A. Supplementary data
A.6 Supplemental: Chapter 8
PtTRX F natural protein sequence:
MESAFDQTIASAGGSLVVVDYSTTWCGPCKVIAPKFEELSEKYGDAVFLKVIGDASPDASKLMKREGVRSVPSFH YFKNGEKVDVVNGANAEAIEAAIAKHGAER
PtTRX F (CS) protein sequence:
MESAFDQTIASAGGSLVVVDYSTTWCGPSKVIAPKFEELSEKYGDAVFLKVIGDASPDASKLMKREGVRSVPSFH YFKNGEKVDVVNGANAEAIEAAIAKHGAER
PtTRX F (CS) E. coli optimised nucleotide sequence including flanking restriction sites:
CATATGGAATCCGCCTTTGACCAGACCATTGCGAGTGCTGGTGGCTCGTTAGTTGTTGTCGACTATTCGACGACT TGGTGCGGTCCTAGCAAAGTCATTGCGCCGAAATTCGAGGAATTGAGCGAGAAGTATGGCGATGCCGTGTTTCTG AAAGTGATCGGCGATGCATCACCAGATGCGTCTAAACTGATGAAACGCGAAGGTGTTCGCAGTGTACCGAGCTTT CACTACTTCAAGAACGGGGAGAAAGTGGATGTGGTAAACGGCGCAAATGCGGAAGCCATCGAAGCTGCCATTGCG AAACATGGAGCAGAACGTTAAGAGCTC
CATATG NdeI restriction site GAGCTC SacI restriction site
Supplemental Figure 8-SI Sequence adaption of the natural PtTRX F (46280) sequence: the second cysteine of the catalytic centre (green) is changed into serin (grey). This modified sequence is optimised for improved expression in E. coli K12 by application of the GENEius software (Eurofins MWG). Terminal restriction sites (marked by underlining) and a STOP codon were added to the optimised sequence: at the 5’ end an NdeI restriction site and at the 3’ end a TAA STOP codon. This sequence was produced as synthetic gene (Eurofins MWG, Ebersberg, Germany) and allows the fusion to 5’ terminal tags.
A.7 Supplemental: Chapter 9 A.7.1 Supplemental Figure 9-SI
gi|108707737|gb|ABF95532.1| ---MAT---ILAVAAMATFQAPTTLPGPLAP-TLPVR---- 30
gi|224285472|gb|ACN40458.1| MAAASMAASTVSKAVLFPSNAAAAAAVFSFK---PSPLKFHCKPVVGKGY 47 gi|1617206|emb|CAA96570.1| ---MATISGLS---LSNP-RLLFNSPGFP---QTIKISSASPLST 35 gi|355500984|gb|AES82187.1| ---MATIGGLSS---LSNP-KLLFNSSGFP---QTIKISPATPLQT 36 gi|325512541|gb|ADZ23481.1| ---MATISGMT---LSSPTRVLAQGPEIPQKAQTVKFSPVVRLNQ 39 gi|355480449|gb|AES61652.1| ----MAATMAGVS---LSSP-RVIFKGPESLQKSQAIRSGPVFMLNQ 39 gi|218156192|dbj|BAH03328.1| ---MATIAGLN---LSTP-RVLAKSADSP-KAQTIKSPWLSHSWK 37 gi|151564658|gb|ABS17660.1| --- gi|332646828|gb|AEE80349.1| ---MATIATG-L---NIATQRVFVTSENRPV----CLAGPVHLNNS 35 gi|312282565|dbj|BAJ34148.1| ---MAAIATS-V---NIATQRGVVTSENRPV----RLIGPVRLNNP 35 gi|119720838|gb|ABL97989.1| ---MATIATS-L---NIATQRAVITSKSQPA----RLACPVRLNNP 35 gi|330255742|gb|AEC10836.1| ---MTTIAAAGL---NVATPRVVV----RPVA---RVLGPVRLNYP 33 gi|297824833|ref|XP_002880299. ---MTTIAAAGL---NVATPRVVV----RPVA---RVSGPVRLNYP 33 gi|1617197|emb|CAA96569.1| ---MASIAGVS---ITTPRILSKTSDSP-KVQTLKFQSLNKPWK 37 gi|1617213|emb|CAA96568.1| ---MA----SMS---LSMTPKILLP--NNP----TTNFDAPKLANM 30 Thaps_270287protein ---MKTIASLLAIVITTMSSSSNAFFN----PANLSTKLTPPKPSTTS 41 Thaps_20804protein MK---IFLASLIGSCAA-FAPAPFGKS-PTALFG 29 Phaeo_56708protein MK---FTFALLALPLASGFAPSTFGVQRQTAIFS 31 FragiCyl_287476protein MTNM---TMMTSVLLVLSAGS---TMIMSSCSAFVI 30 FragiCyl_245870protein MTNM---TMMTSVLLVLSAG---TMIMSSCSAFVI 29 Thaps_6605protein MN---TKGILSILLVSANSSISVSAFSVSSISARGH 33 FragilariopsisCylindrus_269073 MN---FSLFL-LLLGPAT---AF---TGT 19 