_SLC11A1 : GLLAAGQSSTMTGTYAGQFVMEGFLRLRWSRFARVLLTRSCAILPTVLVAVFR----DLRD---LSGLNDLLNVLQSLLLPFAVLPILTFTSMPTLMQ-E : 458 Mmu_DCT1 : GILAAGQSSTMTGTYSGQFVMEGFLNLKWSRFARVILTRSIAIIPTLLVAVFQ----DVEH---LTGMNDFLNVLQSLQLPFALIPILTFTSLRPVMS-E : 469 Ath_NRAMP3 : GLLAAGQSSTITGTYAGQFIMGGFLNFKMKKWLRALITRSCAIIPTIIVALVF----DSSE-ATLDVLNEWLNVLQSIQIPFALIPLLCLVSKEQIMG-S : 434 Ath_NRAMP4 : GVLAAGQSSTITGTYAGQFIMGGFLNLKMKKWVRALITRSCAIIPTMIVALVF----DSSD--MLDELNEWLNVLQSVQIPFAVIPLLCLVSNEQIMG-S : 429 Supplemental Figure 3. NRAMP4 homologous sequences are highly conserved in algae, plants and animal species. Protein sequences were aligned using MUSCLE, and the alignment was manually adjusted using GeneDoc multiple alignment editor software. Back-shaded amino acids are conserved in 12 (black), (dark grey), or 9-8 (light grey) of the aligned proteins C. reinhardtii (Cre), A. thaliana (Ath), Coccomyxa sp. C-169 (Coc_C169), Chlorella variabilis (ChlNC64A), V. carteri f. nagariensis (Vca), Ostreococcus tauri (Ostta), Ostreococcus sp.rcc809 (OstRCC809), Ostreococcus lucimarinus (Olu), Micromonas sp. RCC299 (MicpuN3), Mus musculus (Mmu), Homo sapiens (Hsa). TM, transmembrane helices; residues highlighted in red were shown to be essential for metal transport in mouse DCT1. Mutations in the conserved amino acid residues from mouse DCT1 (green) indicated significant changes in uptake activity, in the specificity to the various substrates and in the affinity to protons. Residue highlighted in cyan corresponds to G216R mutation that causes microcytic anemia in mk-/- mice and in Belgrade rats. Residue F212 (purple) corresponds to mutation F227I that almost abolished the proton slip generated by DCT1, without any significant changes in the metal ion transport activity and expression levels of the transporter. Conserved histidine residues (H283 and H288 = in blue) correspond to H267 and H272, whose mutations (H267A and H272A) resulted in decreased uptake activity compared to WT DCT1. The metal uptake by H272A is independent of ph and the H272A mutant was sensitive in the presence of Fe 2+. Regions with low amino acid conservation are not shown. 5

6 Supplemental Figure 4. * 420 * 440 * 460 * 480 * 500 Cre_TEF22 : APRALSSAG EALVHFGVEGPGVSGYVSLGFPENPDLMYDADMVLGWVSADGRGVVETYHVTS--YEMSATDVVSQD--WALGSGVVEKRGADG : 441 Cre19.g : ATATASSSSSSAD-----LVHMALSSP-QAGYVSIAFAAKSGVMSPANALIGRIDASGAALVEAYPLEG--YDVGTAVATS---AWGVEGAGVVFTSAGG : 320 Cre14.g : DSAGKTVLAAGDVAT-DGTIHMAVQAA-TSGYVAIGFSPNPGTMGPSDIVLGWAGSSGSS-IRAFYVTG--EDMDDSNALSGTRDWAYDKGVMESGGV-- : 456 Vca_ : GSSDIISKTQQQQQELGKLVHFAVEAA-VGGYVSLGFPENAARMYDADVILGWVNADGRGVVNTYHVTS--YEMTAADV GV : 330 Dre_FRRS1 : TSTGCYFVAVQASSD-QSEMRIEMFGP-ADGYVAIGFSDDQ-QMGNDDVYICGKDNNGNLQVQHAFNSGRSRPAILSLGNVTDILTAVTNGNINCS---- : 302 Hsa_FRRS1 : KEASCVFLSFTRD---DQSVMVEMSGP-SKGYLSFALSHDQ-WMGDDDAYLC-IHEDQTVYIQPSHLTGRSHPVMDSRDTLEDMAWRLADGVMQCS---- : 304 Mmu_FRRS1 : KEPACVFLSFTRD---NQSVMVEMSGP-SDGYVSFAFSHDQ-WMGDDDAYLC-IREDQTVDIQSSYLTGRSYPVMDSRGTLEDMAWRLADSVIQCS---- : 304 Rno_FRRS1 : NEPACVFLSFTRE---NQSVMVEMSGP-SDGYVSFAFSHDQ-WMGDDDAYLC-IYEDQTVYIQPSYLTGRSYPVMDSRGTLEDMAWRLADGVIQCS---- : 301 * 520 * 540 * 560 * 580 * 600 Cre_TEF22 : SPTTIMC FSRRVAEPLARSSPLLDMNDGT IKYSWAVSPEDALVEHPPNGYGAGLVNLRSGTS-S-AIT : 507 Cre19.g : TVLCFSIPASGLSGVAGRRRGRALAQAASNLGLDATA LQVNWAVNDSPDLVTHMAK--GGLQLNAVSGSA---AMA : 391 Cre14.g : ---TTIC FSRRLREPRALASSDLRAAGGLTSAASGASGSSSRRRLAQALPQMGFNWATFPQDRLVQHLSRTVGGFSLDVASGAA---DVV : 540 Vca_ : LKYSWAVSPEDGLVEHPADGFGGGLVDLRSGVVTELDVV : 369 Dre_FRRS1 : FISRNTISTA-SRAATTNEYY LMIAAGSSSQGNIQFHTNK-YVTSTKVNLLNPSVV-ITS : 359 Hsa_FRRS1 : FRRNITLPGVKNRFDLNTSYY IFLADGAANDGRIYKHSQQPLITYEKYDVTDSP-K-NIG : 362 Mmu_FRRS1 : FRRNITLPEAKNRFVLNESYY IFFAEGPSHDGRIFRHSQQPLITYEKYNVTDTP-K-SVG : 362 Rno_FRRS1 : FRRNITLPEAKNRFVLNESYY IFYAEGAAHDGRIFRHSQQPLITYEKYNVTGTP-Q-NVG : 359 TM2 TM3 * 620 * 640 * 660 * 680 * 700 Cre_TEF22 : VKDNSKIIIAHGVLMAVAWVLLLPLGAMAPAHRWLFRGR MWG-----SKAAWFWVHFVGQLGGFGIFCAGFILAMVAFDRPQGG : 586 Cre19.g : AAEKEKFVQVHGALMALSWNFLLPLGLLLARHRWALNGARCAMPTAANADGSPGDLCANNHCCAKEIWFYLHVSCQCGGLLLFLIGFIIAVVKLE-VEGD : 490 Cre14.g : AVDKGYWVNVHGALMAVAWALLLPLGTFLPAHRWVLRD VRGPA---GKHLWFLLHVGCQYVGISLFVAGFVIAYVKLD--NGG : 618 Vca_ : SNRRRAAVLAHGVLMTIAWVLLLPIGAMVPAHRWLFDG RQVGGKALWYWTHIGMQLGGFGTFAVGFVLAMAYFR--RPG : 446 Dre_FRRS1 : EEEFPPMVKAHGCLMLISWMATGSIGMIIARY---LKG VAKGQGCFGKDFWFVAHVSLMTLSIIATAIAFIIVFVS----AGD : 435 Hsa_FRRS1 : GSHSVLLLKVHGALMFVAWMTTVSIGVLVARF---FKP VWSKAFLLGEAAWFQVHRMLMFTTTVLTCIAFVMPFIY----RGG : 438 Mmu_FRRS1 : GSRSSPLLKAHGALMFVAWMTTVSIGVLVARF---FRS VWSKAFFLREAAWFQVHRMLMVATSLLTCVAFVLPFVY----RGG : 438 Rno_FRRS1 : GSRSSALLKAHGALMFVAWVTTVSTGVLVARF---FRS VWSKAFFFGQAAWFQVHRMLMLATSMLTCVAFVLPFVY----RAG : 435 TM4 TM5 TM6 * 720 * 740 * 760 * 780 * 800 Cre_TEF22 : T--L--TSS-HAIMGYVVAGMAGLQMVVAFMRPDPGTKLRVMLWNPLHMNLGRATTLLAWATCLVGAAVHSESICKAPIVPWVATLGSAMGLILLADWAL : 681 Cre19.g : M--GTPRGA-HKVMGYLVAALAGLQFVVAFIRPAPGA-PRRPVWNFLHHNLGRGALLLAWATVYLGVYLAHLSYEQVL-ASWLTPLVAVLACVLGIDGAL : 585 Cre14.g : VVVGGKAGSAHAPIGIAVMAAAGAQMVVGHVRLDPTHR-RRWLWNLVHHNLGRCTVLLAWANVYIGIYMAHTGYQASY-AAWAAPIAAVMGVLVLADLVL : 716 Vca_ : S--SDSLHFTHAAIGYVVAGLAALQVILAFVRPDPGTKMREFVWNPVHKLGGRACTLVAWCAVLTGIVLHRDMVYRGPVVPWVVPVAIVMGLILLVDSWL : 544 Dre_FRRS1 : W--A--GGA-HPVLGCLVMILSLIQPIVAAFRCEPQHE-RRFVFNWAHSCNAFAIKCLAVAAIFTGLALFEEY---DS-DGWMLKVMGGYLAWEALMYIL : 525 Hsa_FRRS1 : W--SRHAGY-HPYLGCIVMTLAVLQPLLAVFRP-PLHDPRRQMFNWTHWSMGTAARIIAVAAMFLGMDLPGLN---LP-DSWKTYAMTGFVAWHVGTEVV : 530 Mmu_FRRS1 : W--SWRAGY-HPYLGCTVMTLAVLQPLLATFRP-PLHDPRRQVFNWTHWSVGTAARIIAVAAMFLGMDLPGLN---LP-SPQKTYAMMGFVVWHIGTEVI : 530 Rno_FRRS1 : W--SWRAGY-HPYLGCIVMTLAVLQPLLATFRP-PLHDPRRQVFNWTHWSVGTAARIIAVAAMFLGMDLPGLD---LP-SPQKTYAMMGFVVWHIGTEVI : 527 6

