-'V-Oh

J>: - c:

YPK ^^^^ Localization and Functional Characterization of Iridoid Biosynthetic Genes in

Catharanthus roseus

By

Jonathon Roepke, B.Sc.

A Thesis

Submitted to the Centre for Biotechnology

in partial fulfillment of the requirements for the degree of

Masters in Science

11

Abstract '

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high

economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety

which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan.

The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene

indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.

This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid

methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase involved in iridoid biosynthesis loganic acid

Ill

methyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10-

fold enrichment in the leaf epidermis as compared to the whole leaf specific activity.

Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate

specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as

substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and

shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition

and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor

in monoterpene indole alkaloid biosynthesis.

Metabolite profiling of C. roseus organs shows that secologanin accumulates

within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin

does not accumulate within these organs suggesting that the product inhibition of loganin

with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA

biosynthesis seems to be related to the spatial separation of secologanin and the MIA

pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total

secologanin is found in the epidermis while the remaining secologanin is found within

the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the

biochemical specialization of the epidermis for the production of secondary metabolites.

The epidermal cells synthesize metabolites that are sequestered within the plant and

metabolites that are secreted to the leaf surface. The secreted metabolites comprise the

epidermome, a layer separating the plant from its environment.

IV

Acknowledgements

Firstly, I would like to thank my supervisor Dr. Vincenzo De Luca for giving me the opportunity to work in his lab for the past two years. I have appreciated his insightful comments and suggestions which have helped me along the way.

Secondly, I would like to thank my conmiittee members Dr. Doug Bruce and Dr.

Tony (Hongbin) Yan for their help throughout my project.

Additionally, I would like to thank the past and present lab members of the De

Luca lab, especially Dr. Jun Murata. They have made my research experience exciting,

' fresh and interesting.

Lastly, I would like to thank my family and friends who have given me support over that last two years of my project.

Table of Contents *

Page

Abstract ii Acknowledgments iv Table of Contents v List of Tables ix List of Figures X General Introduction 1 Outline 1 Chapter 1 Literature Review- Cellular Localization and Regulation of 3 Monoterpene Indole Alkaloid Biosynthesis in Catharanthus roseus 1.1- Abstract- Catharanthus roseus: A Biological Source for 4'^ Pharmaceutically and Economically Important Compounds 1.2-Early Monoterpene Biosynthesis 5 1.2.1- Gerneral Terpene Biosynthesis 5'

' 1.2.2- Early Monoterpene Biosynthesis within C. roseus 8 1.2.3- Cellular Localization of the Monoterpene Pathway 9 1.2.4- Gene Regulation with Respect to the Monoterpenes of C. roseus 9 1.3- The Iridoid Biosynthetic Pathway 10 1.3.1- General Iridoid Biosynthesis 10 1.3.2- Iridoid Biosynthesis within C. roseus .11 1.3.3- Cellular Localization of the Iridoid Pathway 13 1.3.4- Gene Regulation with Respect to the Iridoids of C. roseus 14 1.4- The Early MIA Biosynthetic Pathway " 14 1.4.1- General Early MIA Biosynthesis 14 1.4.2- Early MIA Biosynthesis within C. roseus 15 1.4.3- Cellular Localization of the Early MIA Pathway 16 1.4.4- Gene Regulation with respect to the Early MIA of C. roseus 16 1.5- The Late MIA Biosynthetic Pathway 19 1.5.1- General Late MIA Biosynthetic Pathway 19 1.5.2- Late Monoterpene Indole Alkaloid Biosynthesis within C. roseus 19 [The Vindoline Biosynthetic Pathway] 1.5.3- Cellular Localization of the Late MIA Pathway [The Vindoline 20 Pathway] 1.5.4- Gene Regulation with respect to the Late MIA of C. roseus [The 22 Vindoline Pathway] 1.6-Conclusion and Perspectus 23 Chapter 2 The Epidermome analysis of Catharanthus leaf reveals its 25 biochemical specialization 2.1- Abstract 26 2.2- Introduction 27 2.3- Methods 31 2.3.1- Plant Material 31 2.3.2- Carborundum abrasion (CA) for cDNA library construction 32 ». VI