gi|108707737|gb|ABF95532.1| RNVVSFAGRRQGRALGRLAVVVAAGSPTPPELAQK---VSESIKQAEE 75 gi|224285472|gb|ACN40458.1| RSVV---GRRNG---IQVVAMSSPTP---AKGNISEVVEESIKKAIE 85 gi|1617206|emb|CAA96570.1| RQT-LTGSGRMKI---VQPVRAAPEQ---ISKKVEESIKSAQE 71 gi|355500984|gb|AES82187.1| RWG-VAGSGRMTV---VRPVRAAPEQ---ISKKVEESIKSAQE 72 gi|325512541|gb|ADZ23481.1| RWPALTSSNRVSS---VRLVRAAPDR---ISERVEKSISDAQE 76 gi|355480449|gb|AES61652.1| RWTGAVSSGRMVS---IRPVQASPD---ITGKVEESIKSAEE 75 gi|218156192|dbj|BAH03328.1| RPTLFGYAGRSMQ---VRPVSAAPEK---ISDKVAESIKDAEE 74 gi|151564658|gb|ABS17660.1| ---GPGRMC---VRPVAAVQEG---LSEKVAESIKSAEE 30 gi|332646828|gb|AEE80349.1| WNLGSRTTNRMMK---LQPIKAAPEGG---ISDVVEKSIKEAQE 73 gi|312282565|dbj|BAJ34148.1| WNLGSRTTNRMVK---IRPVKATPEGG---ITDKVEKSIKEAEE 73 gi|119720838|gb|ABL97989.1| WKLGSR-TNRLVS---FRPVKSTPEGV---ISDKVEKSIKDAKE 72 gi|330255742|gb|AEC10836.1| WKFGSM--KRMV---VVKATSEGE---ISEKVEKSIQEAKE 66 gi|297824833|ref|XP_002880299. WKFGSM--KRMV---VVKATSEGG---ISDKVEKSIQEAKE 66 gi|1617197|emb|CAA96569.1| NSSLVQFGHGKLY---LKAISATPDNK---LSDLVAESVKEAEE 75 gi|1617213|emb|CAA96568.1| VTYKLHGGRRSHG---LVAVRAAPDNR---ISENVEKSIKEAQE 68 Thaps_270287protein RNLYAS---VEEAIAEAQR 57 Thaps_20804protein R---VDTSAA---IEAALD 42 Phaeo_56708protein AP---VDTSSA---VQAALQ 45 FragiCyl_287476protein IPSSPRTIS---SSSSTSSSSLYMA---NSKNIRIAMD 62 FragiCyl_245870protein VQSSPRTISNPSSSSSSSSSSSSSSSLYMA---NSKNIRIAMD 69 Thaps_6605protein VGVVHRQPAQ-VHHALE-RPDASDA---ITDAFQ 62 FragilariopsisCylindrus_269073 KKVILRQRAS-SSFLLKSRPDSSAA---VADALR 49 : * gi|108707737|gb|ABF95532.1| TCAGDP--EGGECAAAWDEVEELSAA-ASHARD--- 105
gi|224285472|gb|ACN40458.1| TCAEDS--TSGECAASWDEVEELSAA-RSHMRE--- 115
gi|1617206|emb|CAA96570.1| TCADDP--VSGECVAAWDEVEELSAA-ASHARD--- 101
gi|355500984|gb|AES82187.1| TCADDP--VSGECVAAWDEVEELSAA-ASHARD--- 102
gi|325512541|gb|ADZ23481.1| ACN-DNPAGS-ECAAAWDEVEELSAA-ASHARD--- 106
gi|355480449|gb|AES61652.1| ACAGD--ATSGECVAAWDEVEELSAA-ASHARD--- 105
gi|218156192|dbj|BAH03328.1| ACAGDP--ASGECVAAWDEVEELSAA-ASHARD--- 104
gi|151564658|gb|ABS17660.1| TCSDDP--VSGECAAAWDEVEELSAA-ASHAGD--- 60
gi|332646828|gb|AEE80349.1| TCAGDP--VSGECVAAWDEVEELSAA-ASHARD--- 103
gi|312282565|dbj|BAJ34148.1| TCAGDP--VSGECVAAWDEVEELSAA-ASHARD--- 103
gi|119720838|gb|ABL97989.1| SCADDP--VSGECVAAWDEVEELSAA-ASHARD--- 102
gi|330255742|gb|AEC10836.1| TCADDP--VSGECVAAWDEVEELSAA-ASHARD--- 96
gi|297824833|ref|XP_002880299. TCADDP--VSGECVAAWDEVEELSAA-ASHARD--- 96
gi|1617197|emb|CAA96569.1| ACAENP--VSGECAAAWDVVEEASAA-ASHARD--- 105
gi|1617213|emb|CAA96568.1| TCSDDP--VSGECVAAWDVVEELSAA-ASHARD--- 98
Thaps_270287protein ICAEDP--NSESCKVAWDIVEELEAA-DSHK-DNVE--- 89 Thaps_20804protein ASKKFG-STSSEARVLWDIVEEMDASDNSVA---SK-APIVDSEYEAKV- 86 Phaeo_56708protein ASKQYG-ATSPEARVAWDAVEEMRANDDSPATQGSL-ADECDID-EDKVS 92 FragiCyl_287476protein LTEQYG-IQSQEAKLAWETVEEFDARSNDNA---AYTQDEIGMSKLT 105 FragiCyl_245870protein LTEQYG-IQSQEAKLAWETVEEFDARSNDNA---AYTQDSEGMSKLT 112 Thaps_6605protein MSEKYG-TTSKEARIAWDVVEEIYET--SP--PLSS---FDEQTST 100 FragilariopsisCylindrus_269073 ISKEFG-GTSSEARIAWETVEEMDAADMSPAITLSSTIKSVEKLHEMEYT 98 . .. *: *** .