7 Supplemental Figure 4. C. reinhardtii TEF22 is similar to animal ferric-reductases. Protein sequences from C. reinhardtii (Cre), V. carteri f. nagariensis (Vca), M. musculus (Mmu), H. sapiens (Hsa), Danio rerio (Dre) and Rattus norvegicus (Rno) were aligned with MUSCLE and edited in GeneDoc. Back-shaded amino acids are conserved in 8 (black), 6-7 (dark grey), or 5 (light grey) of the aligned proteins. DOMON domain is indicated by a green line and cyt b 561 domain by a purple line. TM indicate the putative transmembrane domains (blue line). Conserved histidine residues highlighted in red are involved in heme binding in mammalian homologs. 7

8 Supplemental Figure 5. A. TM1 TM2 * 20 * 40 * 60 * 80 * 100 Ath_Cytb561 : MAVRINAMA----VTFVAHALAVIAAIMVLVWSISYRGGLAWEA TNKNLIFNLHPVLMLIGFIILGGEAIISYKS- : 71 Hsa_Cytb561 : MEGGAAAATPTALPYYVAFSQLLGLTLVAMTGAWLGLYRGGIAW ESDLQFNAHPLCMVIGLIFLQGNALLVYRV- : 74 Hsa_CYBASC3 : MGKNFSDSFLSRECVIRMVSGRF--YLSCLLLGSLGSMCILFTIYWMQYWRGGFAW NGSIYMFNWHPVLMVAGMVVFYGGASLVYRL- : 85 MicpuC2_52667 : MPPSTPPNLDSLGAW----LALLTRSSYLLFVAVLLAWF----GRLAWFDRE TDGAVSFNYHPLFMGLAFVVLMPEALLAYADL : 76 Cre13.g : MSGGSSSEDPHGVQPARWAVGPPAPKAIAWLARATSLAILILIGHWVHVTLGGVGLHPQP--LPDGSGNDTGKLFNWHPILMTLGFAVFMVEALLTYQA- : 97 Vca_ : MTG---IDDPFGIQPARWIIGPPAPRGTAFLARACSLAIMVLVGHWVRVPLHGVGIKPETNGYPDGT-NDTSKLFNWHPVLMTLGFVVFMAEALLSYQA- : 95 Coc_C169_44519 : MSENGPVFWLGPPAPRAFSIFTRVLQVVTLILVAVWVLHDLGGVAFSPKR---INLLVNDTGVIFNWHPLLMTLAFPVLMGEAVIAYRA- : 86 ChlNC64A_12290 : DGES NDTSQLFNWHPLLLTLGFPVLMAEAVLAYRA- : 35 TM3 TM4 * 120 * 140 * 160 * 180 * 200 Ath_Cytb561 : ---LPLEKPVKKLIHLILHAIALALGIFGICAAFKNHNESH---IPNLYSLHSWIGIGVISLYGFQWVYSFIVFFFP---GGSTNLKSGLLPWHAMLGLF : 162 Hsa_Cytb561 : --FRNEAKRTTKVLHGLLHIFALVIALVGLVAVFDYHRKKG---YADLYSLHSWCGILVFVLYFVQWLVGFSFFLFP---GASFSLRSRYRPQHIFFGAT : 166 Hsa_CYBASC3 : PQSWVGPKLPWKLLHAALHLMAFVLTVVGLVAVFTFHNHGR---TANLYSLHSWLGITTVFLFACQWFLGFAVFLLP---WASMWLRSLLKPIHVFFGAA : 179 MicpuC2_52667 : EERRGFGHAAAKKMHAWAHIAASVCILLGLIFVFANHSAKG---IPALYSAHSWIGLTAAMCTFLQAIVGCVAYALPTLFGVGGESRARLLPAHRFAGAA : 173 Cre13.g : PLLASLPRETRKRIHWGCHSAAIICLALGLIAVLQSHRLKLPVPMPDWYSPHSYLGITALVLLALQFVLGAYAYLWP---RLSLPQRMALGPLHRYLGAA : 194 Vca_ : PIIPGLPREVRKRIHLGCHLAATVCVFLGFVAVLQSHRLKLPDPMPDWYSPHSFLGLTAIALVGLQVLVGLYSYVWP---RLGLSHRMALGPLHRFWGAA : 192 Coc_C169_44519 : PVASNLDRGQRKLLHALLQIIAVVAALLGVIAAFQSHNLKKPLPIPNLYSAHSYLGIFTLILLGLQAILGFTAFLYP---TFSKSNREALAPWHTFLGRA : 183 ChlNC64A_12290 : PLVPVKDRAHSKAYHFLLHTAALICTVLGVVAAFKSHTLKRPAPVPNLYSAHSWMGMAVLSLLTFQYLMGAGAYVWP---KLPLGQRRALGPLHSYLGKS : 132 TM5 TM6 * 220 * 240 * 260 * 280 * 300 Ath_Cytb561 : VYILAVGNAALGFLEKLTFLEN----G--GLDKYGSEAFLINFTAIITI-----LFGAFVV---LTASAESP---SPSVSNDDSVDFSYSAI : 237 Hsa_Cytb561 : IFLLSVGTALLGLKEALLFNLG----G--KYSAFEPEGVLANVLGLLLA-----CFGGAVLYILTRADWKRPSQAEEQALSMDFKTLTEGDSPGSQ---- : 251 Hsa_CYBASC3 : ILSLSIASVISGINEKLFFSLK-NTTR--PYHSLPSEAVFANSTGMLVV-----AFGLLVLYILLASSWKRP---EPGILTDRQPLLHDGE : 259 MicpuC2_52667 : TLALGAAACVAGFVEKQSFTKC----APDASDKRCAALSLVNVLVLLVASTATCALGTVYARWVGGGGSGGG---SDGANQGSYQTLGEDF : 257 Cre13.g : TWLAGLGAIATGLQEKITFMQMSKVVT--GAALYGPAVRVPAAVLVLLV-----VLAALVLYFQVPPPAKHA---GAVAVGADGSGAGGGAASTQGLLRG : 284 Vca_ : AWLAALGAVATGLQEKVTFMQM-KTLT--RKDLFGPAVRLPAVVLPLLV-----VLALLVLYHQVPPASKAS---TPEGFGGSPSPGGSGSSGSAGRMAS : 281 Coc_C169_44519 : TFIMGLATMAVGIQEKTQFVAAAKKLA--GAGLYSSALRLPATIEIFLA-----LVGMAVLYHHVPGPSTRR---EADTLLPDVLQDA : 261 ChlNC64A_12290 : IFVAGLATMAAGIQEKQ VGRL : 153 Supplemental Figure 5. Sequence analysis of Cyt b 561 domain containing proteins. A. Conservation around the putative heme binding ligands in Cyt b 561 related sequences. Homologous sequences were aligned as described in Supplemental Figure 2 online. Conserved histidine residues highlighted in red indicate the potential heme binding ligands. Red and green boxes indicate the predicted ascorbate-binding site (ALLVYRVFR) and monodehydroascorbate-binding site (YSLHSW), respectively. TM, transmembrane helices. Backshaded amino acids are conserved in 8 (black), 6-7 (dark grey), or 3-5 (light grey) of the aligned proteins. 8

9 Supplemental Figure 5. B C Supplemental Figure 5. Sequence analysis of Cyt b 561 domain containing proteins. B. Phylogenetic trees of DOMON-cytb 561 domain proteins (TEF22 and its homologs) (left) and cytb only proteins (right) were generated as described in Material and Methods section. Bootstrap values are indicated at each node. Homologous sequences from C. reinhardtii (Cre), A. thaliana (Ath; AT), O. sativa (Os), Ricinus communis (Rco), M. musculus (Mmu), R. norvegicus (Rno), D. rerio (Dre), H. sapiens (Hsa), Coccomyxa sp. C-169 (Coc_C169), C. variabilis (ChlNC64A), V. carteri f. nagariensis (Vca), M. pusilla CCMP1545 (MicpuC2) were used. C. Protein similarity network of Cyt b 561 domain containing proteins. The similarity network was generated using all-vs-all blast analysis (pairwise alignment between all pairs of proteins) of DOMONcyt b 561 domain proteins (TEF22 and its homologs) and cyt b 561 -only proteins on a local sequence database using an e-value of 1e-6. Nodes representing Chlorophytes are colored in light green, those representing Streptophytes in dark green, Stramenopiles in brown and animal proteins in red. C. reinhardtii and human DOMON-cytb 561 and cytb 561 -only domain containing proteins are labeled. 9

