fundamental aspects and practical applications in the production of non-toxic Citrus

F. Bourgaud1, A. Dugrand1, A. Olry1, A. Hehn1, Y. Froehlicher2 1: Laboratoire Agronomie et Environnement - Université de Lorraine/INRA -Nancy (France) 2: Laboratoire AGAP – INRA/CIRAD – Montpellier/San Gugliano (France) R5 R5 R6

O O O O O O R8R6

Linear furanocoumarins Angular furanocoumarins Psoralen: R5, R8: H Angelicin: R5, R6: H Bergapten (5-MOP): R5: OMe Sphondin: R6: OMe Xanthotoxin (8-MOP): R8: OMe Pimpinellin: R5, R6: OMe Isopimpinellin (5-8-MOP): R5, R8: OMe

Skin blisters and burns !

Giant Hogweed (Heracleum mantegazzianum) Blisters after contact with plants + UV/Sun exposure AFERP-STOLON 15-17 juillet 2015 – Lyon O + O O O O O

Moraceae

Rosids Fabaceae

Ficus carica

Rutaceae Cullen cinenea

Core tricolpates

Citrus bergamia

Apiaceae Asterids Apiaceae

Pastinaca sativa

Ammi majus AFERP-STOLON 15-17 juillet 2015 – Lyon Ecological functions of furanocoumarins

• Phytoalexins

•Allelopathy (germination inhibition)

Fusarium sp.

O O O

Genotoxicity (DNA crosslinks)

Enzyme inhibition (MBI P450) Hordeum vulgare Papilio polyxenes •Anti-insect

Grapefruit juice effect (effet pomélo) AFERP-STOLON 15-17 juillet 2015 – Lyon Cinnamic acid p-Coumaroyl CoA Umbelliferone OH HO OH

HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin Multiple steps reaction HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins

O O O O O O

OH H Psoralen OH Angelicin

O O O O O O Xanthotoxol Bergaptol OH Sphondin Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 P-Coumaroyl-CoA 2’Hydroxylase (C2’H) Dioxygenase 2-OGD-Dioxygenase Vialart et al., Plant J, 2012, 70(3), 460-470 P450 Cinnamic acid p-Coumaroyl CoA ? Dioxygenase OH HO OH Umbelliferone HO O O HOOC COSCoA HOOC Osthenol Demethylsuberosin

HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins

O O O O O O

OH H Psoralen OH Angelicin

O O O O O O Xanthotoxol Bergaptol OH Sphondin Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Characterization of p-coumaroyl CoA 2’-hydroxylase

MeO

COSCoA COSCoA HO OH HO p-coumaroyl-CoA Feruloyl-CoA C2’H - Diox Arabidopsis thaliana Kai et al., F6’H - Diox Plant J 2008, 55(6), 989-999

Ruta graveolens MeO Vialart et al. Plant J, 2012, 70(3), 460-470 COSCoA COSCoA HO OH HO OH

Spontaneous lactonization Spontaneous lactonization

MeO

Umbelliferone HO O O HO O O Scopoletin

Ruta graveolens To furanocoumarins Vialart et al. Plant J, 2012, 70(3), 460-470

STOLON/AFERP Symposium, July 15-17th 2015 Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids

Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox

Cinnamic acid - - - - p-Coumaric acid - - - - Ferulic acid - - - - Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - -

In vitro activity / heterologous expression in E. coli

*: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids

Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox

Cinnamic acid - - - - p-Coumaric acid - - - - Diox 1 & 2: Ferulic acid - - - more - than 90% identity Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - -

In vitro activity / heterologous expression in E. coli

*: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids

Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox

Cinnamic acid - - - - p-Coumaric acid - - - - Ferulic acid - - - - Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - -

In vitro activity / heterologous expression in E. coli

*: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Prenyltransferase Cinnamic acid p-Coumaroyl CoA C-Prenyltransferase Umbelliferone OH HO OH

HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin

HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins Umbelliferone prenyltransferases O O O O O Karamat et al., Plant J, 2014, 77(4), 627-638 O OH Munakata et al., Plant Phys, 2014 H

Psoralen OH Angelicin

O O O O O O Xanthotoxol Bergaptol OH Sphondin Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Parsley prenyltransferase C-Prenyltransferase Umbelliferone