gi|108707737|gb|ABF95532.1| ---RKKD---SDP 112 gi|218156192|dbj|BAH03328.1| ---KKKG---SDP 111 gi|151564658|gb|ABS17660.1| ---KMKE---SDP 67 gi|332646828|gb|AEE80349.1| ---KKKADG---SDP 112 gi|312282565|dbj|BAJ34148.1| ---KKKAGG---SDP 112 gi|119720838|gb|ABL97989.1| ---KKKAGG---SDP 111 gi|330255742|gb|AEC10836.1| ---KKKAGG---SDP 105 gi|297824833|ref|XP_002880299. ---KKKAGG---SDP 105 gi|1617197|emb|CAA96569.1| ---KKKES----SDP 113 gi|1617213|emb|CAA96568.1| ---KAKD---VEP 105 Thaps_270287protein ---QRQLPTDINYYPLIQSLDILSQKVERKMDELNKLSLQ 126 Thaps_20804protein ---KSLSQM---LTKTKA----ELDQVKALADDLKGV 113 Phaeo_56708protein AACL---EYGSKIEE---LNKLLADQVPHLNNVKTLADDLQKI 129 FragiCyl_287476protein EEEWNKAYYELQQSMELMERNEYSGLNILQNNQ----QLMKDVAAELSAI 151 FragiCyl_245870protein EEEWNQAYYELQQSMELMERNDYSGLNILQNNQ----QLMKDVAAELSAI 158 Thaps_6605protein AVDAPQDYY---DRIHFLNHLLMESQSNLGQVKELVAQIKEL 139 FragilariopsisCylindrus_269073 AK---MRS--LSRLLTETHENLSQIKILAANLKNL 128
gi|108707737|gb|ABF95532.1| LEEYCKDNPETDECRTYED--- 131
gi|224285472|gb|ACN40458.1| LEEFCKDNPETDECRTYED--- 141
gi|1617206|emb|CAA96570.1| LEDYCKDNPETDECKTYDN--- 127
gi|355500984|gb|AES82187.1| LEDYCKDNPETDECKTFDT--- 128
gi|325512541|gb|ADZ23481.1| LENYCKDNPETDECRAYDN--- 132
gi|355480449|gb|AES61652.1| LEEYCKDNPETDECRTYDN--- 131
gi|218156192|dbj|BAH03328.1| LEEYCKDNPETEECRTYED--- 130
gi|151564658|gb|ABS17660.1| LETLCKDNMDTEECRTYDD--- 86
gi|332646828|gb|AEE80349.1| LEEYCKDNPETNECRTYDN--- 131
gi|312282565|dbj|BAJ34148.1| LEEYCKDNPETNECRTYDN--- 131
gi|119720838|gb|ABL97989.1| LEEYCKDNPETDECRTYDN--- 130
gi|330255742|gb|AEC10836.1| LEEYCNDNPETDECRTYDN--- 124
gi|297824833|ref|XP_002880299. LEEYCSDNPETDECRTYDN--- 124
gi|1617197|emb|CAA96569.1| LENYCKDNPETDECRTYDN--- 132
gi|1617213|emb|CAA96568.1| LEEYCKDNPETDECRTYDN--- 124
Thaps_270287protein LAEAGAG--EEIERLVYASE-EMKGVLRDARARLEQL--- 160 Thaps_20804protein KLAS----PSVGSSAPDDS--VMKEALAAARAATEEFGQSSPQARLAWET 157 Phaeo_56708protein KLIVTKNQP---APD-SPQVR-AALEHAKAMSAEHGTTSPEAKVAWDS 172 FragiCyl_287476protein KLSPPERKP---APKIPG--LWDAKLKARAFSDNYGYDSVEAKLAWED 194 FragiCyl_245870protein KLSPPERKP---APKIPG--LWDAKLKARAFSDNYGFDSVEAKLAWED 201 Thaps_6605protein ELED----PSLARLADDGMGTALKAALAEAKAASEVHGPSSIQAIEAWDK 185 FragilariopsisCylindrus_269073 DIED----PHLSKLPDT--ATGLKKVLQEAKAASEVHGPESAESVAAWNE 172 gi|297824833|ref|XP_002880299. --- gi|1617197|emb|CAA96569.1| --- gi|1617213|emb|CAA96568.1| --- Thaps_270287protein --- Thaps_20804protein V---EEIAAS--PVDIRAP---LDEECLIE-LIEG 183 Phaeo_56708protein L---EEIASSGLSNAMGAG---LDQECLVESAMEA 201 FragiCyl_287476protein V---EELASSGLSNSWLGN-NLDVYDDESCDLVQAAEA 228 FragiCyl_245870protein V---EELASSGLSNS-LGTSNLDVYEDESCDLIQAAEA 235 Thaps_6605protein LDSCVGDENGVLKVSEECDIVST-YRYSAAALKAHHNYDATIDTTLLQES 234 FragilariopsisCylindrus_269073 VDFCTDVMGGVG---CN-VDTMYHYSAAALKAHHVYDAVVDATFFQEA 216
