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

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LOCUS XP_001943938 510 aa linear INV 02-JUL-2008

DEFINITION PREDICTED: similar to phytoene dehydrogenase [Acyrthosiphon pisum].

ACCESSION XP_001943938

VERSION XP_001943938.1 GI:193673878

DBSOURCE REFSEQ: accession XM_001943903.1

SOURCE Acyrthosiphon pisum (pea aphid)

1 mvvkiiiigs gvggtavaar lskkgfqvei yeknsynggr csliyqnghr fdqgpslylm

61 pkifeetfed lgediknhie llkcptnysv hfhdgetfel ttdisklsrs lekyegsges

121 tlinflnylk lthlyyrksv nviqlhlldt vynkvskyfk sdymrkafsf qtmylgmspy

181 dglalysllq yteiaegiwy pkggyhkvle ilekiavqhg akfnynadvq eiiiddkgva

241 kgiklvngdv vnsdivicna dltyaynkll pktsyaekld kkehtsssis fywsmntivs

301 qlnvhnifla ekykesfdqi fkdhtlpddp sfyvnvpsri dptaapegkd sivvlvpvgh

361 lsnepnidfd klvnkareqv idtiekrlki snfrsmidhe kvndprtwrn efnlwkgsil

421 glshtflqvv wfrpslkcni fknlyfvgas ahpgtgvpvv lcgakllenq lcdrflkska

481 klslwskcvs flislltllf lwislffnkt

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LOCUS XP_001946689 526 aa linear INV 02-JUL-2008

DEFINITION PREDICTED: similar to F37C4.6 [Acyrthosiphon pisum].

ACCESSION XP_001946689

VERSION XP_001946689.1 GI:193706960

DBSOURCE REFSEQ: accession XM_001946654.1

SOURCE Acyrthosiphon pisum (pea aphid)

1 mvvkiiiiga gvggtaaaar lskkgfqvei yeknaynggr csliyqnghr fdqgpslylm

61 pkifeeifed lgediknhid llkcpsnysv hfhdgetfel ttdisklsrs lekyegsges

121 tlinflrylk ethvhyqrsv kvalktdfqh wydffnpkfl pdviqlhlld tvynrvckyf

181 ksdymrkafs fqtmylgmsp ydglaaysll qyteiaegiw ypkggfhkvl esleniavqh

241 gakfnynadv qeiivddkgv akgikmvngd vvnsdivicn adlvyaynkl lpktsyadkl

301 gkkeltsssi sfywsmktiv sqlkvhnifl aekykesfdq ifkdhtlpde psfyvnvpsr

361 idptaapegk dtivvlvpvg hisnvpnidf dqhvktareh vidtiekrlk isnfrsmidh

421 eivndprtwq nnfnlwkgsi lglshslfqv lwfrpsmkck ifenlyfvga svqpgtgvpi

481 vlcgtkllek qlcdrfldsk vtksswsmcv sfligiivll ifctlf

LOCUS XP_001943170 608 aa linear INV 02-JUL-2008

DEFINITION PREDICTED: similar to lycopene cyclase / phytoene synthase

[Acyrthosiphon pisum]

ACCESSION XP_001943170

VERSION XP_001943170.1 GI:193648048

DBSOURCE REFSEQ: accession XM_001943135.1

SOURCE Acyrthosiphon pisum (pea aphid)

1 mltyidvhfi ytlpvvavla litwpfisrl elfkigfvct mafvyttpwd nyiifhnawm

61 ykpknilavi gyvpveeymf fviqtlmtsl walvftrwsp acfnfnfnkt sytlirwipi

121 lalvmttiqg yniavpgknt fylgcimwws cpvimflwyg agnyfvkkst ssaiavivpt

181 lylcwvdria lkddvwhine ktslnifvvd dlpfeeclff litnviivlg gmafdksygl

241 adtytfefpl ryssswkyys qqmqqfvrae cdmspspvnd irqclnvlkr asksfnvasl

301 vfpagvrlhl iilyafcrvt ddmidsepkv gvkkqklkli etfidelfad rsadydvkts

361 mtprkpevkw eqyrldltde elscfraisr isfylprkpf yelldgyrwd vdgktvqnet

421 dlllyssyva gsvgtlcvyv mvyksgtqid ddkrhdfvig kaqqmgqvlq ivnisrdivt

481 dsetlgrcyv paeymdnaaa vvntlcsdrd pwtlgseklk syatrmirla nryqlesleg

541 irylpyevrg pvlvatdiyr gvacaveasp typrraslgk wdkilvsins lyfkslkyff

601 qadrckhc

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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%)

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%)

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LOCUS AAT35222 1798 aa linear SYN 12-JUL-2004

DEFINITION fusion of carotene synthesis proteins [synthetic construct].