10 Supplemental Figure 6. A. * 120 * 140 * 160 * 180 * 200 AT3G25190 : RNQKT RPRAEKEEVDYMQRAQWLRAALLGANDGLVTVASLMMGVGSIK : 61 AT1G21140 : DMEMD QEKAFDYSKRAQWLRAAVLGANDGLVSTASLMMGVGAVK : 56 AT1G76800 : EKEST TFDYSKRSQWLRAAVLGANDGLVSTASLMMGVGAVK : 53 Coc_C169_60838 : NRDVE ANQGEHYDGDAHELRAAVLGANDGLVSVASIMLGVGAAS : 56 Vca_ : APERA VAIAVDGDDSSVDEHVHYSHRAPWLRAFVLGANDGLVSVAALMLGVGGGS : 70 Cre02.g : EAPVA LDVAGQPQAQVPESGAAQQQAHAPAQMPDIEAAVVGADVVVKEDSSADEDEHVHYAHRAPWLRAFVLGANDGLVSVAALMLGVGGGS : 192 Cre02.g : SAAAP APRHSGNGAHSHSHHHHDDDIHSHAGSDEHVHYSHRLPWLRAFVLGATDGLVSVAALMLGVGGGS : 82 Vca_ : NDGVS SSDEHEHYIHRAPWLRAFILGANDGLVSVAALMLGVGSGN : 58 Coc_C169_18908 : HFSQRAPWLRAAVLGANDGLVSVASLMLGVEGGT : 34 ChlNC64A_ : GAHGSHPAAPDAYEPLPSTLTRLGRHSLASAGRLHGGTPDGSTHHLPDVIQPLAPGDDNDELCDHMHHSNRAPWLRALVLGANDGLVSIASLMMGVGGGT : 114 ChlNC64A_25013 : HHSHRAPWLRAFVLGANDGLVSTSSLLMGVGAAS : 34 Sce_CCC1 : RGSNS NSSRHDNLSSSSSDIIYGRNSAQDLENSPMSVGKDNRNGDNGSDNEKANLGFFQSVDPRVISDLIIGLSDGLTVPFALTAGLSSLG : 126 Ath_VIT1 : EPEKQ TLLDHHTEKHFT-AGEIVRDIIIGVSDGLTVPFALAAGLSGAN : 60 * 220 * 240 * 260 * 280 * 300 AT3G25190 : E--DVKAMLLVGFAGLVAGACSMAIGEFVSVCTQRDIETAQMKRAIEHKTSLSAIDEQEEEE : 121 AT1G21140 : Q--DVKVMILSGFAGLVAGACSMAIGEFVSVYSQYDIEVAQMKRENGGQ : 103 AT1G76800 : H--DVKAMILSGFAGMVAGACSMAIGEFVSVYSQYDIEVAQME RDS : 97 Coc_C169_60838 : T--DQHTLLLSGLSALVAGAMSMAAGEYISVASQKDTEEADVEKEREQQEGSAETREHELEELTQIYVARGLEYNLARE-VAVALSRTKESAVEAHARDE : 153 Vca_ : A--ELATMRLAG ARDTEEADVEKERQQQLKGPAARARELEELTEIYVKRGLSRGLARE-VATEL--TEKDVIRAHARDE : 144 Cre02.g : D--DLSAMRLAGIASWVAGALSMALGEYVSVASQLDTEEADIEKERQQQLKGPAARAHELQELTEIYIGRGLTPELARQ-VAEQL--TEKDVIRAHARDE : 287 Cre02.g : E--SLTTLRLAGIAAWIAGALSMAVGEYISVASQRDTEEADIEKERQQQLKGPAARAHELQELTEIYIGRGLTPELARQ-VAEQL--TEKDVIRAHARDE : 177 Vca_ : V--SLNTMRLAGVASWIAGALSMAVGEYISVSSQRDTEEADIEKERQQQRKGPAARARELQELTDIYIKRGLDPDLARK-VAEQL--TEKDVIRAHARDE : 153 Coc_C169_18908 : H--ELHPVVLAGVAGLVAGALSMAVGEFVSVSSQRDAEEADIEKERKEQAKGPAARQHELEELTAIYEDRGLSQGLARQ-VAEEL--TAKDVIRAHARDE : 129 ChlNC64A_ : S--DLATLRLAGVAGLVGGALSMAVGEYISVSSQRDAEKADIEQERLEQLKGPVAQARELEELAQIYVARGLPYNLARQ-VAEVL--TEKDVIRAHARDE : 209 ChlNC64A_25013 : SPAPLDPAWPLAPAGLVGGALSMAVGEYISVSSQRDAEAADVEQERLEQLKGAEAQ---LEELSQIYEGRGLPPHLARSYVAEVL--TEKDVVRAHARDE : 129 Sce_CCC1 : ---DAKLVITGGFAELISGAISMGLGGYLGAKSESDYYHAEVKKEKRKFYDNSNLINREIEDIL-LEINPNFSDETIVS-FIKDLQRTPELMVDFIIRYG : 221 Ath_VIT1 : A--SSSIVLTAGIAEVAAGAISMGLGGYLAAKSEEDHYAREMKREQEEIVAVPETEAAEVAEILAQY---GIEPHEYSP-VVNALRKNPQAWLDFMMRFE : 154 * 320 * 340 * 360 * 380 * 400 AT3G25190 : ---KKERLPNPGQAAIASALAFSVGAAMPLLGAVFIENHKVRMVVVA VVATIALVVFGV------TGAVLGKTSVVKSSVRVVIGGWMA : 201 AT1G21140 : --VEKEKLPSPMQAAAASALAFSLGAIVPLMAAAFVKDYHVRIGAIV AAVTLALVMFGW------LGAVLGKAPVFKSSARVLIGGWLA : 184 AT1G76800 : VEIEKEKLPSPMQAAAASALAFSAGAIVPLLAAAFVKEYKMRIISVV VAVTVALMVFGW------LGAALGKAPAVRSSARVLFGGWLA : 180 Coc_C169_60838 : LGIDLDDLANPFQAAAASLLAFSIGGVVPLVGAIFITDPRIRLATVLEQRVSDAECLQVLATFALLTFGA------TGAWLGGAKRVRAALRVLIGGWLA : 247 Vca_ : LGIDLDAMANPLQAAVVSSIAFTAGAMIPLLAGSFATHRVTRLVLVS VLSVAGLAIFGL------TGSLLGGAKPLVGALRVVIGGCLA : 227 Cre02.g : LGIDMDAMANPLQAAVVSCIAFTAGALIPLLAGSFIHDPTGRLVAVA LVAVLGLAVFGL------TGSLLGGAKWFIGASRVVIGGCLA : 370 Cre02.g : LGIDLDEMANPMQAACVSALAFTAGALIPLLGGAFITDARIRLAVVA VAATLGLLAFGL------MGSVLGGAKPLIGSIRVLVGGCLA : 260 Vca_ : LGIDMDELANPLQAAVVSALAFTAGALIPLLAGAFLPEPQMRLVAVA VASLVGLALFGL------VGALLGGAKPIVGALRVVLGGCLA : 236 Coc_C169_18908 : LGIDIDDMTNPYQAAASSALAFCIGAGLPLLAAAFIEDPKWRIVSVV LTSAAALLVFGI------MGAVLGGAGVMKGGTRVLIGGLAA : 212 ChlNC64A_ : LGIDIDELANPLQAAVVSAICFTCGAALPLLSAAWIMDPNLRLGILA GATTIGLCFFGF------LGAYLGGAGVCRGGMRVLLGGWMA : 292 ChlNC64A_25013 : LGIDVDQLARPVQAAVVSAITFSLGAGIPLLAACCV-----RMAVVL ASTTVGLAGFGS------LAAWLGGAHKMQAAARVVLGGWAA : 207 Sce_CCC1 : RGLDEPAENRELISAVTIGGGYLLGGLVPLVPYFFVSDVGTGLIYSI IVMVVTLFWFGYVKTKLSMGSGSSTSKKVTEGVEMVVVGGVA : 310 Ath_VIT1 : LGLEKPDPKRALQSAFTIAIAYVLGGFIPLLPYMLIPHAMDAVVASV VITLFALFIFGY------AKGHFTGSKPLRSAFETAFIGAIA :

11 Supplemental Figure 6. B Cre02.g Vca Vca Cre02.g ChlNC64A Coc C ChlNC64A Coc C AT3G25190 AT1G AT1G76800 Ara Pcl1 Spo Pcl1 Pbe nodulin21 Mlo nodulin21 Ostta OstRCC MicpuC MicpuN Sce CCC1 Lel CCC1 Ath VIT1 Zma CCC1 0.5 Figure 6. C. reinhardtii genome encodes two VIT1-like proteins. A. Sequences from C. reinhardtii (Cre), A. thaliana (Ath, AT), Coccomyxa sp. C-169 (Coc_C169), C. variabilis (ChlNC64A), V. carteri f. nagariensis (Vca) and S. cerevisiae (Sce) were aligned using MUSCLE and edited with GeneDoc. Back-shaded amino acids are conserved in 13 (black), (dark grey), or 7-9 (light grey) of the aligned proteins. B. The phylogenetic tree of VIT1-like proteins was build as described in Material and Methods section using the protein sequences from C. reinhardtii (Cre), Coccomyxa sp. C-169 (Coc_C169), C. variabilis (ChlNC64A), V. carteri f. nagariensis (Vca), O. tauri (Ostta), O. sp. RCC809 (OstRCC809), Micromonas sp. RCC299 (MicpuN), M. pusilla (MicpuC), A. thaliana (AT, Ath), Zea mays (Zma), Acinetobacter radioresistens SK82 (Ara), Mesorhizobium loti MAFF (Mlo), Pelagibaca bermudensis HTCC2601 (Pba), Schizosaccharomyces pombe, (Spo) and S. cerevisiae (Sce). Orthologs of the C. reinhardtii Cre02.g are depicted in blue. 11