gi|108707737|gb|ABF95532.1| --- gi|297824833|ref|XP_002880299. --- gi|1617197|emb|CAA96569.1| --- gi|1617213|emb|CAA96568.1| --- Thaps_270287protein --- Thaps_20804protein CEA---LEKFQAAL---G--SR--- 197 Phaeo_56708protein CLA---LEELNRVLNLEKSKNEGANS---- 224 FragiCyl_287476protein CMA---LEELDRFFTNYYANNNNIGSDDGL 255 FragiCyl_245870protein CMA---LEELDRFFTNYYANNNNIGSDDGL 262 Thaps_6605protein LDAVGVLEALGRFVSLEKRRLDARDSAAGP 264 FragilariopsisCylindrus_269073 EDAIEMLENLRRFVRIENNRLNA--- 239
Supplemental figure 9-SI Protein alignment of plant/green algae CP12 proteins (gene bank IDs) and diatom CP12-like proteins (JGI protein numbers). To characterise the CP12 like proteins found in diatoms their sequences were aligned by ClustalW protein alignment with sequences of 15 plant and green algae CP12 proteins. The highly conserved domains found in the CP12 proteins are marked with yellow letters. Homologous regions in the diatom CP12-like proteins are marked similarly in yellow. Diatom specific homologies are marked with bright blue letters. Cysteines are marked by black letters on turquoise background. The cysteines are important for redox regulation via thioredoxins. Plant CP12 proteins feature a highly conserved domain with two redox sensitive subdomains able to form intramolecular disulphide bonds. The C-terminal double cysteine domain is important for the formation of dimeric complex of CP12 and plastidic GAP-DH and the N-terminal double cysteine domain was shown to be responsible for the formation of the ternary complex with PRK [243].
In diatoms homologies to the N-terminal part of the highly conserved sequence can be found, but they are with the exception of Thaps_270287protein missing the distinct cysteines of the N-terminal subdomain. The Thaps_270287protein is missing a C-terminal double cysteine domain, which can be found for all other diatom protein sequences. The diatom C-terminal cysteine domain features no homology to the plant sequences. In summary there are CP12-like proteins in diatoms but their domain structure is strongly deviant from plant CP12, rendering it unlikely that they act as CP12 proteins.