ACCESSION AAT35222

VERSION AAT35222.1 GI:47531118

DBSOURCE accession AY605097.1

SOURCE synthetic construct

ORGANISM 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 Location/Qualifiers

source 1..1798

/organism="synthetic construct"

/db_xref="taxon:32630"

/note="derived from Paracoccus marcusii"

Protein 1..1798

/product="fusion of carotene synthesis proteins"

/name="CrtE/CrtB/CrtI/CrtW/CrtZ"

1 msdlvltste aitqgsqsfa taaklmppgi rddtvmlyaw crhaddvidg qalgsrpeav

61 ndpqarldgl radtlaalqg dgpvtppfaa lravarrhdf pqawpmdlie gfaidveard

121 yrtlddvley syhvagilgv mmarvmgvrd hpvldracdl glafqlthia rdvidyarig

181 rcylpgdwld hagarvdgpv pspelytvil rlldaaepyy psarvgladl pprcawsipa

241 afriyraigl pirkggpeay rqristskaa kigllgiggw dvadhacrgs gvsrqdlwtr

301 phhaqllnfd llklagdves npgpmrrdvn pihatllqtr ieeiaqgfga vlqplgaama

361 alssgkrfrg mlmllaaeas ggvcdtivda acavemvhaa slifddlpcm ddaglrrgqp

421 athvahgesr avlggialit eamallagar gasgtvraql vrilsrslgp qglcagqdld

481 lhaakngagv eqeqdlktgv lfiaglemla vikefdaeeq tqmidfgrql grvfqsyddl

541 levvgdqaal gkdtgrdaaa pgprrgllav sdlqkvsrqy easraqlegm vgskrlqape

601 ieallervlp yaaraqllnf dllklagdve snpgpmnahs paaktaivig agfgglalai

661 rlqsagiatt lveardkpgg rayvwhdqgh vfdagptvit dpdalkelwa ltgqdmardv

721 tlmpvspfyr lmwpggkvfd yvneadqler qiaqfnpddl egyrrfrdya eevyqegyvk

781 lgtvpflklg qmlkaapalm kleaynsvha kvatfikdpy lrqafsyhtl lvggnpfsts

841 siyalihale rrggvwfakg gtnqlvagmv alferpggqm mlnakvarie tegarttgvt

901 ladgrslrad mvasngdvmh nyrdllghta rgqsraksld rkrwsmslfv lhfglreapk

961 diahhtilfg prykelvnei fkgpklaedf slylhspctt dpdmappgms thyvlapvph

1021 lsraeidwav egpryadril afleerlipn lranltrtri ftpadfasel nahhgsafsv

1081 epiltqsawf rphnrdktir nfylvgagth pgagipgvvg sakataqvml qllnfdllkl

1141 agdvesnpgp msahalpkad ltatslivsg giiaawlalh vhalwfldaa ahpilavanf

1201 lgltwlsvgl fiiahdamhg svvpgrpran aamgqlvlwl yagfswrkmi vkhmahhrha

1261 gtdddpdfdh ggpvrwyarf igtyfgwreg lllpvivtvy alilgdrwmy vvfwplpsil

1321 asiqlfvfgt wlphrpghda fpdrhnarss risdpvsllt cfhfggyhhe hhlhptvpww

1381 rlpstrtkgd taqllnfdll klagdvesnp gpmthdvlla gaglanglia lalraarpdl

1441 rvllldhaag pseghtwsch dpdlsphwla rlkplrranw pdqevrfprh arrlatgygs

1501 ldgaaladav vrsgaeirwd sdialldaqg atlscgtrie agsvldgrgp hpsrhltlgf

1561 hkflgveiet drphgvprpv imdgtvtqrd gygfiyllpf srtriliedt rysdggdldd

1621 dalaaasqdy arqqgwtgae vrrergilpi alahdaagfw adhaegpvpv glragffhpv

1681 tgyslpyaaq vadvvaglsg ppgtdalrga irdyaidrar rdrflrllnr mlfrgcapdr

1741 rytllqrlyr mphglierfy agrlsvadql rivtgkppip lgtairclpe 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.

DMAPPdimethylallyl pyrophosphate IPP isopentenyl pyrophosphate G3P glycerol phosphate crtE geranylgeranyl pyrophosphate synthetase crtI phytoene dehydrogenase crtY lycopene cyclase crtZ beta-carotene hydroxylase crtW beta-carotene oxygenase crtB phytoene synthase Idi 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.

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.

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 byRamanimaging.(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 1:0.880.04 for the orange phenotype and 1:1.200.02 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 theorange and 1:2.5 for the green, show some differences in accordance with the mass spectrum determinations.

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.

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.

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