12 Supplemental Figure 7. A. TM4 TM5 * 320 * 340 * 360 * 380 * 400 Ath_FRO7 : ELTGLRFGMIGLLCMVFLFLPISRGSILLRLIDIPFEHATRYHVWLGHITMTFFSLHGLCY------VVGWTIQGQLLELLFE : 257 Ath_FRO6 : EVTGLRLGMIGLLCMVFLFLPISRGSILLRLIDIPFEHATRYHVWLGHITMAFFSLHGLCY------VVGWIIQGQLLELIFS : 248 Ath_FRO8 : YRVATRFGLLAEACLSLLLFPVLRGLSMFRLLNIEFAASVKYHVWFGTGLIFFSLVHGGST------LFIWTITHHIEEEIWK : 229 Ath_FRO5 : RAFGLRIGYVGHYCWAFLFFPVTRASTILPLVGLTSESSIKYHIWLGHVSNFCFLVHTVVF------LIYWAMVNK-LMETFA : 183 Ath_FRO4 : RAFGLRIGYVGNICWAFLFFPVTRASTILPLVGLTSESSIKYHIWLGHVSNFCFLVHTVVF------LIYWAMINK-LMETFA : 225 Ath_FRO2 : ESAALRLGLIGNICLAFLFLPVARGSSLLPAMGLTSESSIKYHIWLGHMVMALFTVHGLCY------IIYWASMHE-ISQMIM : 252 Ath_FRO1 : DLIAVRIGIVGNICLAFLFYPVARGSSLLAAVGLTSESSIKYHIWLGHLVMIIFTSHGLCY------FIYWISKNQLVSKMLE : 243 Ath_FRO3 : DSIAVRLGLTGNICLGFLFYPVARGSSLLAAVGLTSESSTKYHIWLGNLVMTLFTSHGLCY------CIYWISTNQ-VSQMLE : 242 Cre05.g : MSEGSVRALRIA-----YNCGMAAAWPLLANLFVALLPVERSLLLLAALGAGHEDGVAAHTWAGTAVATWACVHAVLTQFAMLAAGLWA------FIVLP : 220 Cre_FRE1 : PPPPTVRRLKPLPEVVQDSVAKYSGWVGRLDLLLLFFPLPRCNFLHWLLGSDFPTLVKYHRWLGHGTMLINSIHGIVY------MGMWANAGT-LTTMLN : 372 TM6 TM7 TM8 * 420 * 440 * 460 * 480 * 500 Ath_FRO7 : WKAT----GIAVLPGVISLVAGLLMWVTSLHTVRKNYFELFFYTHQLYIVFVVFLALHVGDYLFSIVAGGIFLFILDRFLRFYQSRRTVDVISAKSLPCG : 353 Ath_FRO6 : WNAI----GIAILPGVISLVAGLLMWVTSLHTVRKNYFELFFYTHQLYIVFIVFLALHVGDYMFSIVAGGIFLFILDRFLRFCQSRRTVDVISAKSLPCG : 344 Ath_FRO8 : WQRT----GRVYVAGLISLVTGLLMWITSLPQIRRKNFEVFYYTHHLYIVFLVAFLFHAGDRHFYWVLPGMFLFGLDKILRIVQSRSESCILSANLFSCK : 325 Ath_FRO5 : WNAT----YVPNLAGTIAMVIGIAIWVTSLPSFRRKKFEIFFYTHHLYGLYIVFYAIHVGDSWFCMILPNIFLFFIDRYLRFLQSTKRSRLVSAKILPSD : 279 Ath_FRO4 : WNPT----YVPNLAGTIAMVIGIAMWVTSLPSFRRKKFEIFFYTHHLYGLYIVFYVIHVGDSWFCMILPNIFLFFIDRYLRFLQSTKRSRLVSARILPSD : 321 Ath_FRO2 : WDTK----GVSNLAGEIALAAGLVMWATTYPKIRRRFFEVFFYTHYLYIVFMLFFVLHVGISFSFIALPGFYIFLVDRFLRFLQSRENVRLLAARILPSD : 348 Ath_FRO1 : WDRT----AVSNLAGEIALVAGLMMWVTTYPKIRRRLFEVFFYSHYLYIVFMLFFVFHVGISHALIPLPGFYIFLVDRFLRFLQSRNNVKLVSARVLPCD : 339 Ath_FRO3 : WDRT----GISHLAGEIALVAGLLMWATTFPAIRRRFFEVFFYTHYLYMVFMLFFVFHVGISYALISFPGFYIFMVDRFLRFLQSRNNVKLVSARVLPCE : 338 Cre05.g : PGNGAETKAVTNFMGLLAWLCLCGLAATSLAAVRRACYNLFIWAHVILAPAMFLFAILHSGLVFWYGFLGLLLYGADVVQRLVL-RRRTAAATVTVYPQA : 319 Cre_FRE1 : WGMGA---GVNRLAGLISLCGGWLLWITCIPIIRRRFFNVFYISHILGAIIFLLFGFMHRKDVATWVMPGIFLYLLDVVLRTIQQCFNATRVSLVASPAD : 469 * 520 * 540 * 560 * 580 * 600 Ath_FRO7 : TLELVLSKPPNMRYNALSFIFLQVKELSWLQWHPFSV SSSPLDGNH : 399 Ath_FRO6 : TLELVLSKPPNMRYNALSFIFLQVRELSWLQWHPFSV SSSPLDGNH : 390 Ath_FRO8 : AIELVLPKDPMLNYAPSSFIFLNIPLVSRFQWHPFSI ISSSSVDKH : 371 Ath_FRO5 : NLELTFAKTSGLHYTPTSILFLHVPSISKLQWHPFTI TSSSNLEKD : 325 Ath_FRO4 : NLELTFSKTPGLHYTPTSILFLHVPSISKIQWHPFTI TSSSNLEKD : 367 Ath_FRO2 : TMELTFSKNSKLVYSPTSIMFVNIPSISKLQWHPFTI TSSSKLEPE : 394 Ath_FRO1 : TVELNFSKNPMLMYSPTSTMFVNIPSISKLQWHPFTI ISSSKLEPE : 385 Ath_FRO3 : TVELNFSKNPMLMYSPTSILFVNIPSISKLQWHPFTI TSSSKLEPK : 384 Cre05.g : AAAAAAAAVVDGGRLPAAGEGGKFV----VLRIHTPGPPGCF--AGQHVFLRVPGVSAWQWHPYSV AAADDR : 385 Cre_FRE1 : TPASIKRAVMSMPQGGSGGVAASLSRDGGVLTLTIPCDQSMTWCGGDIVFLNLPCVSWWQWHPFTIANTSATFDHPAAATPSPGAEPHFAKDSSMSSTSA :

13 TM9 * 620 * 640 * 660 * 680 * 700 Ath_FRO7 : HVAVLIKVLGGWTAKLRDQLSTLYEAENQDQLISPESYPKITTCVEGPYGHESPYHLA--YENL VLVAGGIGITPFFA : 475 Ath_FRO6 : HVAVLIKVLGGWTAKLRDQLSNLYEAENQDQLISPQSYPKITTCVEGPYGHESPYHLA--YENL VLVAGGIGITPFFA : 466 Ath_FRO8 : SLSIMMKCEGDWTNSVYNKIEEAANCE NKINNIIVRVEGPYGPASVDFLR--YDNL FLVAGGIGITPFLS : 439 Ath_FRO5 : TLSVVIRKQGSWTQKLYTHLSSS IDSLEVSTEGPYGPNSFDVSR--HDSL ILVGGGSGVTPFIS : 387 Ath_FRO4 : TLSVVIRRQGSWTQKLYTHLSSS IDSLEVSTEGPYGPNSFDVSR--HNSL ILVSGGSGITPFIS : 429 Ath_FRO2 : KLSIVIKKEGKWSTKLHQRLSSS DQIDRLAVSVEGPYGPASADFLR--HEAL VMVCGGSGITPFIS : 458 Ath_FRO1 : TLSVMIKSQGKWSTKLYDMLSSSSS DQINRLAVSVEGPYGPSSTDFLR--HESL VMVSGGSGITPFIS : 451 Ath_FRO3 : KLSVMIKSQGKWSSKLHHMLASS NQIDHLAVSVEGPYGPASTDYLR--HDSL VMVSGGSGITPFIS : 448 Cre05.g : SFALVVKAHGDWERRLAAAAEQDSALEGG------AVSTKMTVCYEGFYGTPDLQAAAASADRM LLFAGGSGITPLAA : 457 Cre_FRE1 : SSGAAAAAASGARVRYMQVHIKKYNAWTRSLIKQLEAS GGASSLALYVSGPYHPPTLTGSNSYSRRLVNGFRRHVFIAGGIGVTPALG : 657 * 720 * 740 * 760 * 780 * 800 Ath_FRO7 : ILSDILHRKR DGKDCLPGKVLVVWAIKNSDELSLLSAIDIP----- : 516 Ath_FRO6 : ILSDILHRKR DGKACLPSKVLVVWAIKNSDELSLLSAIDIP----- : 507 Ath_FRO8 : ILKELASKNR LKSPKRVQLVFAVRTFQDLNML----LP---IA : 475 Ath_FRO5 : VIRELIFQSQ NRSTKLPNVLLVCAFKNYHDLAFLDLI-FP----S : 427 Ath_FRO4 : VIRELISQSQ NKSTKLPDVLLVCSFKHYHDLAFLDLI-FP----L : 469 Ath_FRO2 : VIRDLIATSQ KETCKIPKITLICAFKKSSEISMLDLV-LP---LS : 499 Ath_FRO1 : IVRDLFYMSS THKCKIPKMTLICAFKNSSDLSMLDLI-LP---TS : 492 Ath_FRO3 : IIRDLLYVSS TNAYKTPKITLICAFKNSSDLSMLNLI-LP----- : 487 Cre05.g : LLQSLLKHPAPLLPPPQEPAAAKAPHAARSSSSAAPLHSSSVMGGGMGAVGVQGAAAAVAGGTEDGDSDEAPRVHLVWAVAHESDAAPL----LP---LL : 550 Cre_FRE1 : MIQELIAARR NTNTNNNTNNTNTGTNDDKAHGRVTFVWVSRSADELEAV----LPAEVLE : 713 * 820 * 840 * 860 * 880 * 900 Ath_FRO7 : SICHFFSKKLNLEI HIYVTRQSEPCLEDGMV--HKVVHPSVKTP : 558 Ath_FRO6 : SICPFFSKKLNLEI HIYITRQSEPRLEDGMV--HKVVHPSVKLP : 549 Ath_FRO8 : SIIFNPIYNLNLKL KVFVTQEKKPSNGTTTL--QEFLAQSQVQSIHLG : 521 Ath_FRO5 : DISVSDISKLNLRI EAYITREDKKPETTDD---HKLLQTKWFKP : 468 Ath_FRO4 : DMSASDISRLNLRI EAYITREDKKPETTDD---HRLLQTKWFKP : 510 Ath_FRO2 : GLETELSSDINIKI EAFITRDNDAGDEAK----AGKIKTLWFKP : 539 Ath_FRO1 : GLTTDMASFVDIQI KAFVTREEKTSVKESTHN-RNIIKTRHFKP : 535 Ath_FRO3 : -NSTEISSFIDIQI KAFVTREKVSTCN------MNIIKTLSFKP : 524 Cre05.g : HAAAGRGWWVDLHV-----TRPQQQQAAAPVDVSVKGSGSGDALPADPHPLHLRQAHPHLHTVAVALHSSHHPAHHHPAHGHHPPTQHGHVSIPSRNGSS : 645 Cre_FRE1 : EANRGKDGWLDMQLYITGSSNNSNSKAAAASAITVDVAADANTAAGAADASAAAAAEAKLASLTPAF-SRSYATFSHALSGLSGLRRSGS------NSSS : 806 Supplemental Figure 7. C. reinhardtii ferric reductase-like protein (Cre05.g241400) is closely related to plant ferric reductases. A. Green line below the alignment indicates the ferric-reductase transmembrane domain, the purple line indicates the FAD-binding domain whereas the red indicates the putative NAD + -binding domain. Blue lines over the alignment indicate the putative transmembrane domains (TM, transmembrane helices). Histidine residues highlighted in red indicate putative heme binding ligands. TM, transmembrane helices. Back-shaded amino acids are conserved in 10 (black), 8-9 (dark grey), or 6-7 (light grey) of the aligned proteins. 13

