A Joint Meeting

2nd International Conference on Plant Biology

21st Symposium of the Serbian Plant Physiology Society
COST ACTION FA1106 QUALITYFRUIT Workshop

Dragana Božić, Antigoni Papanikolaou, Dimitra Katsarou,

David Manzano, Ulschan Scheler, Kathleen Brückner, Albert Ferrer,
Alain Tissier, Angelos K. Kanellis
Carnosic acid

Ortho-diphenolic group
-Phenolic diterpene

-In planta role: protects

photosynthetic membranes
against oxidative damage
cascade oxidation:
carnosic acid converts to
carnosol, rosmanol,
rosmariquinone, etc.
Carnosic acid

Rosmarinus officinalis Salvia fruticosa

http://commons.wikimedia.org http://www.botanik-jagel.de
Carnosic acid

Use in food industry:

- Carnosic acid (CA) is the main antioxidant in Rosemary extract

- extends the shelf life of food (oils, meat products) by slowing the development of oxidative
rancidity

http://luirig.altervista.org

Rosmarinus officinalis http://kingherbs.en.made-in-china.com

Carnosic acid

Pharmaceutical use and potential:

- Sage and Rosemary have been used since ancient times in folk medicine, food preparation.
- Recent investigations have determined many biological effects of these plants, their essential oils,
extracts and individual compounds.
- Carnosic acid posseses antioxidative, antiinflamatory, anticarcinogenic, neuroprotective,
antimicrobial and other activities.

http://writingwithlight-bonnard3.blogspot.com
Carnosic acid

Pharmaceutical use and potential:

- Sage and Rosemary have been used since ancient times in folk medicine, food preparation.
- Recent investigations have determined many biological effects of these plants, their essential oils,
extracts and individual compounds.
- Carnosic acid posseses antioxidative, antiinflamatory, anticarcinogenic, neuroprotective,
antimicrobial and other activities.
Trichomes

Secretory trichomes – a place of synthesis and

accumulation of plant natural products.

Molecular aspects of trichome metabolism and

secretion?
Carnosic acid biosynthesis

A more economical alternative to extraction from plants - using genetic engineering (original or
novel hosts).
Knowledge of the biosynthetic pathway of the CA is useful for strategies to enhance it’s
production.

(class II diTPS) (class I diTPS)

?

http://my.import.or.kr
Cytochrome P450s
Gene isolation approach

cDNA sequencing + metabolics

Novel candidate genes can be selected and
identified based on sequence similarity with
homologous genes from other species.

It is possible to profile the transcriptome

from a specific tissue, or from a tissue that
has been subjected to a special treatment.

Our transcriptome analysis approach:

-EST analysis (S. fruticosa cDNA library)

http://www.genengnews.com
-RNA seq (S. fruticosa and R.officinalis 454
sequencing) The workflow of 454 Life Sciences’ Genome
Sequencer FLX System
diTPS candidates identification

GGDP CDP miltiradiene?

Contig 195 Contig 66

(homologous to copalyl (homologous to kaurene
5’ synthases)
diphosphate RACE 5’ synthases)
and 3’ RACE
Glandular trichomes on the
SfCPS full lenth SfKSL surface of Salvia fruticosa leaves.
Sheef et al., Arch. Biol. Sci., 2013
 Alignment of SfCPS and SfKSL
SfCPS 1 ........MTSMSSLNLTPAIIRRLQPAAKVHLPEFYAACAWLNSSSKHAPLT..CQTRRKQLSKVTECRVANLDTSQL.SEKHTSPIQT SfKSL 1 ..........................................................................................
SsLPPS 1 ........MTSVNLSRAPAAIIRRRLQLQ....PEFHAECSWLKSSSKHAPFTLSCQIRPKQLSQIAELRVTSLDASQA.SEKDISLVQT SmKSL 1 ..........................................................................................
SmCPS 1 .....MASLSSTILSRSPAARRRITPASAKLHRPECFATSAWMGSSSKNLSLS..YQLNHKKIS......VATVDAPQV.HDHDGTTVHQ SsSS 1 ..........................................................................................
NtCPS2 1 MQVIITSSHRFFC....HHLHQLKSPTSLSAQKAEFKKHGPRNW.LFQTEGSLLYKPVRLNCATSDASYLGNVNEYLES..DHSKNSEEK ShSBS 1 MIVGYRSTIITLSHPKLGNGKTIS.SNAIFQRSCRVRCSHSTPSSMNGFEDARDRIRESFGKVELSPSSYDTAWVAMVPSKHSLNEPCFP
CcCLS 1 MAFTFTSAHLFLPVTENHSVHVNYSIPPGNWRLWSTAKGGSNKLDIRRLRCSARRTPEPLAQGSNGGRDGVEAIQRLQTIADDKIDGGAN SlPHS 1 MIVGYRSTIITLSHPKLGNGKTIS.SNAIFQRSCRVRCSHSTTSSMNGFEDARDRIRESFGKLELSPSSYDTAWVAMVPSRHSLNEPCFP
HvCPS 1 .......MLLLLPLACPPSGYGHGPRTRALAVKGPCRFRGKGDAALVETAGLRVALQITQASSVSSSNGLQTNLVRNDIRILEQTEEPCE NtABS 1 MVLGLRSKIIPLPDHKLGNIKLGSVTNAICHRPCRVRCSHSTASSM...EEAKERIRETFGKIELSPSSYDTAWVAMVPSRYSMNQPCFP

