Light- induced electron transfer and ATP synthesis in a carotene synthesizing insect Jean Christophe Valmalette1, Aviv Dombrovsky2,4, Pierre Brat3, Christian Mertz3, Maria Capovilla4, Alain Robichon41 – IM2NP UMR 7334 CNRS, Université du Sud Toulon Var, P.O. Box 20132, 83957 La Garde CEDEX, France 2 - Volcani Center, Institute of Plant Protection, P.O. Box 6, 50250 Bet Dagan, Israel 3 - CIRAD UMR QualiSud, 73 rue J.F. Breton, TA B-95/16, 34398 Montpellier CEDEX 5, France 4 - UMR7254 INRA/CNRS/UNS, Institut Sophia Agrobiotech, 400 route des Chappes, P. O. Box 167, 06903 Sophia Antipolis, France ******************* LOCUS XP_001943938 510 aa DEFINITION pisum]. ACCESSION VERSION DBSOURCE SOURCE PREDICTED: similar to phytoene dehydrogenase 1 61 121 181 241 301 361 421 481 linear INV 02-JUL-2008 [Acyrthosiphon XP_001943938 XP_001943938.1 GI:193673878 REFSEQ: accession XM_001943903.1 Acyrthosiphon pisum (pea aphid) mvvkiiiigs pkifeetfed tlinflnylk dglalysllq kgiklvngdv qlnvhnifla lsnepnidfd glshtflqvv klslwskcvs gvggtavaar lgediknhie lthlyyrksv yteiaegiwy vnsdivicna ekykesfdqi klvnkareqv wfrpslkcni flislltllf lskkgfqvei llkcptnysv nviqlhlldt pkggyhkvle dltyaynkll fkdhtlpddp idtiekrlki fknlyfvgas lwislffnkt yeknsynggr hfhdgetfel vynkvskyfk ilekiavqhg pktsyaekld sfyvnvpsri snfrsmidhe ahpgtgvpvv csliyqnghr ttdisklsrs sdymrkafsf akfnynadvq kkehtsssis dptaapegkd kvndprtwrn lcgakllenq fdqgpslylm lekyegsges qtmylgmspy eiiiddkgva fywsmntivs sivvlvpvgh efnlwkgsil lcdrflkska ======================== LOCUS DEFINITION ACCESSION VERSION XP_001946689 526 aa linear INV 02-JUL-2008 PREDICTED: similar to F37C4.6 [Acyrthosiphon pisum]. XP_001946689 XP_001946689.1 GI:193706960 1 DBSOURCE SOURCE 1 61 121 181 241 301 361 421 481 LOCUS REFSEQ: accession XM_001946654.1 Acyrthosiphon pisum (pea aphid) mvvkiiiiga pkifeeifed tlinflrylk ksdymrkafs gakfnynadv gkkeltsssi idptaapegk eivndprtwq vlcgtkllek gvggtaaaar lgediknhid ethvhyqrsv fqtmylgmsp qeiivddkgv sfywsmktiv dtivvlvpvg nnfnlwkgsi qlcdrfldsk XP_001943170 lskkgfqvei llkcpsnysv kvalktdfqh ydglaaysll akgikmvngd sqlkvhnifl hisnvpnidf lglshslfqv vtksswsmcv yeknaynggr hfhdgetfel wydffnpkfl qyteiaegiw vvnsdivicn aekykesfdq dqhvktareh lwfrpsmkck sfligiivll 608 aa linear DEFINITION PREDICTED: similar to lycopene cyclase [Acyrthosiphon pisum] ACCESSION XP_001943170 VERSION XP_001943170.1 GI:193648048 DBSOURCE REFSEQ: accession XM_001943135.1 SOURCE Acyrthosiphon pisum (pea aphid) 1 61 121 181 241 301 361 421 481 541 601 mltyidvhfi ykpknilavi lalvmttiqg lylcwvdria adtytfefpl vfpagvrlhl mtprkpevkw dlllyssyva dsetlgrcyv irylpyevrg qadrckhc ytlpvvavla gyvpveeymf yniavpgknt lkddvwhine ryssswkyys iilyafcrvt eqyrldltde gsvgtlcvyv paeymdnaaa pvlvatdiyr litwpfisrl fviqtlmtsl fylgcimwws ktslnifvvd qqmqqfvrae ddmidsepkv elscfraisr mvyksgtqid vvntlcsdrd gvacaveasp csliyqnghr ttdisklsrs pdviqlhlld ypkggfhkvl adlvyaynkl ifkdhtlpde vidtiekrlk ifenlyfvga ifctlf fdqgpslylm lekyegsges tvynrvckyf esleniavqh lpktsyadkl psfyvnvpsr isnfrsmidh svqpgtgvpi INV 02-JUL-2008 / phytoene synthase elfkigfvct walvftrwsp cpvimflwyg dlpfeeclff cdmspspvnd gvkkqklkli isfylprkpf ddkrhdfvig pwtlgseklk typrraslgk mafvyttpwd acfnfnfnkt agnyfvkkst litnviivlg irqclnvlkr etfidelfad yelldgyrwd