HO O O

Osthenol Demethylsuberosin

HO O O

HO O O

100:5 To linear To angular furanocoumarins furanocoumarins

O O O

Umbelliferone prenyltransferase

Karamat et al., Plant J, 2014, 77(4), 627-638

STOLON/AFERP Symposium, July 15-17th 2015 Parsley prenyltransferase C-Prenyltransferase Umbelliferone

HO O O

Osthenol Demethylsuberosin

HO O O

HO O O

100:5 To linear To angular furanocoumarins furanocoumarins

O O O

Ruta graveolens

Umbelliferone prenyltransferase

Karamat et al., Plant J, 2014, 77(4), 627-638

STOLON/AFERP Symposium, July 15-17th 2015 P450s P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Apiaceae : CYP71 AJ OH HO OH

HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin

HO O O

HO O O Marmesin (+) Columbianetin Psoralen synthase Angelicin synthase CYP71AJ1, Ammi majus CYP71AJ4, Pastinaca sativa CYP71AJ2, Apium graveolens (celery) O O O O O O CYP71AJ3, Pastinaca sativa (parsnip) OH Larbat et al., JBC, (2009) celery Ammi majus H Psoralen Larbat et al., JBC, 281, 542 (2007) OH Larbat et al., JBC, 284, 4776 (2009) Angelicin

Dueholm et al. BMC Evol Biol 15:122 (2015) O O O parsnip Thapsia O O O Sphondin Xanthotoxol Bergaptol OMe Pimpinellin OH MeO MeO

O O O O O O O O O OH O O O Bergapten OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Apiaceae : CYP71 AJ OH HO OH CYP71 xx Non apiaceae : CYP82 HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin

HO O O

HO O O Marmesin (+) Columbianetin

O O O O O O

Ammi majus OH H Psoralen OH Angelicin

O O O parsnip O O O Xanthotoxol Psoralen 8-monooxygenase Sphondin Pimpinellin CYP71xx4, Pastinaca sativa (parsnip) OMe MeO MeO C.Krieger (PhD) O O O O O O O O O OH

Xanthotoxin Xanthotoxin 5-monooxygenase CYP71xx1, Ammi majus Isopimpinellin CYP71xx6, Pastinaca sativa (parsnip) O O O OH OMe OMe C.Krieger (PhD), S. Rosselli (Post-doc) Biosynthetic routes CYP82 , Non apiaceae to furanocoumarins O O O O O O A. Dugrand (PhD) OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Crucial steps required to close the road ? P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Dioxygenase OH HO OH C-Prenyltransferase

HO O O O-Methyltransferase HOOC Multiple steps HOOC COSCoA Osthenol reaction Demethylsuberosin

HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins

O O O O O O

OH H Psoralen OH Angelicin

O O O O O O

OH Sphondin Xanthotoxol Bergaptol OMe Pimpinellin MeO MeO

O O O O O O O O O O O O OH Bergapten OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Demethylallyllated compounds Geranylated compounds

8-MOP-Derived 5-MOP-Derived Psoralen O OH HO O O O O O O O O O O O O O O O O O O OH OH HO O O O O O O O O O O Isoimperatorin Oxypeucedanin Oxypeucedanin Heraclenol Heraclenin O O O O O O Imperatorin hydrate OH Xanthotoxol Bergaptol O OH OH

O O O OMe O O O O

O O O O O O O O O O O O O O O 8-geranyloxypsoralen Bergamottin Epoxybergamottin 6’,7’-dihydroxy OMe Xanthotoxin Bergapten 8-MOP bergamottin 5-MOP

OMe

Citrus fruit O O O OMe Isopimpinellin 5-8-MOP

5-8-MOP-Derived C. paradisi C. maxima C. medica O O OMe OMe OMe

O O O O O O O O O O O O O O O OMe O O O O

OH HO C. aurantium C. aurantifolia C. limon O Cnidilin Cnidicin Phellopterin Byakangelicol Byakangelicin

C. bergamia C. micrantha C. jambhiri

STOLON/AFERP Symposium, July 15-17th 2015 Quantitative distribution of furanocoumarins in Citrus pulp