SfCPS
SsLPPS
SmCPS
80
78
77
PD..EVNEKMGDYIEYVKNLLMTSGDGRISVSPYDTSIIALIKDLKGRDA...PQFPSCLEWIARNQRADGSWGDQ.FFCIYDRILNTLA
PHKVEVNEKIEESIEYVQNLLMTSGDGRISVSPYDTAVIALIKDLKGRDA...PQFPSCLEWIAHHQLADGSWGDE.FFCIYDRILNTLA
GH..DAVKNIEDPIEYIRTLLRTTGDGRISVSPYDTAWVAMIKDVEGRDG...PQFPSSLEWIVQNQLEDGSWGDQKLFCVYDRLVNTIA
SfKSL
SmKSL
SsSS
1
1
1
γ-domain (aproximately 210 aa)
..........................................................................................
..........................................................................................
..........................................................................................
NtCPS2 84 DIQVSRTIQMKGLTEEIKHMLNSMEDGRLNVLAYDTAWVSFIPNTTNNGNDQRPMFPSCLQWIIDNQLSDGSWGEEIVFCIYDRLLNTLV ShSBS 90 QCLDWIIENQREDGSWGLNPSHPLLLKDSLSSTLACLLALTKWRVGDEQIKRGLGFIETQSWAIDNKDQISPLGFEIIFPSMIKSAEKLN
CcCLS 91 ELGIVVWDLIRDGVDAVKSMFDSMGDGDISISAYDTAWVALVKDVNGSGG...PQFPSSLQWIVDNQLPDGSWGDSEVFSAYDRLLKTLA SlPHS 90 QCLDWIIENQREDGSWGLNPTHPLLLKDSLSSTLACLLALTKWRVGDEQIKRGLGFIETYGWAVDNKDQISPLGFEVIFSSMIKSAEKLD
HvCPS 84 LDDYCVTEFEQPLVDKVRMMLGSMSDGEINVSAYDTAWVALVPSLDDGDS...PQFPTTLRWILDNQLPDGSWGDAALFSAFDRVINTLA NtABS 88 QCLDWILENQREDGSWGLNPSHPLLVKDSLSSTLASLLALRKWRIGDNQVQRGLGFIETHGWAVDNKDQISPLGFEIIFPCMINYAEKLN

SfCPS 164 CVVALQSWNLHAHMIQKGVTYVNENVHKLKDGNIE...HMTSGFEIVVPALVQRARXLGIQGLPYDHPLIKEIANTKQGRLKKIPKDMIY SfKSL 1 ..........................MLLTFNITDATLSHGVILRRRENFP.RHAFQIFPAIAATKRSFNAVCSLGTPPTDLIGKIKEKF

SsLPPS 164 CVVALKSWNLQSDIIEKGVTYIKENVHKLKGANVE...HRTAGFELVVPTFMQMATDLGIQGLPYDHPLIKEIADTKKQRLKEIPKDLVY SmKSL 1 ..........................MSLAFNPAATAFSGNGARSRRENFPVKHVTVRGFPMITNKSSFAVKCNLTT..TDLMGKIAEKF
SmCPS 162 CVVALRSWNVHAHKVKRGVTYIKENVDKLMEGNEE...HMTCGFEVVFPALLQKAKSLGIEDLPYDSPAVQEVYHVREQKLKRIPLEIMH SsSS 1 ..........................MSLAFNVGVTPFSGQRVGSRKENFPVQG....FPVTTPNRSRLIVNCSLTT..IDFMAKMKENF
NtCPS2 174 CVIALTLWNTCLHKRNKGVMFIKENLSKLETGEVE...NMTSGFELVFPTLLEKAQQLDI.DIPYDAPVLKDIYARREVKLTRIPKDVIH ShSBS 180 LNLAINKR.DSTIKRALQNEFTR...........NIEYMSEGVGELCDWKEIIKLHQRQNGSLFDSPATTAAALIYHQHDKKCYEYLNSI
CcCLS 178 CVVALKSWNIRPDKCQKGLKFFRDNISKLEKENVEASAQMLSGFEVVFLSLIEVARRLDI.QIPLHSPVFEDLIARRNLKFAKIPLDLMH SlPHS 180 LNLPLNLHLVNLVKCKRDSTIKR...........NVEYMGEGVGELCDWKEMIKLHQRQNGSLFDSPATTAAALIYHQHDQKCYQYLNSI
HvCPS 171 CVVALTKWSLGPDKCRRGLSFLEENMWRLAEEDLE...SMPIGFEIAFPSLLEVAKSLSI.GFPYDHHALQRIYANREVKLKRIPMEMMH NtABS 178 LDLPLDPNLVNMMLCERELTIERALKNEFEGNMANVEYFAEGLGELCHWKEMM.LRQRHNGSLFDSPATTAAALIYHQYDEKCFGYLNSI

SfCPS 251 QTPTTLLFSLEGLED..LDWEKILKLQSGDGSFLTSPSSTAQVFMKTKDEKCLKFIENAVRNCDGGAPHTYPVHVFPRLWAVDRLQRLGI SfKSL 64 NGKDDNPLAAAAAIQSAANIPSSLCIIDTLQRLGVDRYFQSEIDSILEETYRSWREKDSEIYSDVTTHAMAFRLLRVKGYEVSSEELAPY