kaqqmgqvlq syatrmirla wdkilvsins nyiifhnawm sytlirwipi ssaiavivpt gmafdksygl asksfnvasl rsadydvkts vdgktvqnet ivnisrdivt nryqlesleg lyfkslkyff ============================== ref|XP_001943938.1| PREDICTED: similar to phytoene dehydrogenase [Acyrthosiphon pisum] Length=510 GENE ID: 100169245 LOC100169245 | similar to phytoene dehydrogenase [Acyrthosiphon pisum] Identities = 130/472 (27%), Positives = 226/472 (47%), Gaps = 27/472 (5%) ref|XP_001950764.1| PREDICTED: similar to phytoene dehydrogenase [Acyrthosiphon pisum] Length=528 GENE ID: 100161380 LOC100161380 | similar to phytoene dehydrogenase [Acyrthosiphon pisum] ref|XP_001946689.1| PREDICTED: similar to F37C4.6 [Acyrthosiphon pisum] Length=526 GENE ID: 100169110 LOC100169110 | similar to F37C4.6 [Acyrthosiphon pisum] Identities = 127/472 (26%), Positives = 221/472 (46%), Gaps = 14/472 (2%) 2 ref|XP_001943225.1| PREDICTED: similar to phytoene dehydrogenase, partial [Acyrthosiphon pisum] Length=373 GENE ID: 100159050 LOC100159050 | similar to phytoene dehydrogenase [Acyrthosiphon pisum] ref|XP_001943170.1| PREDICTED: similar to lycopene cyclase / phytoene synthase [Acyrthosiphon pisum] Length=608 GENE ID: 100161104 LOC100161104 | similar to lycopene cyclase / phytoene synthase [Acyrthosiphon pisum] Identities = 78/301 (25%), Positives = 140/301 (46%), Gaps = 40/301 (13%) ref|XP_001950868.1| PREDICTED: similar to lycopene cyclase / phytoene synthase [Acyrthosiphon pisum] Length=589 GENE ID: 100164140 LOC100164140 | similar to lycopene cyclase / phytoene synthase [Acyrthosiphon pisum] Identities = 70/216 (32%), Positives = 109/216 (50%), Gaps = 37/216 (17%) ============================= LOCUS AAT35222 1798 aa linear SYN 12-JUL-2004 DEFINITION ACCESSION VERSION DBSOURCE SOURCE ORGANISM fusion of carotene synthesis proteins [synthetic construct]. AAT35222 AAT35222.1 GI:47531118 accession AY605097.1 synthetic construct synthetic construct other sequences; artificial sequences. REFERENCE 1 (residues 1 to 1798) AUTHORS Xiong,A.S.,Yao,Q.H.,Peng,R.H.,Li,X.,Fan,H.Q.,Cheng,Z.M.Li,Y. TITLE A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences JOURNAL Nucleic Acids Res. 32 (12), E98 (2004) PUBMED 15240836 FEATURES source Protein 1 61 121 181 241 301 361 421 Location/Qualifiers 1..1798 /organism="synthetic construct" /db_xref="taxon:32630" /note="derived from Paracoccus marcusii" 1..1798 /product="fusion of carotene synthesis proteins" /name="CrtE/CrtB/CrtI/CrtW/CrtZ" msdlvltste ndpqarldgl yrtlddvley rcylpgdwld afriyraigl phhaqllnfd alssgkrfrg athvahgesr aitqgsqsfa radtlaalqg syhvagilgv hagarvdgpv pirkggpeay llklagdves mlmllaaeas avlggialit taaklmppgi dgpvtppfaa mmarvmgvrd pspelytvil rqristskaa npgpmrrdvn ggvcdtivda eamallagar rddtvmlyaw lravarrhdf hpvldracdl rlldaaepyy kigllgiggw pihatllqtr acavemvhaa gasgtvraql crhaddvidg pqawpmdlie glafqlthia psarvgladl dvadhacrgs ieeiaqgfga slifddlpcm vrilsrslgp qalgsrpeav gfaidveard rdvidyarig pprcawsipa gvsrqdlwtr vlqplgaama ddaglrrgqp qglcagqdld 3 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 lhaakngagv levvgdqaal ieallervlp rlqsagiatt tlmpvspfyr lgtvpflklg siyalihale ladgrslrad diahhtilfg lsraeidwav epiltqsawf agdvesnpgp lgltwlsvgl gtdddpdfdh asiqlfvfgt rlpstrtkgd