14 Supplemental Figure 7. B Ath FRO1 Ath FRO3 Ath FRO2 Ath FRO5 Ath FRO4 Ath FRO8 Vvi Ptr Sce Fre7 Ptr Ppa Ppa Rco Ath FRO7 Ath FRO6 Cre FRE1 Cre05.g Coc C ChlNC64A Sce Fre8 97 Sce Fre5 Cli HCR2 Sce Fre6 Sce Fre4 Sce Fre2 Sce Fre3 Vca Sce Fre1 0.5 Supplemental Figure 7. C. reinhardtii ferric reductase-like protein (Cre05.g241400) is closely related to plant ferric reductases. B. Phylogenetic tree of ferric reductase-like. Protein sequences from C. reinhardtii (Cre05.g241400), V. carteri f. nagariensis (Vca), C. variabilis (ChlNC64A), Coccomyxa sp. C-169 (Coc_C169), A. thaliana (Ath), R. communis (Rco), Physcomitrella patens (Ppa), Vitis vinifera (Vvi), Populus thichocarpa (Ptr), Chlorococcum littorale (Cli), and S. cerevisiae (Sce) were used to build the phylogenetic tree as described in Materials and Methods section. 14

15 Supplemental Figure 7. C. Supplemental Figure 7. C. reinhardtii ferric reductase-like protein (Cre05.g241400) is closely related to plant ferric reductases. C. Protein similarity network of ferric-reductase like proteins. The similarity network was generated using allvs-all blast analysis (pairwise alignment between all pairs of proteins) of ferric-reductase like and respiratory-burst oxidase sequences on a local sequence database using an e-value of 1e-17 (inset). To emphasize the separation between ferric-reuctases and respiratory-burst oxidases the e-value was lowered to 1e-30. Nodes representing Chlorophytes are colored in light green, those representing Streptopytes in dark green, Stramenopiles in brown, animal proteins in red, Euglenozoa in yellow, Rhodophyta in magenta while nodes representing Cyanobacteria are colored in cyan, Bacteria in dark blue and Fungi in light blue. 15

16 Supplemental Figure 8. A TM1 * 120 * 140 * 160 * 180 * 200 Cme_CMG212 : NRTGEPATQH--SRQAKPSILAFLP-LYTAYALICGVQRAFTFVVPIFLMIVSQRALNRTDGSFVPAAIYMLSSR : 152 Ath_IREG3 : HLDTNISVTESLTLLTECTYVDTVLTALPVLSEEEQTVIAATPAHPEGLYVLYASCLVGNLVEQLWNFAWPSAIAMLYP SLLPVAVMGFVTK : 192 Hsa_S40A1 : NRQRGCCGSL-----ADYLTSAKFL-LYLGHSLSTWGDRMWHFAVSVFLVELYG NSLLLTAVYGLVVA : 69 Mmu_IREG1 : THQEGCCGSL-----ANYLTSAKFL-LYLGHSLSTWGDRMWHFAVSVFLVELYG NSLLLTAVYGLVVA : 69 ATth_IREG1 : QREEGEDESQPQNPPPALRRRFVIY-LYVGYFLARWSARTWEFSVALYMIHLWP NSLLLAAIYGAIES : 82 Ath_IREG2 : HQEEEEEEEE----EPSLPRSMVIS-LYLGYFLARWGARTWEFSVALYMIYLWP NSLFLTAMYGVVES : 78 Cre_FPN1 : QRLPADSESE--TPSDTIPKAARFY-LCSSYAFAAWAWRSWEFLVALVLIELYP NSLLMVSAYGLLDN : 68 Vca_ : MEPQVPVRAKVF-LCASYALAAWAWRSWEFIVALVLIELYP DSLLMVSAYGLLDN : 54 ChlNC64A_59298 : TRETLETEVP--AVEKTGHVPASRA-LQASHFLSTWGQRGWEFAVGLVMLELHP SSLLLVAVWGLLDA : 84 Coc_C169_55598 : MA-LYGSHFLSTWGQRMWEFAIGLILLELRP GSLALVSAFGLVDS : 44 TM2 TM3 * 220 * 240 * 260 * 280 * 300 Cme_CMG212 : GACFLLGPTVGKMLDWGWRFRTAAVASVLYGVSTSLCAIWMPLLARYRPNDQATAIPEGTLWFCVF-----GTIAALSEFALEISLWKRWLPLTVHAQQY : 247 Ath_IREG3 : LAIIAGGPVVGKFMDYSPRVPTYISLNVIQAAAQVLSAG-MIIHAYTVPSTSASSI---LLQPWFFALLFAGAIDSLCGIASGVAIERDWVV : 280 Hsa_S40A1 : GSVLVLGAIIGDWVDKNARLKVAQTSLVVQNVSVILCGI-ILMMVFLHKHELLTMYHGWVLTSCYILIITIANIANLASTATAITIQRDWIV : 160 Mmu_IREG1 : GSVLVLGAIIGDWVDKNARLKVAQTSLVVQNVSVILCGI-ILMMVFLHKNELLTMYHGWVLTVCYILIITIANIANLASTATAITIQRDWIV : 160 ATth_IREG1 : GSTAIFGPIVGQWVEGMDYVKVLRLWLLFQNLSYTIAGG-AVIKLLLVSDLKSRNLP--VFAILIVLTNLAGAIGVLSTLAGTILIERDWAV : 171 Ath_IREG2 : GSATLFGPIVGQMIDGMSYVKVLRLWLVTQNLSFIVAGG-AVVALLVVPDLKSQNFP--VFATLVVLTNLSGAIGVLSTLAGTVLIERDWVV : 167 Cre_FPN1 : LTRVVLGPTVGGYVDRHERMPGCKLMLRLQNCCISGSAA-AALALLWPGSAALTNPA--LYWPLIWVLTVLGAASSAGSTGVQIAVEREAVK : 157 Vca_ : MARVLLGPAVGSYVDRHERMPGAQAMLRLQNLCIGGSAA-AALVLLWPRSAATEHKA--VYWSLMWLLTVLGSASSAGSTGVSISVEREAVK : 143 ChlNC64A_59298 : ALSVVAGTAVGRYVDGLPRLAAASRMYLLQNGMLALSAA-AALGLLASDARAGA-----AFWAGRALTMGAGAVSTLGALGSTLSVEREWTR : 170 Coc_C169_55598 : GAQVIAGPHIGAYIDRTPRLAAACNMYILQNCGVALSAS-SALAASLVGSNAA------LFWPCAVLTIALGSASSVGCQGSALSVEREWTK : 129 TM4 TM5 * 320 * 340 * 360 * 380 * 400 Cme_CMG212 : QSGAIDGSKTEEQLAIANARMRRITMTIDIGMPLLVGWLLSSRG---EVQGSVLVARFGAFLLAIQLVALFRASQICARGE : 325 Ath_IREG3 : ---LLAGINRPIALAQANAVLHRIDLLCEIAGTMLFGILLSKYD---P-VTCLKFAATLMVGSLPTMTALIWLTNKFSSGV : 354 Hsa_S40A1 : ---VVAGEDRSK-LANMNATIRRIDQLTNILAPMAVGQIMTFGS---PVIGCGFISGWNLVSMCVE-YVLLWKVYQKTPAL : 233 Mmu_IREG1 : ---VVAGENRSR-LADMNATIRRIDQLTNILAPMAVGQIMTFGS---PVIGCGFISGWNLVSMCVE-YFLLWKVYQKTPAL : 233 ATth_IREG1 : ---VMSEGHPPAVLTKMNSVIRGIDLSSKLLSPVITGLIISFVS---LKASAITFAAWATITAWVE-YWLFISVYSGVPAI : 245 Ath_IREG2 : ---VMSEGHSPAVLTRMNSVIRGIDLSSKLLSPVITGLIISFVS---LRASAITFAAWATITVWIE-YWLFISVYNGVPAI : 241 Cre_FPN1 : ---ALCGDDAGA-LASTNATMRAIDLTALLLAPLVAGVLMTAAG---PFVAAAAMSAYCGGAYIPE-VFFLGAAFRAAPALMKPKATAATGPAADDDD-- : 247 Vca_ : ---TLCGADGRA-LAALNSVMRAIDLTALLCAPLAAGLLMTAAG---PFTAVAAMAAYCGVAYVPE-VLLLGAAFRAAPVLGQPKVRAASDSAVSGGQAA : 235 ChlNC64A_59298 : ---ALCGGDSAA-LASLNAAMKRIDLTCLIASPILVGLVMQHGGGRPMVAATLALLAWNLASWLPE-VALLRYAQRCSPAL : 246 Coc_C169_55598 : ---ALCPGDSTA-LTILNAGMRRIDLTCLIASPIMAGLLLTYGN---LQVAILAVMAWNMFAWLPE-CWFLRMAQQLSPAL :