SsLPPS 251 QMPTNLLYSLEGLGD..LEWERLLKLQSGNGSFLTSPSSTAAVLMHTKDEKCLKYIENALKNCDGGAPHTYPVDIFSRLWAIDRLQRLGI SmKSL 63 KGEDSN.FPAAAAVQPAADMPSNLCIIDTLQRLGVDRYFRSEIDTILEDTYRLWQRKERAIFSDTAIHAMAFRLLRVKGYEVSSEELAPY
SmCPS 249 KIPTSLLFSLEGLEN..LDWDKLLKLQSADGSFLTSPSSTAFAFMQTKDEKCYQFIKNTIDTFNGGAPHTYPVDVFGRLWAIDRLQRLGI SsSS 59 KREDDK.FPTTTTLRSE.DIPSNLCIIDTLQRLGVDQFFQYEINTILDNTFRLWQEKHKVIYGNVTTHAMAFRLLRVKGYEVSSEELAPY
NtCPS2 260 TIPTTVLFSLEGLRD.DLDWQRLLKLQMPDGSFLISPASTAFAFMETNDEKCLAYLQNVVEKSNGGAR.QYPFDLVTRLWAIDRLQRLGI ShSBS 258 LQQHKNWVPTMYPTK....IHSLLCLVDTLQNLGVHRHFKSEIKKALDEIYRLWQQKNEQIFSNVTHCAMAFRLLRMSYYDVSSDELAEF
CcCLS 267 NVPTSLLNSLEGMTGVELDWEKLLKLQSQDGSFITSPSSTAFALMQTNDTKCLGYLKFVVQKFNGGAPGQYPVEIFERIWVVDRLQRLGI SlPHS 259 FQQHKNWVPTMYPTK....VHSLLCLVDTLQNLGVHRHFKSEIKKALDEIYRLWQQKNEQIFSNVTHCAMAFRLLRMSYYDVSSDELAEF
HvCPS 257 KIPTSILHSLEGMPG..VDWQKILRLQSSDGSFLYSPSATACALMQTGDAKCFEYIDRIVKKFNGGVPNVYPVDLFERIWSVDRLERLGI NtABS 267 LKLHDNWVPTICPTK....IHSNLFLVDALQNLGVDRYFKTEVKRVLDEIYRLWLEKNEEIFSDVAHCAMAFRLLRMNNYEVSSEELEGF

SfCPS 339 SRFFQKEIKYF.LDHINSVWTENG.VFSGRDSEFCDIDDTSMGIRLLKMHGYNVDPNALNHFKQPDGKFSCYGGQMIESASPTYNLYRAA SfKSL 154 AEQERVDL....QTIDVATVIELYRAAQERISEEESS.LEKLLGWTTTFLKQQLLTNSIPDNKLHKQVEYYLKNYHGILDRMGVRRNLDL

SsLPPS 339 SRFFQHEIKYF.LDHIESVWEETG.VFSGRYTKFSDIDDTSMGVRLLKMHGYDVDPNVLKHFKQQDGKFSCYIGQSVESASPMYNLYRAA SmKSL 152 ADQEHVDL....QTIEVATVIELYRAAQERTGEDESS.LKKLHAWTTTFLKQKLLTNSIPDKKLHKLVEYYLKNXHGILDRMGVRQNLDL
SmCPS 337 SRFFEPEIADC.LSHIHKFWTDKG.VFSGRESEFCDIDDTSMGMRLMRMHGYDVDPNVLRNFKQKDGKFSCYGGQMIESPSPIYNLYRAS SsSS 147 GNQEAVSQ....QTNDLPMIIELYRAANERIYEEERS.LEKILAWTTIFLNKQVQDNSIPDKKLHKLVEFYLRNYKGITIRLGARRNLEL
NtCPS2 348 SYYFAEEFKEL.LNHVFRYWDEENGIFSGRNSNVSDVDDTCMAIRLLRLHGYDVSPDALNNFK.DGDQFVCFRGEVDGSPTHMFNLYRCS ShSBS 344 VDEEHFFAIS.GKYTSHVEILELHKASQLAIDHEKDDILDKINNWTRTFMEQKLLNNGFIDRMSKKEVELALRKFYTISDLAENRRCIKS

class II diTPS
CcCLS 357 SRYFQLEIKECCLDYAFKHWTQYGSSWA.RNTPVYDLDDTCMAFRILRLHGYDVSAEAFRHFE.KNGVFFCFGWETTQSVTVNFNLYRAT SlPHS 345 VDEEHFFATN.GKYKSHVEILELHKASQLAIDHEKDDILDKINNWTRAFMEQKLLNNGFIDRMSKKEVELALRKFYTTSHLAENRRYIKS
HvCPS 345 SRYFKQEIKQC.LDYVHRHWTDEGICWA.RNSTVIDVDDTSMAFRLLRLHGYDVSPSVFGKFE.KDGEFFCFVGQSTQAVTGMYNLNRAS NtABS 353 VDQEHFFTTSSGKLMNHVAILELHRASQVAIHERKDHILDKISTWTRNFMEQKLLDKHIPDR.SKKEMEFAMRKFYGTFDRVETRRYIES