rvllldhaag ldgaaladav hkflgveiet dalaaasqdy tgyslpyaaq rytllqrlyr eqeqdlktgv gkdtgrdaaa yaaraqllnf lveardkpgg lmwpggkvfd qmlkaapalm rrggvwfakg mvasngdvmh prykelvnei egpryadril rphnrdktir msahalpkad fiiahdamhg ggpvrwyarf wlphrpghda taqllnfdll pseghtwsch vrsgaeirwd drphgvprpv arqqgwtgae vadvvaglsg mphglierfy lfiaglemla pgprrgllav dllklagdve rayvwhdqgh yvneadqler kleaynsvha gtnqlvagmv nyrdllghta fkgpklaedf afleerlipn nfylvgagth ltatslivsg svvpgrpran igtyfgwreg fpdrhnarss klagdvesnp dpdlsphwla sdialldaqg imdgtvtqrd vrrergilpi ppgtdalrga agrlsvadql vikefdaeeq sdlqkvsrqy snpgpmnahs vfdagptvit qiaqfnpddl kvatfikdpy alferpggqm rgqsraksld slylhspctt lranltrtri pgagipgvvg giiaawlalh aamgqlvlwl lllpvivtvy risdpvsllt gpmthdvlla rlkplrranw atlscgtrie gygfiyllpf alahdaagfw irdyaidrar rivtgkppip tqmidfgrql easraqlegm paaktaivig dpdalkelwa egyrrfrdya lrqafsyhtl mlnakvarie rkrwsmslfv dpdmappgms ftpadfasel sakataqvml vhalwfldaa yagfswrkmi alilgdrwmy cfhfggyhhe gaglanglia pdqevrfprh agsvldgrgp srtriliedt adhaegpvpv rdrflrllnr lgtairclpe grvfqsyddl vgskrlqape agfgglalai ltgqdmardv eevyqegyvk lvggnpfsts tegarttgvt lhfglreapk thyvlapvph nahhgsafsv qllnfdllkl ahpilavanf vkhmahhrha vvfwplpsil hhlhptvpww lalraarpdl arrlatgygs hpsrhltlgf rysdggdldd glragffhpv mlfrgcapdr rpllkena Figure S1. Genes involved in carotenoid metabolism retrieved from the pea aphid (A. pisum) genome. A synthetic construct from enterobacteriacae representing the cluster of genes coding the cascade of enzymes involved in the carotene metabolism was used to Blast the aphid genome. The sequences and accession numbers are retrieved from the public site NCBI. We observed that the phytoene dehydrogenase gene and the lycopene cyclase/phytoene synthase fused gene are represented by several copies located in different places of the genome. 4 crtE crtE crtE Idi crtX crtY crtI crtB crtZ Idi crtI crtB crtZ crtY crtX crtY crtI crtB crtZ GENES AND ENZYMES OF CAROTENOID BIOSYNTHESIS IN PLANTS Annual Review of Plant Physiology and Plant Molecular Biology Vol. 49: 557-583 1998 F. X. Cunningham, Jr. and E. Gantt DMAPP IPP G3P crtE crtI crtY crtZ crtW crtB Idi dimethylallyl pyrophosphate isopentenyl pyrophosphate glycerol phosphate geranylgeranyl pyrophosphate synthetase phytoene dehydrogenase lycopene cyclase beta-carotene hydroxylase beta-carotene oxygenase phytoene synthase isopentenyl pyrophosphate isomerase Figure S2. Biosynthesis of carotenoids in plants (from Cunningham et al., 1998). This flowchart represents the steps of biosynthesis of carotenoids from the pyruvate and glycerol phosphate precursors. On the right, are represented the clusters of genes involved in this metabolic cascade. 5 A embryon blanc signal faible Embryon orange 30 000 Intensity Intensity (a.u.) (a.u.) 30 000 25 000 20 000 1515 000 000 10 000 5 000 5 000 0 500 1 000 Shift Raman (cm-1 ) 1 500 2 000 Shift raman cm1 B Figure S3. Raman imaging of carotene signature in aphid embryos. The spectra corresponding to three stages of embryonic development were obtained with a laser beam at 488 nm. A strong orange color appears in the older embryos and correlates with the apparition of intense Raman vibration shift signals which sign carotene molecules. (A) Raman imaging of two embryos: a white (blue) and an emerging orange (green). (B) Raman imaging of three orange embryos (intensively coloured). Each line represents one individual embryo. 