2.3.3- Construction and sequencing of leaf epidermis-enriched cDNA 32 library 2.3.4- Sequence analysis 33 2.3.5- Extraction and analysis of triterpenes in Catharanthus leaves 33 2.3.6- Capillary HPLC MS analysis of triterpenes 34 2.3.7- Scanning electron microscopy 35 2.3.8- Laser capture microdissection for the isolation of RNA from 35 single cells 2.3.9- LAMT full-length cDNA cloning and protein expression in E. 35 coli 2.3.10- Enzyme assays for recombinant LAMT (rLAMT) 36 2.3.11- Computation methods for LAMT sequence alignment and 38 modelling of the active site 2.4- Results 40 2.4.1- Construction of the leaf epidermis-enriched Catharanthus cDNA 40 library 2.4.2- Data mining of the CROLFING dataset and categorizing the 41 genes of interest

2.4.3- The CROLF 1 NG dataset contains virtually all known MIA 41 biosynthetic genes 2.4.4- The CROLFING dataset contains the genes for the mevalonic 43 acid and triterpenoid biosynthesis pathways. 2.4.5- The CROLFING dataset contains the genes for flavonoid 45 biosynthesis-related genes 2.4.6- The CROLFING dataset contains many genes for lipid 48 biosynthesis 2.4.7- The CROLFING dataset contains genes for S-adenosyl-L- 49 methionine-dependent methyltransferase' s 2.4.8- The CROLFING dataset contains acyltransferase's. Cytochrome 49 P450 monooxygenase's glycosyltransferase's, dioxygenase's and transcription factors 2.4.9- Functional characterization of a novel LAMT obtained from the 50 CROLFING dataset. 2.4.10- Recombinant LAMT is a highly specific 0-methyltransferase 54 2.4.11- LAMT enzyme activity is preferentially expressed in 56 Catharanthus leaf epidermis 2.4.12- Biosynthesis of pentacylic triterpenes with an oleanane 58 skeleton may be exclusively localized to the epidermal cells of Catharanthus leaf 2.5- Discussion 61 2.5.1- Towards a complete understanding of ML\ biosynthesis in 61 Catharanthus roseus

2.5.2- Use of the CROLF 1 NG dataset to elucidate the pathway for 63 secologanin 2.5.3- Features of the active site of LAMT based on the crystal structure 65 ofSAMT i ,(

-';, ' . . . (

.;.' ^

2.5.4- Kinetic analysis and substrate specificity studies with rLAMT 69 suggests a preferred route of secologanin biosynthesis in Catharanthus roseus 2.5.5- Catharanthus leaf epidermis as the exclusive site of oleanane 70 triterpene biosynthesis 2.5.6- The possible roles of the plastidic MEP and cytosolic MVA 71 pathways in secologanin biosynthesis

2.5.7- The versatility of CA technique and its possible use for 75 epidermome analysis 2.6- Acknowledgements 7# 2.7- Accession Numbers 17'- 2.8- Supplemental Data 77 Chapter 3- Spatial Separation of Early and Late Iridoid Biosynthesis within 78 C. roseus Leaves 3.1- Abstract .79 3.2- Introduction 79 3.3- Materials and Methods 81 3.3.1- Metabolite Extractions -81 3.3.2- Iridoid Analysis by Ultra Performance Liquid Chromatography- 82 Mass Spectroscopy Detection 3.3.3- Alkaloid Analysis by Ultra Performance Liquid 83 Chromatography-Photodiode Array Detector (305; 280 nm) 3.3.4- Monoterpene Analysis by High Performance Liquid 83 Chromatography-Photodiode Array Detector (205 nm) 3.3.5- Expression of pET30b-LAMr in E. coli 83 3.3.6- Isolation of LAMT from E. coli cells harbouring pET30b-LAMr 84 3.3.7- Enzyme Assays rLAMT 84 3.3.8- Geraniol 10-Hydroxylase (GIOH)/ loganic acid 85 methyltransferase {LAMT), 16-hydroxytabersonine O- methyltransferase (16-OMT), N-methyltransferase (NMT) and deacetylvindoline acetyltransferase (DAT) Enzyme Preparation and Assays

3.4- Results 89 3.4.1 There are important pools of secologanin different organs of C. 89 roseus 3.4.2- Large secologanin pools are found in leaves of different ages 90 and in hairy roots compared to those of vindoline and catharanthine. 3.4.3- The terminal two steps in secologanin biosynthesis occur in the 94 leaf epidermis but secologanin accumulates elsewhere in the

leaf. 3.4.4- Feedback inhibition of Iridoid Biosynthesis 98 3.5- Discussion 98

3.5.1- Biological Function of Iridoids 98

vm

3.5.2- The Iridoid-alkaloid ratio remains constant as the leaf matures 101 but varies as the roots mature 3.5.3- Different levels of secologanin are detected in Catharanthus 101 organs 3.5.4- Cellular Localization of Iridoid Biosynthesis 102 3.5.5- Iridoids as a limiting factor in monoterpene indole alkaloid 105 biosynthesis Conclusion 106 Future Work 107 References 109 Appendix I- Supplementary Tables 1-10 123