SfCPS
SsLPPS
SmCPS
427
427
425
motif
QLRFPGEEILEEATKFAYNFLQEKIANNQFQEKWVISEHLIDEVKLGLKMPWYATLPRVEAAYYLQYYAASDNVWIGKVFYRMPEISNDT
QLRFPGEEVFEEATKFAFNFLQEMLVKDRLQERWVISDHLFDEIKLGLKMPWYATLPRVEAAYYLDHYAGSGDVWIGKSFYRMPEISNDT
QLRFPGEEILEDAKRFAYDFLKEKLANNQILDKWVISKHLPDEIKLGLEMPWLATLPRVEAKYYIQYYAGSGDVWIGKTLYRMPEISNDT
SfKSL
SmKSL
SsSS
239
237
232
YDISHYRSLRAW..FPNLCNEDFLSFARQDFNMCQAQHREELEQLQRWYSDCRLDTLKFGRNVLRVANFLTSAIIGDPELSEVRIVFAQH
YDISYYRTSKAANRFSNLCSEDFLAFARQDFNICQAQHQKELQQLQRWYADCKLDTLKYGRDVVRVANFLTSAIIGDPELSDVRIVFAQH
YDMTYYQALKSTNRFSNLCNEDFLVFAKQDFDIHEAQNQKGLQQLQRWYADCRLDTLNFGRDVVIVANYLASLIIGDHAFDYVRLAFAKT

class I diTPS
NtCPS2 436 QVLFPGEKILEEAKNFTYNFLQQCLANNRCLDKWVIAKDIPGEIWYALEFPWYASLPRVEARYYIEQYGGADDIWIGKTLYRMPDVNNNV ShSBS 433 YEENNFKILKAAYRSPNIYNKDLFIFSIRNFELCQAQHQEELQQFKRWFEDYRLDQLGIAERYIHDTYLCAVIVVPEPELSDARLLYAKY
CcCLS 445 QVAFPGENILKEAKQFSFNFLMKKQAAREFQDKWVILKDFPGELKYALEFPWYASLPRVETRFYVEQYGGDNDVWIGKTLYRMPYINNNV SlPHS 434 YEENNFKILKAAYRSPNINNKDLLAFSIHDFELCQAQHREELQQLKRWFEDYRLDQLGLAERYIHASYLFGVTVIPEPELSDARLMYAKY
HvCPS 432 QVRFPGEDLLQRAGRFSYEFLREREAHGTIRDKWIIAKDLPGEVKYTLDFPWYASLPRVEARVYLDQYGGDNDVWIGKTLYRMLLVNNNT NtABS 442 YKMDSFKILKAAYRSSGINNIDLLKFSEHDFNLCQTRHKEELQQMKRWFTDCKLEQVGLSQQYLYTSYFIIAAILFEPEYADARLAYAKY

SfCPS
SsLPPS
SmCPS
517
517
515
YKXLAVXDFNRCXAQHQFEWIYMQEWYHRSSVSEFGISKKELLRAYFLAAATIFEPDRTQEELVWAKTQIVSKMITSFVT.NGTTLSLDQ
YKELAILDFNRCQTQHQLEWIHMQEWYDRCSLSEFGISKRELLRSYFLAAATIFEPERTQERLLLAKTRILSKMITSFVNISGTTLSLD.
YHDLAKTDFKRCQAKHQFEWLYMQEWYESCGIEEFGISRKDLLLSYFLATASIFELERTNERIAWAKSQIIAKMITSFF..NKETTSEED
SfKSL
SmKSL
SsSS
327
327
322
motif
IILVTLIDDLFDHGGTKQESYKILELVKEWKEKSAAEYGCEEVEILFTAVYNTVNEVVERAHAAQGRSVKDFLIKLWVQILSIFKIELDT
IVLVTRIDDFFDHRGSREESYKILELIKEWKEKPAAEYGSEEVEILFTAVYNTVNELAERAHVEQGRSVKDFLIKLWVQILSIFKRELDT
SVLVTIMDDFFDCHGSSQECDKIIELVKEWKENPDAEYGSEELEILFMALYNTVNELAERARVEQGRSVKEFLVKLWVEILSAFKIELDT
NtCPS2 526 YLQAAKLDYNRCQSQHRFEWLIMQEWFEKCNFQQFGISKKYLLVSYFLAAASIFEVEKSRERLAWAKSRIICKMITSYYNDEATTWTTRN ShSBS 523 VLLLTIVDDQFDSFASTDECLNIIELVERWDDYASVGYKSEKVKVFFSTLYKSIEELVTIAEIKQGRSVKNHLLNLWLELVKLMLMERVE
CcCLS 535 YLELAKLDFNNCQALHRKEWETMQKWFMESKLDEFGVSSKTLLESYFLAAASIFEPERSTERLAWAKTAFLMETIGSYFDDEMNSKDLRK SlPHS 524 VMLLTIVDDHFESFASKDECFNIIELVERWDDYASVGYKSEKVKVFFSVFYKSIEELATIAEIKQGRSVKNHLINLWLELMKLMLMERVE
HvCPS 522 YLELAKRDFNRCQVQHQLEWHGLQKWFIENGLETFGVTLRDVLRVYFLAAACIFEPSRATERLAWAKVSVLANIITKNLHSDLSGNEMME NtABS 532 AIIITAVDDFFDCFICKEELQNIIELVERWEGYSTVGFRSERVRIFFLALYKMVEEIAAKAETKQGRCVKDHLINLWIDMLKCMLVELDL