6 A B 7 Comparison between two aphids C 2 500 C 2 000 C C Adulte rose 4 SLB adultes verts fort 2 SLB CH3 (a. u.) Intensity Intensity (a.u.) C C 1 500 1 000 500 0 500 1 000 1 500 Shift Raman (cm-1) -1 2 000 Raman shift cm Figure S4. Chromatograms of the carotene content and comparative Raman imaging in the green and orange adult aphids. The differences in carotenoid composition can be assessed by HPLC isolation of the molecules. These variations are also quantified by the ratio of intensity of the peaks obtained by Raman imaging. (A) HPLC chromatograms (15-60 min segments at 470 nm) of an extract of green aphids. Peak assignment refers to Table 1. (B) HPLC chromatograms (15–60 min segments at 470 nm) of an extract of orange aphids. Peak assignment refers to Table 1. (C) Raman imaging of two different individual aphids (green and orange aphid). The ratio C-C/C=C is stronger in the green phenotype. The ratio C=C torulene/carotene is 11:13. The ratio C-C/C=C is 1:0.880.04 for the orange phenotype and 1:1.200.02 for the green. The ratio C-CH3/½(C-C + C=C) is 0.33:1 for the green and 0.36:1 for the orange. The peak C-CH3 as reference, C-CH3/C-C is 1:2.8 for the orange and 1:3 for the green. By contrast, the ratio C-CH3/C=C 1:3.2 for the orange and 1:2.5 for the green, show some differences in accordance with the mass spectrum determinations. 8 Tetrazolium (MTT) reduction by visible light in presence of beta carotene A B C Beta carotene+MTT+light (15min) Beta carotene+light ( 30 min) Beta carotene+MTT+light (30 min) 0,6 A 0,5 OD 550 nm 0,4 B 0,3 0,2 0,1 k ne te ro ca ca ro te ne ,d ar ,l ig h t k m ,d ar t Te tr az ol iu m ,l ig h iu ol tr az Te az tr Te Te tr az ol ol iu iu m m + + ca ca r ro ot te en C ne e, ,d ar lig h k t 0,0 Figure S5. OD measure of the tetrazolium reduction by visible light. β-carotene was dissolved in acetone and spotted on a glass slide. Soluble tetrazolium salts (1 mM) were solubilised in water/NaCl/HCl (1 mM, pH 4-5) and deposited on the dry carotene layer. The light exposure shows the blue precipitation of formazan resulting in the reduction of MTT. The reduced form of tetrazolium (formazan) was solubilized in ethanol/ acetic acid (90/10) (β-carotene after light exposure was extracted with the same solvent to determine the blank control). We notice that carotene is not soluble in ethanol/acetic acid except for some more hydrophilic esterified derivatives. This series of experiment was repeated three times and the bars represent the mean +/- S.E. 9 Erythroaphin :chemistry of aphid colouring matters (Lord Todd) Nature (1948) 162, 79, J.Chem.Soc. (1950) 477;(1953) 477;(1954)107;(1955 )409 Figure S6. Molecular structure of polycyclic compounds found in aphids. We notice strong electron delocalisation due to the abundance of conjugated double bonds and powerful redox potentials that might account for the oxydo-reduction process in cells. Up to date little is known about the physiological functions of these compounds. 10
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