IX

List of Tables

Table Title Page Chapter 2

1. CROLFING genes with significant similarity to known 45 MIA biosynthetic genes 2. CROLFING is enriched in ESTs for enzymes in the MIA 59 biosynthetic pathway Chapter 3 L UPLC Elution Gradient -Iridoids 82 2. UPLC Elution Gradient- Alkaloids 83 3. HPLC Elution Gradient- Monoterpenes 83 Appendix I

1. CROLFING genes related to isoprenoid biosynthesis 123 2. CROLFING genes related to downstream terpenoid 123 biosynthesis 3. CROLFING genes related to flavonoid biosynthesis 123 4. CROLFING genes related to lipid biosynthesis 125 5. CROLFING genes related to methyltransferases 127 6. CROLFING genes related to acyltransferases 128 7. CROLFING genes related to Cytochrome P450- 129 dependent monooxygenases (CYPs)

8. CROLFING genes related to glycosyltransferases 130 9. CROLFING genes related to 2-oxoglutarate dependent 131 dioxygenases 10. CROLFING genes related to transcription factors. 132

1

List of Figures

Figure Title Page Chapter 1

1 . Metabolic Pathway of Plant Derived Terpenes 7 2. Iridoid Biosynthetic Pathway 12

3. Chemical structure of all the characterized classes of 17 monoterpene indole alkaloids 4. The Vindoline Pathway 21 Chapter 2

1 Vindoline biosynthesis and its proposed localization 44 within the leaf in Catharanthus roseus 2. The proposed pathway for secologanin biosynthesis in 5 Catharanthus roseus

3. Identification and functional characterization of loganic 53 acid methyltransferase from Catharanthus. 4. Production of loganin by E.coli cell free extracts 55 expressing recombinant LAMT 5. Substrate specificity of rLAMT 57 6. Differential distribution of LAMT enzyme activity in 60 Catharanthus leaf epidermis

7. The oleanane type triterpenes are restricted to 62 Catharanthus leaf epidermis

8. Computational model for accommodating two 67 conformations of loganic acid in the active site of LAMT 9. Catharanthus leaf epidermal cells express the MIA, 72 mevalonic acid/triterpene, very long chain fatty acid and flavonoid biosynthetic pathways Chapter 3

1. Iridoid analysis from different aerial organs of C roseus 91 2. Quantification of secologanin, vindoline and 93 catharanthine in C. roseus leaves

3. Metabolite analysis of hairy root cultures of C. roseus 96

4. Localization of iridoid biosynthesis 97

5. Putative inhibition of secologanin with LAMT 99

General Introduction

Plants like all organisms are in constant contact with their environment. The

environment is comprised of both biotic and abiotic stresses which the plant must overcome. Plants have evolved both chemical and physical strategies to combat these environmental stresses. Plants produce a variety of secondary metabolites such as polyphenols, flavonoids, terpenes and alkaloids each having a different mode of action.

Plants produce these compounds within the organs and cells where they will have the most biological effect. The epidermis is the outer most cell layer and is the first physical barrier between the plant and the outside environment. The epidermis can be comprised of at least three cell-types that include the pavement cells, the guard cells and trichome

cells.

Catharanthus roseus produces monoterpene indole alkaloids and the biochemical elucidation of the pathway to synthesize MIAs has economical value because their medicinal properties. The epidermis is the site of synthesis for at least 6 biochemically characterized genes involved in MIA biosynthesis and at least two other characterized steps are found within the leaf body. Elucidation of which biosynthetic steps that are localized to the epidermis would aid in the cloning and functional characterization of these enzymes as well as having a better understanding of how the biosynthetic pathway has been spatially separated within C. roseus.