17 TM6 TM7 * 620 * 640 * 660 * 680 * 700 Cme_CMG212 : -IPSTTTRVAVA ARTVYQHLRELFSSWRLYYEQETFVPSLAHVSLYFT-VLSPGGLLFGFLTYCGVNGSTIGIFRAASAIIGILATFSFEIL : 444 Ath_IREG3 : -EGSRTNTDSIF DIGMETIKLGWKEYIQQPVLPASLAYVLLYFNIVLTPGSLMTAFLTQRCVNPSVIGGFSGLCAVMGVAATFLSANL : 454 Hsa_S40A1 : -MGVKDSNIHELEHEQEPTCASQMAEPFRTFRDGWVSYYNQPVFLAGMGLAFLYMT-VLGFDCITTGYAYTQGLSGSILSILMGASAITGIMGTVAFTWL : 363 Mmu_IREG1 : -MGEKDSNIRELECEQEPTCASQMAEPFRTFRDGWVSYYNQPVFLAGMGLAFLYMT-VLGFDCITTGYAYTQGLSGSILSILMGASAITGIMGTVAFTWL : 363 ATth_IREG1 : -TGNPPSRTGIL VILDRMSKSSFVGAWRIYFNQEVVLPGVSLALLFFT-VLSFGTLMTATLQWEGIPTYIIGIGRGISATVGLAATLVYPLM : 370 Ath_IREG2 : -YRNTQSRSRIL RILERISESSFVSAWRNYLNQEIVLPGVSLALLFFT-VLSFGTLMTATLEWKGIPTYIIGIGRGISAGVGLAATVLYPLM : 366 Cre_FPN1 : -GASVASRARAL LAGLGGAVAQYGDSWRVYVQQPLLLPCLALALIYMT-VLSLGFLMTSFLKWTGLTEAEVSVYRGIGALTGLAATALFPPL : 504 Vca_ : CGGGGLARLRVL LRAVRSQFGLYCDSWRVYLQQSVLLLCVALALLYMT-VLSLGFLMTSFLKWSGLSEAEVSGYRGIGALTGLAATAIFPPL : 513 ChlNC64A_59298 : -PGDGDGRWHRL ARPLRQQAQAWALYARQPTAAAAAALALLYLT-VLSWGTLMTAYLKALGLPEAELAVYRGLGAVSGILATLTFPPL : 358 Coc_C169_55598 : IEKLIKKSTTGWRTYARQEVFLAAVALALLYLT-VMSFGLLMTAYMKWRGLPETVLSLYRGAGAVSGIASTFVFPML : 294 TM8 * 720 * 740 * 760 * 780 * 800 Cme_CMG212 : VTRLHWSVLQVGRVGVLCQLACLLPAQLLLWLVRVPEVPLPSAAQNSVAQITYQ QPPLLIGV : 506 Ath_IREG3 : VKRV--GILKAGAVGLFFQASLLAVAVAVYCSSSLS HKSPLFFF : 496 Hsa_S40A1 : RRKC--GLVRTGLISGLAQLSCLILCVISVFMPGSPLDLSVSPFEDIRSRFIQGESITP-TKIPE--ITTEIYMSNGSNSANIVPETSPESVPIISVSLL : 458 Mmu_IREG1 : RXKC--GLVRTGLFSGLAQLSCLILCVISVFMPGSPLDLSVSPFEDIRSRFVNVEPVSPTTKIPETVFTTEMHMSNMSN----VHEMSTKPIPIVSVSLL : 457 ATth_IREG1 : QSRL--STLRTGLWSFWSQWSCLLVCVGSIWVKK DKIASYML : 410 Ath_IREG2 : QSRI--SPLRTGVWSFWSQWTCLLVCVGSIWVEK EKIASYML : 406 Cre_FPN1 : SKWA--GLRFCAAAGITYQMACLASGVLPVALTIMSQGGG-KP SVGEVRVM : 552 Vca_ : SARA--GLLFCAVAGVTYQLACLAAGVLPVVTPTMAGGDGQRP SVPQVRIL : 562 ChlNC64A_59298 : HRAL--GLVATGGLSIWLQLACVAGATLPSVAAAAGAAVG TSARLYAV : 404 Coc_C169_55598 : KERI--GLERAGALGICCQLVSLLAALAVSAALQ SKAATHAL : 334 * 820 * 840 * 860 * 880 * 900 Cme_CMG212 : LVFVTLSRWGLWCWDTAETEIMQTMVDASAVGEVSAVEAALCSLAELLMYGVSFTL : 562 Ath_IREG3 : LSMIVLSRLGHMSYGVVGAQILQTGIPSSKANLIGATEISVASLAESLMLGVAIAA : 552 Hsa_S40A1 : FAGVIAARIGLWSFDLTVTQLLQENVIESERGIINGVQNSMNYLLDLLHFIMVILA : 514 Mmu_IREG1 : FAGVIAARIGLWSFDLTVTQLLQENVIESERGIINGVQNSMNYLLDLLHFIMVILA : 513 ATth_IREG1 : MAGVAASRLGLWMFDLAVIQQMQDLVSESDRCVVGGVQNSLQSALDLMAYLLGIIV : 466 Ath_IREG2 : MAGVAASRLGLWMFDLAVIQQMQDLVPESDRCVVGGVQNSLQSALDLMANLLGIIV : 462 Cre_FPN1 : VGGLVASRTGLWMYDLAVTQLIQQGVAEDHMGAVYGVQSSLQASFEMLSFVAGLLS : 608 Vca_ : VAGLVSSRTGLWLYDLAVTQLIQEEVRQDQLGSVYGVQSSLQASFEMMSFVAGLIGGCFPFATMMSFRVSTLLILAVATARCLAQYEYDYGYGYDFYDTE : 662 ChlNC64A_59298 : VWGLVLSRFGLWSFDLAVNQLIQESVGSSSLGAVSGVQGSMQSLCQMLAYLAGVLV : 460 Coc_C169_55598 : ALGLVASRFGLWTFDLCVSQLLQERVDSLELGVVNGVQSSLQSLLQSFSYLMGLFV : 390 Supplemental Figure 8. C. reinhardtii ferroportin shows high sequence similarity to algal, plant and animal ferroportins. A. Ferroportin homologous sequences from C. reinhardtii (Cre), A. thaliana (Ath), V. carteri f. nagariensis (Vca), Cyanidioschyzon merolae (Cme), V. vinifera (Vvi), P. thichocarpa (Ptr), R. communis (Rco), H. sapiens (Hsa) and M. musculus (Mmu) were used for alignment. Dark and light brown lines indicate the conserved ferroportin (FPN1) domains. TM indicate the putative transmembrane domains (blue lines). Regions with low amino acid conservation are not shown. Back-shaded amino acids are conserved in 10 (black), 8-9 (dark grey), or 6-7 (light grey) of the aligned proteins. 17

18 Supplemental Figure 8. B Ath IREG1 Ath IREG2 Rco Rco Ptr Vvi LOC Vvi LOC Os06g Zma LOC Zma LOC Sbi 10g Coc C ChlNC64A Cre16.g Vca Dre LOC Dre SLC40A1 Rno SLC40A1 94 Hsa SLC40A1 Mmu IREG1 Zma LOC Ath IREG3 Ptr Supplemental Figure 8. C. reinhardtii ferroportin shows high sequence similarity to algal, plant and animal ferroportins. B. Protein sequences from C. reinhardtii (Cre), V. carteri f. nagariensis (Vca), C. variabilis (ChlNC64A), Coccomyxa sp. C-169 (Coc_C169), A. thaliana (Ath), Z. mays (Zma), Sorghum bicolor (Sbi), Oryza sativa (Os), R. communis (Rco), V. vinifera (Vvi), P. thichocarpa (Ptr), D. rerio (Dre), R. norvegicus (Rno), H. sapiens (Hsa) and M. musculus (Mmu) were used to build the phylogenetic tree as described in the Materials and Methods section. Orthologs of C. reinhardtii ferroportin are shown in blue. 18

19 Supplemental Figure 8. C. Supplemental Figure 8. C. reinhardtii ferroportin shows high sequence similarity to algal, plant and animal ferroportins. C. Protein similarity network of ferroportin-like proteins. The similarity network was generated on a local sequence database using an e-value of 1e-26 (top panel). Lowering the e-value to 1e- 35 (bottom panel) shows a clear separation of IREG3-like proteins as suggested by the phylogenetic tree. Nodes are colored as described in Supplemental Figure 7C, Nodes representing Amoebozoa are colored in light pink. 19

20 Supplemental Figure 9. Iron limitation induces changes in abundances of transcripts encoding enzymes required for carotenoid biosynthesis. The enzymes catalyzing each step are indicated by gene name abbreviations. Transcript abundance(s) of the genes encoding each enzyme are indicated with squares and circles for the photoheterotrophically- and photoautotrophically-grown cells, respectively. Full names of the enzymes of the carotenoid biosynthetic pathway are indicated in Supplemental Data Set 6. Enzymes labeled with a question mark are not known. 20