SfCPS 606 KTALLSQIGHNFDGLDEIIS.AMKDHGLA.....RTLLTTFQQLLDGFDRYTRHQLKNAWSQWFMKLQQGEANGGEXAX........... SfKSL 417 W.SDETELSLDEYLSNSWVSIGCRICILMSMQFMGIKLTDEMLLSEECIDLCRHVSMVDRLLNDVQTFEKERKEN.TGNSVSLLLAANKD

SsLPPS 606 .........YNFNGLDEIISSANEDQGLA.....GTLLATFHQLLDGFDIYTLHQLKHVWSQWFMKVQQGEGSGGEDAV........... SmKSL 417 W.SDDTALTLDDYLSASWVSIGCRICILMSMQFIGIKLSDEMLLSEECIDLCRHVSMVDRLLNDVQTFEKERKEN.TGNSVTLLLAANKD
SmCPS 603 KRALLNELG.NINGLNDT.NGAGREGGAG.....SIALATLTQFLEGFDRYTRHQLKNAWSVWLTQLQHGEA...DDAE........... SsSS 412 W.SNGTQQSFDEYISSSWLSNGSRLTGLLTMQFVGVKLSDEMLMSEECTDLARHVCMVGRLLNDVCSSEREREENIAGKSYSILLATEKD
NtCPS2 616 SL....LMEFKVSHDPTRKNGNETKEILVLKNLRQFLRQLSEETFEDLGKDIHHQLQNAWETWLVFLREEKNACQEETE........... ShSBS 613 WFSGKTIPSIEEYLYVTSITFGARLIPLTTQYFLGIKISEDILESDEIYGLCNCTGRVLRILNDLQDSKKEQKED.SVTIVTLLM..K..
CcCLS 625 AF....VQEFKNIYE.RRMEAKGTKWNLII.....ILLTTLNHLTEVCGRDINSYLCHSWEKWMMMWEPEGDRYKGAAE........... SlPHS 614 WCSGKTIPSIEEYLYVTSITFCAKLIPLSTQYFLGIKISKDLLESDEICGLWNCSGRVMRILNDLQDSKREQKEV.SINLVTLLM..K..
HvCPS 612 RFMHGGIYEGNSNISWRKGDAKEDSLVGALEQLIDLLAQEALLVGEE.PMYINNLLRRAWIEWMMEQINREDDTSDTSVIQAGSCMVHDK NtABS 622 WKIKSTTPSIEEYLSVACVTIGVPCFVLTSLYLLGPKLSKDVIESSEVSALCNCTAAVARLINDIHSYKREQAES.STNMVSILI..TQS

SfCPS 679 ....LQANMLNICAGLIAFNEDVLSHHEYTALSTLTNKICKRLTQIQDXKMLEV.....VEGSIKDKELEQDMQALVKLVLEEN.GGGMD SfKSL 505 DSAFTEEEAITKAKEMAEWNRRQLMKIVY..IKGNKFPRKCRDMFLKVCRIGCYLYASGDEFTSPQQMMEDMKSLVYEPVGE........

SsLPPS 671 ....LLANTLNICAGL...NEDVLSNNEYTALSTLTNKICNRLAQIQDNKILQV.....VDGSIKDKELEQDMQALVKLVLQEN.GGAVD SmKSL 505 DSSFTEEEAIRIAKEMAECNRRQLMQIVY..KTGTIFPRQCKDMFLKVCRIGCYLYASGDEFTSPQQMMEDMKSLVYEPLTIHPLVANNV
SmCPS 672 ....LLTNTLNICAGHIAFREEILAHNEYKALSNLTSKICRQLSFIQSEKEMGVEGEIAAKSSIKNKELEEDMQMLVKLVLEKY.GG.ID SsSS 501 GRKVSEDEAIAEINEMVEYHWRKVLQIVY..KKESILPRRCKDVFLEMAKGTFYAYGINDELTSPQQSKEDMKSFVF.............
NtCPS2 691 ....LLVRTINLSGGYMTH.DEILFDADYENLSNLTNKVCGKLNELQ..NDKVTGGS.......KNTNIELDMQALVKLVFGNT.SSNIN ShSBS 698 ..SMSEEEAIMKIKEILEMNRRELLKMVLVQKKGSQLPQICKDIFWRTSNWADFIYLQTDGYRIAEEMKNHIDEVFYKPLNH........
CcCLS 694 ....LLSNSINLSSGRLFS.NDTLSHPNYEKLVTLSNKLCHQLGNSRRGNHNEDSDI.......KDTKIEIAMQELVQLVHQNS.SDDIS SlPHS 699 ..SMSEEEAIMKIKEILEMNRRELLKMVLVQKKGSQLPQLCKDIFWRTSKWAHFTYSQTDGYRIAEEMKNHIDEVFYKPLNH........
HvCPS 701 QTYLLLVKIIEICGGRYGEASSMINNMDCAWFIELASSICDSLHRKRLLSQDTEKNEVMVRH..MDEKIEAGMQELARNVLQSAHGDGAS NtABS 709 QGTISEEEAIRQIKEMMESKRRELLGMVL.QNKESQLPQVCKDLFWTTINAAYSIHTHGDGYRFPEEFKNHINDVIYKPLNQYSP.....

SfCPS 759 RSIKQTFLSVFKTFYYSAYHDDETTDAHIFKVLFGPVV SfKSL ...