Outline

This thesis contains a list of references and three chapters, beginning with a literature review on metabolic pathways of monoterpene indole alkaloids and its regulation and cellular localization. Followed by a chapter that demonstrates the i'-"V-t':-tji

'<> ^v^ym > 'u jfii.

hnu t'ui'.x. %r! '<-.;|;* ?/ "Y vtAij' -jU. iv!j.. .,r '• '?^' ii>'' '-'

> 0'

.• 's r ^ '•'M 'S 'u vf'itu, :.; :v:JiJtM V, Vfi

•uik!;

!;; :,r' il -V/'ift , /•.:?!

vj r;,. •« ,; ,:|-.-V

I";? .(;».''>.. -jf^ 'U,'

;., -tlh;' [,V ) il ^i!^ iL ' (i */'/y

*!; ft.'';^<''"W J'^'IJ'' 3^iJ Ji'f: f'OX'in.. >i''Iirii -6i«X>;*>|)'»ftf>rrj >.».-,'/;:;, ',-;;)'< *

i;7l;: ^^

/'i ;' .)>-.»»:) ?'i ik >;^.» "''>! Iv ;:»/-»

.^';i-^;?f \ii''i'i>Vvit^\Ci t- i\A-: '•xi i".•^\:^^<-\'X:^ •'AT

!i •'•>»'(. • /G' Mi :^.7 •Ji50*i^^^U/f'l M<^ V(>(( i»lib'(i;;*-.-.iji.,'Uf V' ., . I y*. i'

'U-')- ) ,:,!• , -(Ji

iT.j,

i. <*r,'-..f> »'. ! J,

'./•i'A'- '(-iO'i ' ;i, bti. biochemical specialization of the epidermis of C. roseus and an additional chapter illustrating the spatial separation of iridoid biosynthesis and the putative role of iridoids other than as metabolic precursors of monoterpene indole alkaloid.

Chapter 1 is a literature review describing the spatial separation of the monoterpene indole alkaloid biosynthetic pathway, emphasing the coordinate regulation of the methylerythritol 4-phosphate, iridoid, and monoterpene indole alkaloid pathways.

Chapter 2 is a manuscript that was published in The Plant Cell in 2008 and describes the use of carborundum abrasion technique to produce an EST library of epidermal enriched mRNA. This library was produced by Dr. Jun Murata, a post-doctoral fellow from the De Luca Lab. The library was sequenced and the bioinformatics were analysed at the Plant Biotechnology Institute Saskatoon, SK under the supervision of

Larry Pelcher and David Cramm. The computational modelling of loganic acid methyltransferase LAMT was performed by Dr. Heather Gordon Brock University (BU).

The scanning electron micrograph was performed by Glenda Hooper (BU). The LC-MS was preformed by Tim Jones (BU). The cloning and biochemical characterization of

LAMT and the biochemical analysis of the triterpenes was performed by Jonathon Roepke

(BU).

Chapter 3 describes the spatial separation of the early and late iridoid biosynthetic pathway. The genes involved in the early biosynthetic pathway are enriched within the leaf body while the later steps have a 10-fold enrichment in the epidermis. In addition this manuscript illustrates that the flowers have an alkaloid-iridoid ratio that is

70-fold less than the leaves suggesting that the iridoids have an alternative biological role other than being a precursor for monoterpene indole alkaloids within the flower. .., nn, ':- /w*..iv:-,-:*^-, ', ;!.:di;>'>fu %; n?;,i7 'iSiL'

n'i ?t» C"

• !- --. ^'

fc>;c^t.'''si Sid tan 'iTtrj -s-ii •'_/ no;? iron-, (5;'tiifTS' •>tii /i-..-:^ , ':. i*jK{

.'. *' -': ') '^; '^.f.^' ; .' .•' ;*,! - i ''Vl, a

Chapter 1- Literature Review

Cellular Localization and Regulation of Monoterpene Indole Alkloid Biosynthesis in Catharanthus roseus

Jonathon Roepke

1.1- Abstract

Catharanthus roseus: A Biological Source for Pharmaceutically and Economically

Important Compounds

Catharanthus roseus (L.) G. Don, the Madagascar periwinkle, is a perennial

semishrub of the Apocynaceae family which is grown in both tropical and sub-tropical

regions [Sreevalli, et al, (2002)]. Catharanthus produces the monoterpene indole

alkaloids (MIAs) of medicinal value, including the commercially important anticancer

dimeric MIAs, vinblastine (VBL) and vincristine (VCR), that have been used clinically

since the 1960's as chemotherapeutic agents [van der Heijden, et al, (2(X)4)]. Vinblastine

and vincristine are bisindole alkaloids derived from the MIAs catharanthine and

vindoline. VBL and VCR are produced in very low amounts in the leaf with yields as low

as 0.0002% of the fresh weight [Dewick (2004)], which may be due to the low production

of vindoline rather than catharanthine [De Carolis, et al, (1990)]. Catharanthus also

accumulates hundreds of other MIAs that differentially accumulate in roots and in the

aerial organs of the plant [van der Heijden et al. (2004)]. Some of these compounds have

medicinal properties as well; yohimbine is a vasodilator and ajmalicine is used to treat

hypertension [Dewick, (2004)].