21 Supplemental Figure 10. A. * 20 * 40 * 60 * 80 * 100 Rco_ : MGGGDSPKCPPDKEEEALSS----SSMPAVESEPLSHVSLTDAPILLLVY--FHKAMREELSELYRLAVLASESLPNGR-QLIVELRRR : 82 AT1G74770 : MGGGNLHSLPPEN ASVSASYAVTVGNTKLSDAPVLFFVY--CHKAFRAQLVELRRFATDAAEADSFSG-DLAVELSRK : 75 AT1G18910 : MGVGDPLPLPPEKNRREVNKPPDIASTSSSSASAVNNARLSDAPILLFVY--FHKAFRAQLAELQFLAGDTVRSGS----DLAVELRSK : 83 Cre05.g : MAGAAAQLVPRT AFLEPPTVSSAAAPARAFPPI-NFIYGHFHNSIRAEL-GLLAERVRSLEAPGEGVGEMLADLRER : 75 Vca_60154 : MSSATAHLVPIS AHVSSSVC---VATQACFPPI-NFIYGHFHNSIRAEL-HLLADRVRSLEVPAEGVELMLSDLRER : 72 Smo_86556 : MASPVIVDLASA VPIVEPAA---ESEDDVMPPIMQFMY--FHKAIRAELEKLHGDALQIAKGSDQEI----KGLIRR : 68 Osa_03068 : MPRS PSPTASAA----AGSAAEAPMLIFLY--FHKAIRAELEGLHAAAVRLATERAGDLGGVCRALPLF : 63 Ath_BTS : MATPLPDFETARGGGAVASSSTTVLPSSVSSSSS-SSRPLPVANSFSDDAEEISPILIFLF--FHKAVCSELEALHRLALEFATGHHVDL----RLLRER : 93 Lja_LjnsRING : MATPL------DGGGGVTVLSNSVNN VASSSALNGAFKCSDVDSSPILIFLF--FHKAMRNELDALHRLAMAFATGNRSDI----QPLSDR : 79 * 120 * 140 * 160 * 180 * 200 Rco_ : FDFFKH VQKYHS-AFEDEVIFLELDAHIKNIVYTYSLEHNSIDDIFDSIFHCLSTLEENKDGAK--TFQELLSCIGTMDSSICKHMLKEEEQ : 171 AT1G74770 : FEFLKL VYKYHS-AAEDEVIFLALDKRVKNIVSNYSLEHAGTDDLFTSIFHWLH-VLEEEIGSRSDVLREVILCIGTIQSSICQHMLKEERQ : 165 AT1G18910 : FEFLKL VYKYHS-AAEDEVIFSALDTRVKNIVFNYSLEHDATDDLFTSVFHWLN-VLEEEQGNRADVLREVVLCIGTIQSSICQHMLKEERQ : 173 Cre05.g : YKFLEQ VYKYHS-TVEDEVLYPVLDSKVRNVTLAYSIEHQDEEILFEQLSKLISAALEEPEARRKCTIRTLICKVEEIHTTLRKHLAKEEEQ : 166 Vca_60154 : YKFLEQ VYKYHS-TVEDEVLYPALDAKVRNVTLAYSIEHQDEEILFEQLSQLLTTALEESESKRKGTIRALICKVEEIHTTLRKHLAKEEEQ : 163 Smo_86556 : YQFLRS VYEHHS-SAEDEVILPALDLRVKNVAHSYSLEHQVEGNLFEQLFELLNAVLAVKDDGLKESIRNVVCCTEALLTTLVQHLCKEEEQ : 159 Osa_03068 : WKNLKAPLGTPGPEVYVFKCKNVSVAVIFPALDIRVKNVAGTYSLEHKGENDLFSQLFALLQLDIQNDDSLR----RELASCTGAIQTCLSQHMSKEEEQ : 159 Ath_BTS : YRFLRS IYKHHC-NAEDEVIFSALDIRVKNVAQTYSLEHKGESNLFDHLFELLNSATETDESYR----RELARSTGALQTSVSQHLAKEQKQ : 180 Lja_LjnsRING : YHFLSA IYRHHC-NAEDEVIFPALDIRVKNVAQAYSLEHKGESNLFDHLFELLNSSIHNDESFA----RELASCAGALQTSVSQHMAKEEEQ : 166 * 220 * 240 * 260 * 280 * 300 Rco_ : VFPLLIQHFSPKEQALLVWQFFCSIPVILLVELLPWLTSFLTPEKRLNVTRCIEGVVPQEKSLQEVVVSWLHMNGQSSL : 250 AT1G74770 : VFPLLIEKFSFREQASLVWQFICSVPVMVLEDFLPWMISHLSHEEKIEVENCIKDVAPNEDSLQQVISSWLLDDSQSSC : 244 AT1G18910 : VFPLMIENFSFEEQASLVWQFICSVPVMVLEEIFPWMTSLLSPKEKSEVETCFKEVVPNELSLQLVINSWLIDDSQSSL : 252 Cre05.g : LLPLLLQHFSFAEQAELVAQFLYSIPLETVERVLSRLKPRIPRDEQERLLEEIQAVIP-DNLLSQLLTTWLQPEQQQALRMEREGLREAPAEAAEPQSRQ : 265 Vca_60154 : LLPLLLQHFSFAEQAELVAQFLYSIPLETVERVLSQLKPRIPRDEQEHLLVEIRNVIP-DNLLSQLLMTWLRPAAEMNLPHTNVSRQERPAD : 254 Smo_86556 : VFPLLVKHFTYDEQAALVWQFLCSIPVNLMELFLPWLASSLSLEDRDQMIACMRRIVPSEHLLQQVIFAWLKGRE : 234 Osa_03068 : VFPLLTKKFSYEEQADLVWQFLCNIPVNMMAEFLPWLSSSVSSDEHEDIRSCLCKIVPEEKLLQQVVFAWIEGKTTRKVTEN : 241 Ath_BTS : VFPLLIEKFKYEEQAYIVWRFLCSIPVNMLAVFLPWISSSISVDESKEMQTCLKKIVPGEPELTNF : 246 Lja_LjnsRING : VFPLLIEKFSLEEQASLVWQFLCSIPVNMMAEFLPWLSTSIPPDESQDLQNCLIKIVPEEKLLQKVIFTWMEGRSCINK : 245 * 520 * 540 * 560 * 580 * 600 Rco_ : YLTRNSRS---FSDIDSTIVRLKFLADVIIFYSNALKKFFYPV LNELAN---KTCSSEQFSIESRVESIHQLLQSKAENGFPFCK---- : 401 AT1G74770 : CQAKFQTL---ILDLDVLMARLNFLADVLVSYSNAFKKFFHPV LEEMTA--RRSSTAKQFNIDDCLENFQRLLYKSADDKTKTDN---- : 398 AT1G18910 : CQLTFPSL---SLDLNVLVVRLNFLADVLIFYSNAFKTFFYPVFEDM VDQQHS-----SSSKQFTIDGHVENFKKSLDLETRAGSDN : 406 Cre05.g : RSLQAGREPITAGHLQSLLERHRFLRAVYVFHSISEEEVLFPEVQRLAAANVQLAGGAAAAHQQQCEKDHAAELSSFEDLGRLLADVRAFARRGRKEVAG : 565 Vca_60154 : RALQLGRD-VTAAHLSALLERHRFLRSVYVFHSISEEEVLFPEVQRLAAN----VGGRAA---HQCEKDHQAELSSFEDLGRMLADVRSFARRGRK-VAS : 417 Smo_86556 : KLLRSASP----STLSSFVDRLRFLVEVCIFHSAAEDKVLSPA VGQRAN VEEDNQHLEMVQRLADGILAIGHLEGK---- : 356 Osa_03068 : RRMQQSGN---FSDISSFNARLQFIADVCIFHSIAEDQVVFPA VDSELS-----FVHEHAEEERRFNNFRCLIQQIQIAGAKSTA--LD : 392 Ath_BTS : RKIQLSGD---FSDLSAFDERLQYIAEVCIFHSLAEDKIIFPA VDGEFS-----FSEEHDEEENQFNEFRCLIENIKSAGASSTS-AAE : 356 Lja_LjnsRING : RKIQQLGD---FTNISAFNERLQFVAEVCIFHSIAEDKVIFPA VDGEFS-----FFQEHAEEESQFNDFRSLIEGIQNEGASSNS-EVE :

22 * 620 * 640 * 660 * 680 * 700 Rco_ : FVEKLCQELEFLAMDVSKKFSFQETEVLPLISKKFSNDTQQQLLYMSLHLMPLGLLKCVIPWFAAHLSENEFSSFLHGINLGNNLTNSYFASLLLEWFCT : 501 AT1G74770 : FLLQLQEELESLIIQVTKQFAIQRTEVFPIISKNCNHEMQKQLLYTSIHVLPLGLLKCVILWFSAHLSEEESQSILHFLSLEDSSPKKSFPRLLLQWLRF : 498 AT1G18910 : FVITLQEKLESLILTVAKQFSIEETEVFPIISKNCNIEMQRQLLYRSIHFLPLGLLKCVIMWFSAQLPEDECQSIIHYLSSEDSFPNKPFAHLLLQWFRF : 506 Cre05.g : MLEKLCCSVEAVAASIEHHMQREEADVFPLLEAHLCQAQQRALLYRTIRAMPLRLLERVMPWVVSRLDASAAAALLANIALGAPRSDQALVELLSRWARR : 665 Vca_60154 : MLEKLCCSAEAVHASIQHHMQREETDVFPLLEMHLCEAQQRMLVYRTIRAMPLRLLERVMPWVVSGLDDVAAASLLSNIRLGAPPGDLPLVELLSRWARR : 517 Smo_86556 : -ADELCIEVEYVIQTIREHYFNPELKLISLAQEHCTIEEQRVLLYRSLRVMPLRMLERVLPWFVEMQSDKETRDMLHHMCLAAPVSDKHLVALFSGWACK : 455 Osa_03068 : FYSELCSHADQIMETIEKHFCDEETKVLPQARMLFSPEKQRQLLYKSLCVMPLKLLERVLPWLVSKLSDEEASSFLENMRLAAPSSETALVTLFSGWACK : 492 Ath_BTS : FYTKLCSHADQIMETIQRHFHNEEIQVLPLARKNFSFKRQQELLYQSLCIMPLRLIERVLPWLTASLTEDEAKNFLKNLQAGAPKSDVALVTLFSGWACK : 456 Lja_LjnsRING : FYSKLCSHADHIMETIQRHFHNEEVQVLPLARKHFSFKRQCELLYQSLCMMPLKLIERVLPWLVGSLTEKEAKIFLNNMQLAAPATDSALVTLFCGWACK : 500 * 1220 * 1240 * 1260 * 1280 * 1300 Rco_ : HFHLLW LRYQFHSETEDEIAFPALEAKGNVQNISYSYTIDHKLEVKLFNE : 710 AT1G74770 : RFHMIK FLYQIHSDAEDEIAFPALEAKGQLKNISHSFSIDHELETKHFDK : 703 AT1G18910 : RFHLIK FLYQIHSDAEDEIAFPALEAKGKLQNISQSYSIDHELEVEHLNK : 698 Cre05.g : QQQQGQQQQQGQQQQPGPHAHLGVGWPAADGSGAAYTRGGQLAVVQHLHGRFQFLQGIYRAHSKSEDEIVFPALESKQALRNVSHAYTLDHRQEEQLFAD : 1258 Vca_60154 : HLVLAW GIYRAHSKSEDEIVFPALEAKQALRNVSHAYTLDHRQEEQLLPD : 715 Smo_86556 : RFHFLW GLYKAHSNAEDDIVFPALEEKEALHNVSHSYSLDHKQEQHLFEE : 662 Osa_03068 : RFRLLW GLYRAHSNAEDEIVFPALESRETLHNVSHSYTLDHKQEEQLFGD : 731 Ath_BTS : RFHLLW GFYKAHSNAEDDILFPALESKETLHNVSHSYTLDHKQEEKLFGD : 690 Lja_LjnsRING : RFRLLW GLYRAHSNAEDDIVFPALESKEALHNVSHSYMLDHKQEEQLFED : 734 * 1320 * 1340 * 1360 * 1380 * 1400 Rco_ : I-----SLILEKMSKLHVS LSTVDSGMLDQTV AKYNQQCKKLHLTCKSMHKLLSDHIHHEEIELWPLFRECF-SIEEQEKII : 786 AT1G74770 : V-----SFILNEMSELNML VSTINTTAADHDRK------MKYERLCLSLREICKSMHKLLSEHIQHEETELWGLFRNCF-SIEEQEKII : 780 AT1G18910 : V-----SFLLNE LAELNMLVLDHKN VKYEKLCMSLQDICKSIHKLLSEHLHREETELWCLFRDCF-TIEEQEKII : 767 Cre05.g : L-----EAVIDN LRAVDLTAQGAD AELSRQVMAVRRMCAAIRASLETHIRSEESELWPLFTEHFSSREEQQYLV : 1327 Vca_60154 : AMPPDKSPTSTNIAFYHSNGPSSRRQAIDQFRAVDLLKPGHE EELSRQVMGVRRMCAAIRASLETHIRSEESELWPLFTEHF-SREEQQYLV : 806 Smo_86556 : I-----ARFLEELSRLY IPDCLLEKSNINAIR-----EKRGDLAQKLQGMCKSVRCSLDHHVAREELELWPLFDRHF-TFEEQDRIV : 738 Osa_03068 : I-----SDALAELSQLHER--LTHPH----IEVSEAEKNDFNSSDEIDWTRKYNELATKLQGMCKSIRAALTNHVHREELELWPLFDEHF-SVEEQDKLV : 819 Ath_BTS : I-----YSVLTELSILHEK--LQSDSMMEDIAQTDTVRTDIDNGDCN---KKYNELATKLQGMCKSIKITLDQHIFLEELELWPLFDKHF-SIQEQDKIV : 779 Lja_LjnsRING : I-----SCVLSELSVLHEA--LQMTHMSEELSESNFGTSDANGTDDI---KKFNELATKLQGMCKSVRVTLDQHIFREERELWPLFGRHF-TVEEQDKIV : 823 * 1720 * 1740 * 1760 * 1780 * 1800 Rco_ : DIPG QYPSYRDRLKVNLGCK : 1034 AT1G74770 : TVHG QHPSYRDPHSLIFGCN : 1026 AT1G18910 : AVPG QNPSYRDPHKLIFGCK : 1021 Cre05.g : AAAGGCSSSSSTFGAGAVAGSSSSIMSSGAAGATCGHGTPAAPEAAAAAAAAAAATVSPAAATATAAAAAATATAAAGATAARQLHQTWHDAAGGVLGCG : 1716 Vca_60154 : AI IHRTCHDAAAGILGCQ : 971 Smo_86556 : DIPG RSRSYHDPVKGIYGCE : 902 Osa_03068 : GIPG CAPSYRDQEKQIFGCE : 988 Ath_BTS : VELG CSPSFRDPEKQIYGCE : 947 Lja_LjnsRING : EIEG YSPSFRDPGKHVFGCE :