A.7.2 Supplemental Figure 9-SII
A
>Cr_EDP05117
MLVVNKVVITSVLPAPTLVLTVQLLVAVVVVLSGDALGWIKVDKLEWDKVQKFALVVGGFLGTLFANIKVLQYANVETFITFRSSTPLLLSVCD YIWLGRALPNARSWGCLLLLLAGSVGYVLVDADFRLSAYTWLALWYAFFTFDTVYAKHVVDTVQMTNWGRVYYGNFLALIPLAVMVPVMAEHNI LAAVVWTAPKAFILALSCLMGVGMSHASYLLREAVSATFFTIIGILCKVLTVIINVFIWDKHASPEGIACLMVCVVAGTFYQQAPRRQPQAPVV SNADSGAAGQAQKQSGAEVPKQASGSRNSSSGGPPADATGGQLLQERQSLLPKTTSSQLASRPSVSNNSHR
>Pt_45630
MNLKSEVFLSVVAYSLCSGTLVLLNKLTLHHLPYPSLVVSFQLLAALIFIYGAKHTGRLQVDALEWQYVLPYLFYILLFSVGVFCNMKSLSMSN VETVIVFRALSPCIVAFLDVLFLGREYPSLQSWTGLSLIALGAYGYASFDAQFQTQGLAAYAWPGLYLFIISLEMAYGKRIIQSVNLKTLSGPV LYTNLLGLPPMLMFAAMGHEYRSFVYDHMVEQKPVGGVAVSLLLLGCAAGTGIGYAGWWCRGNVSATSFTLIGVINKCLTILLNVMIWDQHAPP KGILSLALCLVGGSIYRQSPLRNNTLKTSSSVAVDDGDDKNGRDDSDSVATDAEDINEQVELLEAEKGMKRRS
CLUSTAL 2.1 multiple sequence alignment
Cr_EDP05117 ---MLVVNKVVITSVLPAPTLVLTVQLLVAVVVVLSGDALGWIK Pt_45630 MNLKSEVFLSVVAYSLCSGTLVLLNKLTLHHLPYPSLVVSFQLLAALIFIYGAKHTGRLQ **: : : ** *:**::.***.*::.: ... * ::
Cr_EDP05117 VDKLEWDKVQKFALVVGGFLGTLFANIKVLQYANVETFITFRSSTPLLLSVCDYIWLGRA Pt_45630 VDALEWQYVLPYLFYILLFSVGVFCNMKSLSMSNVETVIVFRALSPCIVAFLDVLFLGRE ** ***: * : : : * :*.*:* *. :****.*.**: :* :::. * ::***
Cr_EDP05117 LPNARSWGCLLLLLAGSVGYVLVDADFR---LSAYTWLALWYAFFTFDTVYAKHVVDTVQ Pt_45630 YPSLQSWTGLSLIALGAYGYASFDAQFQTQGLAAYAWPGLYLFIISLEMAYGKRIIQSVN *. :** * *: *: **. .**:*: *:**:* .*: ::::: .*.*:::::*:
Cr_EDP05117 MTNWG-RVYYGNFLALIPLAVMVPVMAEHN---ILAAVVWTAPKAFILALSCLMGVG Pt_45630 LKTLSGPVLYTNLLGLPPMLMFAAMGHEYRSFVYDHMVEQKPVGGVAVSLLLLGCAAGTG :.. . * * *:*.* *: ::..: *:. :: . . :* *.* *.*
Cr_EDP05117 MSHASYLLREAVSATFFTIIGILCKVLTVIINVFIWDKHASPEGIACLMVCVVAGTFYQQ Pt_45630 IGYAGWWCRGNVSATSFTLIGVINKCLTILLNVMIWDQHAPPKGILSLALCLVGGSIYRQ :.:*.: * **** **:**:: * **:::**:***:**.*:** .* :*:*.*::*:*
Cr_EDP05117 APRRQPQAPVVSNADSGAAGQAQKQSGAEVPKQASGSRNSSSGGPPADATGGQLLQERQS Pt_45630 SPLRNNTLKTSS---SVAVDDGDDKNGRDDSDSVATDAEDINEQVE :* *: . * .*. :.. ... : **..: ::*: . Cr_EDP05117 LLPKTTSSQLASRPSVSNNSHR
Pt_45630 LLEAEKGMKRRS---
B
At2g13650_GDP-Man-transporter MKLYEHDGVDLEDGKTVKSGGDKPIPRKIHN--- Pt_protein_9609+ MPPSKKEMMDVELAQPLVHHKHGDYSDSTHNPPSHPPANHNAVVKHGGGL * ::: :*:* .:.: . . . **
At2g13650_GDP-Man-transporter ---RALLSGLAYCISSCSMILVNKFVLSSYNFN----AGIFLMLYQN Pt_protein_9609+ MSPSNVKAVTACTLYSFCSVSMILVNKSLASRYVPNDGCDLNVLLVVFQA :*: : *.:.* ******* : * * * .::*:::*
At2g13650_GDP-Man-transporter FVSVIIVVGLSLMGLITTEPLTLRLMKVWFPVNVIFVGMLITSMFSLKYI Pt_protein_9609+ VTAVVCVEICRKAGWVEYPPLTWAVAKSWAPVNIFFCLMLFTGMASLQFN ..:*: * * : *** : * * ***::* **:*.* **::
At2g13650_GDP-Man-transporter NVAMVTVLKNVTNVITAVGEMYLFNKQHDNRVWAALFLMIISAVSGGITD Pt_protein_9609+ SVPMVTVFKNVTNILTTAGDYVCFGARPEGLVYVAFGVMLSGAVAAAWND .*.****:*****::*:.*: *. : :. *:.*: :*: .**:.. .*
At2g13650_GDP-Man-transporter LSFNAVGYAWQIANCFLTASYSLTLRKTMDTAKQVTQSGNLNEFSMVLLN Pt_protein_9609+ VEITLVGLFWMAMNCVATCGYVLYM---KFATQSVKMSKFGMVYVN :.:. ** * **. *..* * : * .*** ::.:*.** :*
At2g13650_GDP-Man-transporter NTLSLPLGLLLSYFFNEMDYLYQTPLLRLPSFWMVMTLSGLLGLAISFTS Pt_protein_9609+ NVLCIVFLLPAAYALGQVDMFWNTPDLHTIDYGIKNFWAGFVGFFLNFAS *.*.: : * :* :.::* :::** *: .: : :*::*: :.*:*
At2g13650_GDP-Man-transporter MWFLHQTGATTYR--- Pt_protein_9609+ LNCVQTTGPTTYAIVGSLNKVPVAMLGFFLFDNVITPQTWFFIGVSMCGG : :: **.***
At2g13650_GDP-Man-transporter --- Pt_protein_9609+ FLYSFAKIFGGRPKVTARQDSE