SfCPS SfKSL
SsLPPS 748 RNIRHTFLSVFKTFYYDAYHDDETTDLHIFKVLFRPVV SmKSL 593 RGK
SmCPS 756 RNIKKAFLAVAKTYYYRAYHAADTIDTHMFKVLFEPVA SsSS ...
NtCPS2 766 QDIKQTFFAVVKTFYYSAHVSEEIMNFHISKVLFQQV. ShSBS ...
CcCLS 771 MDLKQTFFAVVRSFYYAAHCDRGTINSHIVKVLFESVV SlPHS ...
HvCPS 789 SETKQTVLSIVRSCYYAANCPLHVLDGHVLKVIFEHVF NtABS ...
 Phylogenetic analysis of SfCPS and SfKSL

SsLPPS – Salvia sclarea

labda-13-en-8-ol diphosphat synthase
SmCPS – Salvia miltiorrhiza
copalyl diphosphat synthase class II
NtCPS2 – Nicotiana tabacum
8-hydroxy-copalyl diphosphate synthase
CcCLS – Cistus creticus
copal-8-ol diphosphate synthase
class I/II

SmKSL – Salvia miltiorrhiza

miltiradiene synthase class I
SsSS – Salvia sclarea
sclareol synthase
Functional characterization of SfCPS
E. coli expression

ptacHA-SfCPSm – enzymatic assays

- His-tag purified enzyme incubated

?
overnight with 60µM GGPP

- Alkaline phosphatase: 0,025 units/µl

SfCPSm
- Incubation: 4h, 37°C

- Reaction products extracted with GGDP

hexane

- Hexane extracts analysed with GC-MS

Functional characterization of SfCPS
E. coli expression
GC-MS profile (275 m/z extracted chromatogram):
ptacHA-SfCPSm – enzymatic assays
copalol
dephosphorylated
product of SfCPS
- His-tag purified enzyme incubated incubated with
GGDP
overnight with 60µM GGPP

- Alkaline phosphatase: 0,025 units/µl

control reaction
- Incubation: 4h, 37°C (GGDP omitted)
control
- Reaction products extracted with
hexane
copalol
copalol
- Hexane extracts analysed with GC-MS mass spectrum of
the reaction product
Functional characterization of SfCPS
E. coli expression

ptacHA-SfCPSm – enzymatic assays

- His-tag purified enzyme incubated

overnight with 60µM GGPP

- Alkaline phosphatase: 0,025 units/µl

SfCPSm
- Incubation: 4h, 37°C

- Reaction products extracted with GGDP CDP

hexane

- Hexane extracts analysed with GC-MS

Functional characterization of SfKSL
E. coli expression

ptacHA-SfKSLm – enzymatic assays

- His-tag purified enzyme incubated overnight with 200µM GGPP and purified SfCPS
- Incubation: overnight, room temperature
- Reaction products extracted with hexane
- Hexane extracts analysed with GC-MS
Functional characterization of SfKSL
E. coli expression
GC-MS profile (272 m/z
extracted chromatogram)
ptacHA-SfKSLm – enzymatic assays
miltiradiene? product of SfCPS
- His-tag purified enzyme incubated and SfKSL coupled
overnight with 200µM GGPP and activity
purified SfCPS

- Incubation: overnight, room temperature

control reaction
- Reaction products extracted with hexane (SfCPS omitted)

- Hexane extracts analysed with GC-MS

miltiradiene?
mass spectrum of
the reaction product
Functional characterization of SfCPS and
SfKSL in yeast (Saccharomyces cerevisiae)

Functional characterization of SfCPS and SfKSL in

yeast (Saccharomyces cerevisiae)
Functional characterization of SfCPS and
SfKSL in yeast (Saccharomyces cerevisiae)

Head space-solid phase microextraction

(HS-SPME)

GC-MS chromatogram representing products of coupled SfCPS

and SfKSL activity in yeast (Saccharomyces cerevisiae):

miltiradiene-like
abietatriene ?
compound
 SfCPS and SfKSL: transient expression assays
in Nicotiana benthamiana
GC-MS profile of agroinfiltrated leaves:

SfCPS
and
SfKSL

Promoter P 1
Rep Origin 1 nos-nptII
Terminator 1 empty
Promoter P 2
vector
pGA643
tetR
Hin dIII
pGA643 Xba I
11601 bp
Hpa I

Bgl II

tetA Terminator 2
miltiradiene-
oriT 1 like
traJ compound

trfA

abietatriene ?
Scale-up production of miltiradiene-like
compound for the NMR analysis

- 3, 75 l of yeast liquid culture extracted with

hexane.
- the extract purified using silica-gel column.
- the compound of interest detected in the
fraction eluted with pure hexane.
- the mass of the fraction was 20 mg -
enough for NMR.
Scale-up production of miltiradiene-like
compound for the NMR analysis

1D and 2D NMR spectroscopy

(1H, 13C, DEPT, COSY H-H, HMQC и HMBC):

- 3, 75 l of yeast liquid culture extracted with

hexane.
- the extract purified using silica-gel column.
- the compound of interest detected in the
fraction eluted with pure hexane.
- the mass of the fraction was 20 mg -
enough for NMR.