23 * 1820 * 1840 * 1860 * 1880 * 1900 Rco_ : HYKRNCKLFTACCNKLYTCIRCHDEEADHTTDRKGITKMMCMKCLAIQPIGKACSSPSCNNLSMAKYYCSICKLFDDD--REIYHCPYCNLCRVGKGLGI : 1132 AT1G74770 : HYKRNCKLLAPCCDKLFTCIRCHDEEADHSVDRKQITKMMCMKCLLIQPIGANCSNTSCKS-SMGKYFCKICKLYDDE--RKIYHCPYCNLCRVGKGLGI : 1123 AT1G18910 : HYKRSCKLLAPCCNKLYTCIRCHDEEVDHLLDRKQITKMMCMKCMIIQPVGASCSNISCSS-SMGKYYCKICKLFDDD--REIYHCPYCNLCRLGKGLSI : 1118 Cre05.g : HYQRKCQLVAPCCGGVYTCRLCHDEAADHRMDRYAVSEMRCMSCGERQPVAAACR---ACAANMARYYCNICHLFDDEPGKDIYHCPFCNVCRRGRGLGV : 1813 Vca_60154 : HYQRKCQLVAPCCGKIFTCRLCHDAASDHRMDRYAVTEMRCMMCGTRQPVGAVCR---CCSTTMARYVCNICHLFDDEPGKDIYHCPFCNVCRRGRGLGV : 1068 Smo_86556 : HYKRNCKIRAACCGSLFVCRFCHDRASDHPMNRYSCAEMLCMQCLEVQPVAESCHNPECSGFRMARYYCNICKFFDDDT-RDIYHCPFCNLCRKGKGLGI : 1001 Osa_03068 : HYKRNCKLVAACCNKLFTCRFCHDKISDHTMERKATQEMMCMVCLKVQPVGPNCQTPSCNGLSMAKYYCNICKFFDDE--RTVYHCPFCNLCRLGKGLGV : 1086 Ath_BTS : HYKRNCKLRAACCDQLFTCRFCHDKVSDHSMDRKLVTEMLCMRCLKVQPVGPICTTPSCDGFPMAKHYCSICKLFDDE--RAVYHCPFCNLCRVGEGLGI : 1045 Lja_LjnsRING : HYKRNCKLRAACCGKLFTCRFCHDNVSDHSMDRKATSEMMCMRCLNIQPVGPICMTPSCNGLSMAKYFCNICKFFDDE--RNVYHCPFCNLCRVGRGLGI : 1091 * 1920 * 1940 * 1960 * 1980 * 2000 Rco_ : DYFHCMNCNACMSKSL-LVHVCREKCLEGNCPICHEYIFTSSNPVKALPCGHLMHSTCFQEYTCTHYICPICSKSLGDMQVYFKMLDALLAEEKMPDEYS : 1231 AT1G74770 : DYFHCMKCNACMSRTL-VEHVCREKCLEDNCPICHEYIFTSSSPVKALPCGHLMHSTCFQEYTCSHYTCPVCSKSLGDMQVYFKMLDALLAEEKMPDEYS : 1222 AT1G18910 : DYFHCMKCNACMSRLI-VEHVCREKCLEDNCPICHEYIFTSNSPVKALPCGHVMHSTCFQEYTCSHYTCPICSKSLGDMQVYFRMLDALLAEQKMPDEYL : 1217 Cre05.g : DFFHCMNCNACMSLSLFSSHKCREKCIEGNCPVCHEALFDSSQPIKELPCGHFMHSTCFSTYTRYNYTCPLCCKSVGDMSVYFQMLDSLLAAERLPPEYS : 1913 Vca_60154 : DFFHCMNCNACMSLTLFSSHKCREKCIEGNCPVCHEALFDSSQPIKELPCGHFMHSSCFLTYTRYNYTCPLCCKSVGDMSVYFQMLDSLLASERLPPEYS : 1168 Smo_86556 : DYFHCMTCNACMHVSL-KDHKCREKGLESNCPICHDFLFTSSSPVKALPCNHFMHSDCFQAYSCCHYTCPVCCKSLGDMAVYFGMLDALLAAEQLPEEYQ : 1100 Osa_03068 : DFFHCMKCNCCLGMKL-TEHKCREKGLETNCPICCDFLFTSSAAVRALPCGHFMHSACFQAYTCSHYTCPICCKSLGDMAVYFGMLDALLAAEELPEEYR : 1185 Ath_BTS : DFFHCMTCNCCLGMKL-VNHKCLEKSLETNCPICCEFLFTSSEAVRALPCGHYMHSACFQAYTCSHYTCPICGKSLGDMAVYFGMLDALLAAEELPEEYK : 1144 Lja_LjnsRING : DYFHCMKCNCCLGIKS-TSHKCLEKGLEMNCPICCDDLFTSSAPVRALPCGHYMHSACFQAYTCSHYTCPICSKSLGDMAVYFGMLDALLAAEELPEEYR : 1190 * 2020 * 2040 * Rco_ : GKTQVILCNDCEKKGPAAFHWHYHKCPFCDSYNTRLL : 1268 AT1G74770 : NKTQVILCNDCGRKGNAPYHWLYHKCTTCGSYNSRLL : 1259 AT1G18910 : NQTQVILCNDCGRKGNAPYHWLYHKCSSCASYNTRLF : 1254 Cre05.g : GRMQQVLCNDCGKMGFAPFHFVYHSCPHCRSYNTRVL : 1950 Vca_60154 : GRMQQVLCNDCGKMGFAPFHFVYHSCPHCRSYNTRVL : 1205 Smo_86556 : GRVQDILCNDCEQKGTAPFHWLYHKCQKCGSYNTRTI------YDGGSHR : 1144 Osa_03068 : DRCQDILCNDCERKGRSRFHWLYHKCGSCGSYNTRVIKTDT--ADCSTPN : 1233 Ath_BTS : NRCQDILCNDCERKGTTRFHWLYHKCGSCGSYNTRVIKSETIPPDCSTSS : 1194 Lja_LjnsRING : DRNQDILCHDCNRKGTSRFHWLYHKCGFCGSYNTRVIKSET----YSSCS : 1236 Supplemental Figure 10. Plant and algal hemerythrin-e3 ligase like proteins are highly conserved. A. Alignment of plant and algal hemerythrin-e3 ligase like proteins. Proteins sequences from C. reinhardtii (Cre), V. carteri f. nagariensis (Vca), A. thaliana (Ath, AT), R. communis (Rco), Oryza sativa (Osa), Selaginella moellendorffii (Smo) and Lotus japonica (Lja) were aligned using MUSCLE. Red lines indicate the putative hemerythrin domains, green line indicates the conserved CHY-type zinc finger domain and the cyan line, the putative E3 ubiquitin-protein ligase domain. Back-shaded amino acids are conserved in 9 (black), 7-8 (dark grey), or 5-6 (light grey) of the aligned proteins. The regions with low sequence similarity are not shown. 23

24 Supplemental Figure 10. B. Lja LjnsRING 83 Rco Ptr 0012s05140 Ath BTS Rco Ptr 0008s12230 Ptr 0010s13190 Os01g Os05g AT1G74770 AT1G Rco Smo Ptr 0015s07790 Ppa Smo Cre05.g Vca ChlNC64A OstRCC MicpuC MicpuN Supplemental Figure 10. Plant and algal hemerythrin-e3 ligase like proteins are highly conserved. B. Phylogenetic tree of hemerythrin containing E3 ubiquitin ligase like proteins from plants and algae. Hemerythrin-containing E3 ubiquitin ligase like proteins from C. reinhardtii (Cre), V. carteri f. nagariensis (Vca), C. variabilis (ChlNC64A), V. carteri f. nagariensis (Vca), Ostreococcus sp. RCC809 (OstRCC809), Micromonas sp. RCC299 (MicpuN), M. pusilla (MicpuC), A. thaliana (Ath), R. communis (Rco), O. sativa (Osa), Selaginella moellendorffii (Smo), Lotus japonica (Lja), P. trichocarpa (Ptr) and P. patens (Phypa) are compared. The C. reinhardtii protein is depicted in red and its orthologs in blue. 24

25 Supplemental Figure 10. C. Supplemental Figure 10. Plant and algal hemerythrin-e3 ligase like proteins are highly conserved. C. Protein similarity network of hemerythrin-e3 ligase like proteins. The similarity network was generated on a local sequence database using an e-value of 1e-80. The nodes representing animal FBXL5 proteins are linked in the network with the plant and algal proteins (inset) when the e-value was 1e-5. Nodes are colored as described in Supplemental Figure 7C. 25