Supplemental Figure 9-SII Putative plastid targeted sugar nucleotide transporters in P. tricornutum. Protein BLAST at the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with sugar nucleotide transporters of Arabidopsis thaliana (At) and Chlamydomonas reinhardtii (Cr) identified two putative sugar nucleotide transporters (JGI protein Ids: 45630 and 9609) in P. tricornutum (Pt). A The protein sequence of a putative Cr sugar nucleotide transporter (NCBI Accession number EDP05117) and its Pt homologue (45630) are given, as well as a ClustalW protein alignment (http://www.genome.jp/tools/clustalw/). The Pt_45630 protein sequence was tested with SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP-3.0/) for plastid targeting (standard settings for eukaryotes).
The results of the neural networks and Hidden Markow model predictions are shown, predicting a high probability for plastid targeting and signal anchor, which might indicate, that this transporter is integrated in the outermost plastid membrane. B The protein sequence of the At UDP-mannose transporter (NCBI Accession number At2g13650) is given together with an extended (indicated by +) nucleotide and corresponding protein sequence of the nearest matching Pt gene model (JGI protein ID 9609) and a ClustelW alignment between both protein sequences. The Pt gene features EST backed (the EST supported parts are marked in yellow) potential extensions of the 5’-terminal sequence. The original gene model 9609 is marked in red (translated) and blue (untranslated) letters. There are multiple potential START codons, one (marked by red background) is not coding for a sugar nucleotide transporter due to a frameshift, three more (marked by blue background) in the possible gene extension are coding for such transporters, but SignalP 3.0 analyses predicts no plastid targeting for these (data not shown), and two more (marked by green background) are found in the original sequence of 9609, for which SignalP 3.0 is predicting a potential plastid targeting sequence with a low signal anchor probability, the result of the better prediction corresponding to the first green START codon is shown. The corresponding methionins are marked in the same colours in the protein sequence of 9609+. An amino acid motif similar to the ASAFAP cleavage motif of bipartite signal peptides [21] is marked in dark green. The situation which gene model(s) are expressed is unclear as well as the exact targeting, but a plastid targeting to an inner membrane of the plastid is conceivable.
A.7.3 Supplemental Figure 9-SIII
(TC)TGACG(TGA) consensus sequence of Vfaurechrome1
(TC)TGACG(TGA) homology of the highly conserved 6 inner positions (TC)TGACG(TGA) homology of the absolutely conserved 5 inner positions
green GAP C1/PRK gene
grey adjacent genes
darkGrey START codon
blue letters untranslated regions
red letters nucleotide corresponds to likeliest nucleotide at that position of the Vfaurechrome1 recognition site [51]
GAP C1 (22122) “sense orientation“ (chr_3|747953|751058 (3105bp))
ATTGTGTTTCATACCAATACTGTTGGTTGCTTGATATGTTTCCTTCACTTTAGAGTAGGGAACGGAAATGGTTACTGTAATGTTATTGCGTTCG
GAP C1 (22122) “antisense orientation“ (chr_3|751058|747953 (3105bp))