C. Maxima C. Paradisi (pummelo) (grapefruit)

C. Aurantifolia (lime) How to annihilate the Cinnamic acid p-Coumaroyl CoA biosynthesis of OH HO OH furanocoumarins in Umbelliferone Citrus ? HO O O HOOC COSCoA HOOC

Demethylsuberosin

HO O O Linear Marmesin furanocoumarins C. Paradisi (Grapefruit) O O O Gene editing strategy OH (CRISPR / Cas9) Psoralen

O O O

Xanthotoxol Bergaptol OH C. Maxima (pummelo)

O O O O O O OH Bergapten OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe

O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Alain Hehn, Romain Larbat, Guilhem Vialart, Karine Lièvre, Célia Krieger, Audray Dugrand, Sandro Rosselli, Hélène Jugdé, Cédric Paris, Alexandre Olry (Univ. Lorraine-INRA, Nancy, F) : functionnal characterization of CYP71 AJ1, CYP71xx1, C2’H isolation and characterization of CYP71AJ2,3,4, CYP71xx2-6, prenyltransferases

Ulrich Matern, Joachim Hans, Sylvia Hubner, Sandra Specker (Univ Marburg, G) : isolation of CYP71AJ1

Bernd Schneider & Wilhem Boland (MPI, Jena, G) : NMR characterization, supply of deuterated compounds

Masaharu Mizutani, Bun-Ichi Shimizu, Kyoko Ito (Univ. Kobe, J) functionnal characterization of C2’H

Kazufumi Yazaki, Ryosuke Munakata, Akifumi Sugiyama, Takao Koeduka (Univ. Kyoto, J) Isolation and functionnal characterization of prenyltransferases

Henrik Tof Simmonsen, Björn Duelhorm (Univ. Copenhagen, D) Isolation of CYP71AJ5 and other 71AJs from Thapsia…

AFERP-STOLON 15-17 juillet 2015 – Lyon

B STOLON/AFERP Symposium, July 15-17th 2015 Hierarchical ascendant classification (HAC) based on occurrence of molecules in peel

A

Clade 1

Clade 2

Clade 3

C

Clade 4

Group IV Group III Group II Group I P450s first ! P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone OH HO OH

HO O O HOOC HOOC COSCoA Osthenol Cinnamate 4-Hydroxylase Demethylsuberosin Ruta graveolens CYP73A32, Ruta graveolens HO O O Gravot et al, 2004, ABB, 422, 71 (2004) HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins

O O O O O O

OH H Psoralen OH Angelicin

O O O O O O

OH Sphondin Xanthotoxol Bergaptol OMe Pimpinellin MeO MeO

O O O O O O O O O O O O OH Bergapten OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe Kyoto P450 congress Sept. 25th, 2014 C4H from plants producing furanocoumarins – Resistance to MBI

Cinnamate 4-Hydroxylase CYP73A32, Ruta graveolens

Gravot et al, 2004, ABB, 422, 71 (2004)

O O O

Jerusalem artichoke Arabidopsis Parsley Ruta graveolens CYP73A1 CYP73A5 CYP73A10 CYP73A32

KI (µM) 4,6 ± 2 3 ± 0,4 8 ± 5,1 8 ± 1,4

kinact (min-1) 0,18 ± 0,02 0,113 ± 0,01 0,036 ± 0,009 0,066 ± 0,03

kinact/KI 41 37 4 8

C4H from furanocoumarin producing-plants are 5 to 10 times less sensitive to MIB

C4H structurally adapted to furanocoumarins ? P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Dioxygenase OH HO OH C-Prenyltransferase

HO O O O-Methyltransferase HOOC Multiple steps HOOC COSCoA Osthenol reaction Demethylsuberosin

HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins

O O O O O O

OH H Psoralen OH Angelicin

O O O O O O

OH Sphondin Xanthotoxol Bergaptol OMe Pimpinellin MeO MeO

O O O O O O O O O O O O OH Bergapten OMe Xanthotoxin

Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe Kyoto P450 congress Sept. 25th, 2014 Cytochrome P450

Meander Heme-binding domain

I helix Proline hinge

Heme Meander : P-E/D-R/H-F/W Heme-binding domain: F-x-x-G-x-R-x-C-x-G I helix : A/G-G-x-D/E-T-T/S Proline hinge :P-P-x-P

AFERP-STOLON 15-17 juillet 2015 – Lyon