The analysis was performed by dr Konstantinos Tsoleridis (Department of

Chemistry, Laboratory of Organic Chemistry, Aristotle University of
Thessaloniki, Greece).
spontaneous:
Zi and Peters,
Org. Biomol. Chem., 2013
Transcriptome analysis of Cretan sage and
Rosemary trichomes using the 454 sequencing

- In order to retrieve the remaining genes responsible for the biosynthesis of carnosic acid, new
sequencing was performed.
- Glandular trichomes of Cretan sage young leaves (genotype Kavousi) exposed to mild stress, and
glandular trichomes on the Rosemary leaves of the younger developmental stage (commercial
genotype B&T World Seeds), were chosen for the transcriptome analysis.
- Glandular trichomes were isolated using the dry-ice abbrasion method.
- High quality RNA was isolated using the Spectrum Plant Total RNA Kit (Sigma, USA) and PureLink
RNA Mini Kit (Ambion, USA).

Salvia Rosmarinus
fruticosa officinalis
Transcriptome analysis of Cretan sage and
Rosemary trichomes using the 454 sequencing

- 454 GS FLX Titanium platform was used for sequencing.

- Newbler runAssembly module was used for reads assembly into contigs
- FastAnnotator online service was used for transcripts annotation
- GO (Gene ontology) terms assigned
- EC (Enzyme Commission) numbers determined
- functional domains retreived.
Species Salvia fruticosa Rosmarinus officinalis
Number of contigs 29912 57254
Total reads length 27782418 78835481
Average reads length 1099 1377
Maximum read length 10338 12058
N50 length 1230 1831

Previous sequencing - 904 contigs

0,75 % of S. fruticosa contigs and 0,55 % R. officinalis contigs belong to the secondary
metabolism .
But, for 30 % of S. fruticosa sequences and 22 % R. officinalis sequences - GO terms
were not assigned  probably secondary metabolism genes belong to this group!
Isolation of candidate genes

Eighteen candidate sequences from S. fruticosa and eighteen from R. officinalis could were
identified as candidates and isolated. Three had high similarity to known ferruginol
synthases.

SfFS 1 MDPFPLVAAALFI.AATWFITFKRRRNLPPGPFPYPIVGNMLQLGSQPHETFAKLSKKYGPLMSIHLGSLYTVIISSPEMAKEIMHKYGQ
RoFS1 1 MDSFPLLAALFFILAATWFISFRRPRNLPPGPFPYPIVGNMLQLGTQPHETFAKLSKKYGPLMSIHLGSLYTVIVSSPEMAKEIMHKYGQ
RoFS2 1 MDSFPLLATLFFILAATWFISFRRRRNLPPGPFPYPIVGNMLQLGSQPHETFAKLSKKYGPLMSIHLGSLYTVIVSSPEMAKEIMHKYGQ
SmilCYP76AH1 1 MDSFPLLAALFFIAATITFLSFRRRRNLPPGPFPYPIVGNMLQLGANPHQVFAKLSKRYGPLMSIHLGSLYTVIVSSPEMAKEILHRHGQ
RoCYP76AH4 1 .................MAKKTSSKGKLPPGPFPLPIVGNMLQLGTQPHETFAKLSKKYGPLMSIHLGSLYTVIVSSPEMAKEIMHKYGQ

SfFS 90 VFSGRTIAQAVHACDHDKISMGFLPVGAEWRDMRKICKEQMFSHQSMEDSQNLRKQKLQQLLEYAQKCSEEGRGIDIREAAFITTLNLMS
RoFS1 91 VFSGRTVAQAVHACGHDKISMGFLPVGGEWRDMRKICKEQMFSHQSMEDSQWLRKQKLQQLLEYAQKCSERGRAIDIREAAFITTLNLMS
RoFS2 91 VFSGRTVAQAVHACGHDKISMGFLPVGGEWRDMRKICKEQMFSHQSMEDSQGLRKQKLQQLLDYAQKCSERGRAIDIREAAFITTLNLMS
SmilCYP76AH1 91 VFSGRTIAQAVHACDHDKISMGFLPVASEWRDMRKICKEQMFSNQSMEASQGLRRQKLQQLLDHVQKCSDSGRAVDIREAAFITTLNLMS
RoCYP76AH4 74 VFSGRTVAQAVHACGHDKISMGFLPVGGEWRDMRKICKEQMFSHQSMEDSQWLRKQKLQQLLEYAQKCSERGRAIDIREAAFITTLNLMS

SfFS 180 ATLFSMQATEFDSKVTMEFKEIIEGVASIVGVPNFADYFPILRPFDPQGVKRRADVYFGRLLGLIEGYLNERIEFRKANPNAPKKDDFLE

RoFS1 181 ATLFSMQATEFDSKVTMEFKEIIEGVASIVGVPNFADYFPILRPFDPQGVKRRADVYFGRLLAIIEGFLNERVESRRTNPNAPKKDDFLE
RoFS2 181 ATLFSMQATEFDSKVTMEFKEIIEGVASIVGVPNFADYFPILRPFDPQGVKRRADVYFGRLLAMIEGFLNERVESRRTNPNAPKKDDFLE
SmilCYP76AH1 181 ATLFSSQATEFDSKATMEFKEIIEGVATIVGVPNFADYFPILRPFDPQGVKRRADVFFGKLLAKIEGYLNERLESKRANPNAPKKDDFLE
RoCYP76AH4 164 ATLFSMQATEFDSKVTMEFKEIIEGVATIVGVPNFADYFPILRPFDPQGVKRRADVYFGRLLAIIEGFLNERIESRRTNPNAPKKDDFLE