GGTCGTTCAAGGTTATTTGCAAAAAGCTCGGAAGCGACTCTTATCTCTGCAGACGCGCTACAGGGAACTTATTGTGGGCGGTGATAATGATCGA CGTGCCGGTGGACGGAGTGCGGCCAGTTCGGGTCCGGGAGATTATAATTCCTACGCGAAAACAAGTTCTCCGAATGCTGGCGAAGCGAATGACT CGAAGGGTTCTACGGCCTACGGTACCGGTTCGACGTCTTCGTCATCTCGTTCGCGGACATCTTCCAACAGCAATTCTGAGGACGCCTGGAAAGA CGAAGGTTTTGGTTCGTTTGGACGTGGCCGAGGTAGCACTCGACGCGGTCGTACCGGAACCCATCGACGAAGCACATCTGCTTCCGACCAGGCG TCGGCGACGTCGCCCGGTAAGGAAACGTCCTACGGTGGGTCGTCATCCTCATCTTCCTCGCCATATTCCACGCGATCCAGTACACCACCGCCGC CGCCGCCTCGTTCGTCCCCGGAGCGACCGCAGCCCACCAGTTACACGCAAGTACCGCCACACCGACGAACGAGCGGTTCAACGTGGTCCATCGA AGACGATAAACGACGACTACGGGAGATGCAAGTAGATGACGACTTTGAAAAACTCAAAAAAGACTTGGGTCTATAATTGTCTGATATAGAAACC TTAGTGTCCTTTTTTCCACAGTCCTGATTTACTTTCCTATTTGCATCCGTCAAATAGGTTGCTGCTGGAAGTACAAACACGATTGCATACATAC ATGAGTACATGTATACCTAGGCGTACCGTATGGAGGTGCTCAACTCCATGTTTCGCAACAATTTGTTCGCTAGTAAGTGTTTATTCCCAAGTCG GCTGTGCGTTTAGGCCTTGATCTTGGCGTCGACGGCCGCGACGTGCTTCATGAGGTCGACGACACGGCCGGAGTAACCGTATTCGTTGTCGTAC CAGGCGATGAGCTTGACAAAGTTGGGGTTGAGCATGATACCGGCATCGGCATCAAAGATGGAGGAGCGCAAGTCACCTTCAAAGTCGGTGGAGA CCAACGGTTCGTCGGAGTATCCGAGGAATCCCTTCATTTCACCCTCGGACTTGGCCTTGATGACGGCACAGATTTCTTCGTAAGTGGTGGACTT TTCGAGTTTTGCGGTCAAGTCGACGACGGAGACGTCAATGGTGGGGACACGGAAGGCCATGCCGGTGATCTTTCCAACGAGGGAAGGAATGACC TTGGTGACGGCTTTGGCGGCTCCGGTGGAGGATGGGATGATATTTCCCGAGGCCGCACGTCCTCCGCGCCAGTCCTTGCGGGATGAGGAGTCGA CAACGGCCTGGGTGGCGGTCATGGCGTGGACGGTGGTCATGAGGGCTTCCTCAATGACGAATTCGTCGTGAATGGCCTTAACCATAGGGGCAAG TCCGTTGGTGGTGCAAGAAGCGCAGGAGATGAAATCTTCCGAACCATCGTAGGCTTCCTGGTTGACTCCCATGACAATGGTGAGTGAGTCGTCT TTGGCGGGTGCCGAGTAGATGACCTTCTTGGCGCCTCCGTCAATGATGGACTGTGCGGATTCCTTGGTGAGGAAGACTCCGGTGGATTCGCAGA CGTAGTCGGCTCCGAGTGCGCCCCAGCCCACTTCCTTGGGGTCACGGCAGCGCGAAGTGATGATCTTCTTGCCGTCCAAGACGAGGAAGTCGCC ATCGATTTCGACCGTCTGCTTGGCCTTGCCGTGGATGGTATCGTACTTGTACTGGTAGGCCATGTAGTCCGGAGTGGCGGAACCGGCGTTGATT CCGACCAAATCGCATTCGTCGTCTTCCATCATGATGCGGGTGACGAGACGTCCGATACGTCCGAATCCGTTGACTCCCATACCGGTAGCCATCG ACATGGAGGCATCGTTGGACGCCGAGCTCTTGAGGGCCGATCCGGTAAAGCTGTCAAAGCAACGAGAGTGAGTGGATACGTGTGAGTACTTGAA TGAGCAAATGAATAAGCGCCTGCGTTTGCGTAATACAGGCTAGAGACCAAAGGGAACTACGGTGCGGAGATGCTTTTACAGTTGACGCGTATTC CTTCCAACCAACCGCGGTAGGCTTGGATCCGAACCCTTCCTTCGGCTCTTCGTCGTCGCGACGTGTCTCGATACGTGCCACAACAGCAGCAGCA GGCAAGATATCCACTTACGAAGAACTGGAGTAGGCAGCGGCAGATCCTACAAGGGCAGCAAAAGTGGCGGCAGAGAACTTCATGATGGAGTCAA AAAAGAAAGTAGAGTGAACTAACTGTCAGGTACAAAGTTATCGACGAACTCGGAGGATTTGTGAGACTCGTGTGTGTGGAGCGACGTGAGACCG GGAAGATTTGCATTTTGTTAGAAGCCTCTTTTCTCGCCCCATGCTCTTCCACCGGCTTTCGTACGTCAGTGGTCTCGTCCCCCGAAAAACCAAC CAAGTCAAAACGACTCCGGATGGCTCAGGCCCAATCGATGAGATACATTGCAGTCCCGTACCGACGAGACCACCCTCACAGGTCACCAAATCTC ATACACAGACACACGTTTTCGCGGAAACCAGTACGTATCACGCGGAACACGTATGTGGATGGGGAGCAGCTGCCAGACCGACACACGAGTGGAA TGACAGACGATATCTTTTCATTCTTATTTCTATAATTCTTCAAATATCATCCTAGGGAGTAAACCGGATGTGACAGAACATGTGACGTGTCCAA AATGGATTGCTTCAATTCCAACCAGCTCAGGAGTGGGTCGGGTCGGACCCAACACGCGTGTGCGTTTCCCAGTCGTGCTGTAGAACGGCAGACG GACGATAGATACGTCCAACGCAGTCCGAATCGGTTTGGAAAGGCGAAATTTACGGTACGCAAGGAGACAGGCAGTGAACTATTGCGCAATCCCG CAAACAATACATACAATCGAGAAGCACCGGCTTCGGTTCGAGAGGGTAGGAAACGCTCTACAGAGCATGACGAAAATTGACGGAACGGACTGTG AATACGAACGCAATAACATTACAGTAACCATTTCCGTTCCCTACTCTAAAGTGAAGGAAACATATCAAGCAACCAACAGTATTGGTATGAAACA CAAT
PRK (50773) “sense orientation“ (chr_3|1051600|1047200 (4401bp))
PRK (50773) “antisense orientation“ (chr_3|1047200|1051600(4401bp))
Supplemental Figure 9-SIII Analysis of the GAP C1 (22122) and PRK (50773) genes for potential AUREO1a (49116) recognition sites (cis elements). The chromosomal sequence is investigated in sense and antisense orientation. The gene sequence of PRK and GAP C1 exons are dyed green. Neighbouring genes are marked in grey. Untranslated regions are marked in blue letters, START codons in dark grey. The sequences were screened for the V. frigida aureochrome1 recognition site consensus sequence (TC)TGACG(TGA) [51]. The nucleotides in brackets are not totally conserved, but the T at position 8 is very highly conserved and is putatively very necessary for aureochrome recognition as well. Recognition sites which are featuring identity in these six highly conserved positions are marked in yellow, those which are missing identity at position 8 are marked in turquoise.
Each nucleotide corresponding to the likeliest nucleotide of the predicted recognition sequence is marked in red letters. PRK and GAP C1 all feature many possible aureochrome binding sites and each gene is featuring at least one closely associated highly homologous (yellow) recognition site, making them likely target for AUREO1a blue light sensitive transcription factor.
B. Author contributions
Chapter 2: Identification and evaluation of endogenous reference genes for steady state transcript quantification by qPCR in the diatom Phaeodactylum tricornutum with constitutive expression independent from time and light
Conception of the experiments was done by MS and PGK. Sample design for RNA and preparation of RNA was done by MS, SS and AG. qPCR experiments for determination of endogenous reference genes were conceived, designed and performed by MS. The data was analysed by MS. The Manuscript was written by: MS, PGK and AG.
Chapter 3: Transcriptional regulation of Calvin cycle enzymes in the diatom Phaeodactylum tricornutum
Conception of the experiments was done by MS and PGK. Sample design for RNA was done by SS and AG. RNA preparation was done by SS, AG and MS. Protein sample design was done by MS. Protein sample preparation was done by MS, JR and BSC. qPCR experiments were conceived and performed by MS. PAR-qPCR was developed by MS. Immunoblot quantification experiments were conceived and performed by MS. All data was analysed by MS. The Manuscript was written by MS, PGK, AG, JR and BSC.
Chapter 4: Overexpression, fractioning and adaption of an enzymatic assay for fructose-1,6-bisphosphatases
Conception of the experiments was done by MS and PGK. The experiments for the FBP activity assay, the gel filtration fractioning and intein tag overexpression constructs were designed and performed by MS. Data analysis was performed by MS. The chapter was written by MS.
Chapter 5: Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum
Conceived and designed the experiments: MS BSC PGK CW AJ TJ AG. Performed the experiments: MS BSC AJ CRB AG. Analysed the data: MS BSC AJ AG TJ PGK CW. Wrote the paper: MS BSC PGK CW TJ CRB.
Chapter 6: Conception and generation of aureochrome silencing and overexpression constructs for Phaeodactylum tricornutum
Conception of the experiments was done by MS and PGK. The design, generation and verification of the constructs were performed by MS. The data was analysed by MS. The chapter was written by MS.
Chapter 7: Generation of AUREO1a/LOV/LOV-JĮ overexpression constructs optimised for expression in Escherischia coli
Conception of the experiments was done by MS, TK and PGK. The design, generation and verification of the constructs were performed by MS. The chapter was written by MS.
Chapter 8: Design and generation of a PtTRX F (CS) overexpression construct optimised for expression in Escherischia coli
Conception of the experiments was done by MS and PGK. The design, generation and verification of the constructs were performed by MS. The chapter was written by MS.