SfFS 270 TLVDALDAKDYKLKTEHLTHLMLDLFVGGSETSTTEIEWIMWELLASPEKMAKVKAELKSVMGGEKVVDESMMPRLPYLQAVVKESMRLH

RoFS1 271 TLVDTLQTNDNKLKTDHLTHLMLDLFVGGSETSTTEIEWIMWELLANPEKMAKMKAELKSVMGEEKVVDESQMPRLPYLQAVVKESMRLH
RoFS2 271 TLVDILEANDCKLKTDHLTHLMLDLFVGGSETSTTEIEWIMWELIAHPDKMVKVKAELKSVMGEEKVVDESNMPRLPYLQAVVKESMRLH
SmilCYP76AH1 271 IVVDIIQANEFKLKTHHFTHLMLDLFVGGSDTNTTSIEWAMSELVMNPDKMARLKAELKSVAGDEKIVDESAMPKLPYLQAVIKEVMRIH
RoCYP76AH4 254 TLVDTLQTNDNKLKTDHLTHLMLDLFVGGSETSTTSIEWTMSELVMNPEKMAKLKAELKSVAGDEKIVDESEIAKLPYLQAVIKEVMRIH

SfFS 360 PPGPLLLPRKAESDQVVNGYLIPKGAQVLINAWAMGRDPSLWKNPDSFEPERFLDQKIDFKGTDYELIPFGSGRRVCPGMPLANRILHTV

RoFS1 361 PPGPLLLPRKAESDQVVNGYLIPKGAQVLINAWAIGRDHSIWKNPDSFEPERFLDQKIDFKGTDYELIPFGSGRRVCPGMPLANRILHTV
RoFS2 361 PPGPLLLPRKAESDQVVNGYLIPKGTQVLINAWAMGRDPSIWKNPDSFEPERFLDQKIDFKGQDYELIPFGSGRRVCPGMPLANRILHTV
SmilCYP76AH1 361 PPGPLLLPRKAESDQEVNGYLIPKGTQILINAYAIGRDPSIWTDPETFDPERFLDNKIDFKGQDYELLPFGSGRRVCPGMPLATRILHMA
RoCYP76AH4 344 PPGPLLLPRKAESDQEVNGYLIPKGTQVLINAWAIGRDPSVWKNPDSFEPERFLEQKIDFKGQDFELLPFGSGRRVCPGLPLASRILHMT

SfFS 450 TATLVHNFDWKLERPEASDA.HKGVLFGFAVRRAVPLKIVPIKA.

RoFS1 451 TATLVHNFDWKLERPEASDA.HRGVLFGFAVRRAVPLKIVPFKV.
RoFS2 451 TATLVHNFHWELERPDASHAEHQGVLFGFAVRRAVPLKIVPFKA.
SmilCYP76AH1 451 TATLVHNFDWKLEDDSTAAADHAGELFGVAVRRAVPLRIIPIVKS
RoCYP76AH4 434 AATLVHNFDWKLEDEATAEADHAGELFGLAVRRAVPLRIIPIVKS
Transcriptome analysis of Cretan sage and
Rosemary trichomes using the 454 sequencing
Digital PCR analysis of Salvia fruticosa genes
wounding stages of development
Biomark HD instrument (Fluidigm, USA) SfCPS

SfKSL

SfFS
wounded S. fruticosa plant
 Functional characterization of cytochrome
P450s in Saccharomyces cerevisiae
AM113

Mat a/alpha, GALp-(K6R)HMG2::HOX2, ura3,his3, trp1, PTDH3-

HMG2(K6R)X2-::leu2, PTDH3-HMG2(K6R)::HO1, ERG9/erg9,
UBC7/ubc7, ssm4::/SSM4, mct1/MCT1, whi2/WHI2 PTDH3-
CcGGDPS1-FLO8, PTDH3-SfFDPS1-FLO1 derived from AM108
Candidate p450s
SfCDSm+SfKSm+ PtCPR2
or
RoCLSm+RoKSL1f+ PtCPR2

http://microbewiki.kenyon.edu
Functional characterization of cytochrome
P450s in Saccharomyces cerevisiae

ferruginol
Functional characterization of isolated
ferruginol synthases in Nicotiana benthamiana
1.5
(x10,000,000) A B
1.0
a untreated
a S. fruticosa trichome extract

0.5

17.50 17.75 18.00 18.25 18.50

(x10,000,000)
3.0
non-transformed
2.0 b GV3101 b R. officinalis trichome extract

1.0

17.50 17.75 18.00 18.25 18.50

SfCPS x SfKSL x AtCPR1 x

c SfFS
Ferruginol standard

SfCPS x SfKSL x AtCPR1 x

ferruginol d RoFS1
C
(x100,000)
1.5 271
Ferruginol MS 286
189
175
1.0
69
SfCPS x SfKSL x AtCPR1 x
e RoFS2 0.5 41
55
149
201

229
115 133 159
91
216 243
0.0
50 100 150 200 250

Ferruginol standard
Acknowledgements Many Thanks to…
Summary of project achievements
This work was supported by EU Grant
agreement KBBE-227448 for the
TERPMED project

Aristotle University of Thessaloniki

Department of Pharmaceutical Sciences

Leibniz Institute of Plant Biochemistry

Department of Cell and Metabolic Biology

Centre for Research in Agricultural Genomics (CRAG) &

Department of Biochemistry and Molecular Biology