Catharanthus cells can be engineered to be cell factories which could

accumulation high levels of these compounds, however before this can be obtained the

metabolic pathways which produce these compounds need to be explored. The cellular

localization and transcriptional regulation of these pathways are the pivotal areas of

research to study secondary metabolism within Catharanthus as they may limit the biosynthesis of these compounds. Metabolites may be present within the tissue, however $i

l^-^ ;':u ;V.

.u :.''

J ^n-i

.ri\

-i /' ,-••; -'zii or h <*?!! I I. - H:}..' , bOr/

(!*••• .-!'!'- il'iO:: _ _ ., .2 "

iha»i..o:j &,) .-.-.- >i t>';^i. .?tj«s?oqn"."> voiiJ ci^f'i.'y'iq !t;^f.'.'/ ?,'.e

"'; ''!) i.'.u'*p>.-.wv".)'> :T>!^;i/' . :'ilVi "" ?.; iJfj'iiod,:' jffi vi.,o,j<./ . /bu*" .jS

-•;". :i •! 't.o' >; if the subsequent enzyme is not expressed then this metabolite will accumulate.

Alternatively, the subsequent enzyme may be expressed, however the substrate may be spatially isolated within a vacuole or another cell type, and therefore the metabolic pathway would be halted. Simply, the substrate and the enzyme have to be within the same cell and sub-cellular compartment for metabolic flux to occur.

The goal of this literature review is to describe the metabolic pathway leading to

MIAs, emphasizing factors such as developmental and environmental regulation, limiting substrates, and cellular localization, which may limit the production of the dimeric alkaloids from Catharanthus roseus. The biosynthetic pathway will be divided into four different stages: early steps in monoterpene biosynthesis, iridoid biosynthesis, early MIA biosynthesis and late MIA biosynthesis emphasizing the vindoline pathway.

1.2- Early Monoterpene Biosynthesis

1.2.1- General Terpene Biosynthesis

Terpenes are a class of secondary metabolites which are found ubiquitously throughout the plant kingdom and are derived from C5 isoprenoid units. Isoprene units condense to form Csx moieties consisting of monoterpenes (Cio), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Csx). Terpenes are essential for the normal growth and development of plants as they form the side chain of chlorophyll, and are components of hormones such as cytokinins, gibberellins and abscisic acid [Rodriguez-Concepcion and Boronat (2002)]. Within plants there are two metabolic pathways to produce terpenes; the first pathway to be discovered, in the

1950's, was the mevalonic acid (MVA) pathway, followed by the discovery of the methylerythritol 4-phosphate (MEP) pathway in the mid-1990's [Rohmer, (1999)]. The ^

•: .. ' •) .^ir-:!! i-i'rx ;f'T:.n,j)'/m f.v. <::>. '-..;; t^>i-J > im ;.' ^.. 'v.-j rn'

*?^ ,' « '; . : r

:'(i--'li.v'-^fTf ;- ): ;..»<<, J K'. \'UV '{\t^''-.A'K tf> tit'. if 1), ' •• *?«

:•. -^ !;W -A^ . : ./';,, :„ F/Vi;3 -ifj' Lv.f. &•>'.-•.,: 'X!

"] )|J'>V-,' .'. K i r

/((-;:«.,:, /*'„]<;:, >* '.'• y . *j-f c"i /i--,< -i',|!'

I i.< ':',, ':. ;; / f^ ;

"( -•::! vt'i; j?:'^il Vi.^:i fi.,!(i'- .'" ilt.,. blTi ,x.

;U';'

'!• .-: :.i ,.ri-?'?H;nv-^. :'• .j'^Aj-u •III -. '(,

Vr\;, V ''.S

!• ,.\- >'. .\

^U lillls'V'. :j{ f*,:,;ic' /•'.• '<:•'.. J':". .• i-.iij

. »> .^>r'^•';TJ^^i,!!>;^ jP i") .(- ;i^;./;;r > a

. :--)i \i

jI< r^'o"! v3ii1 ''; -M:.!'; i;. if. ;!i.c;^.r 'iH

,'fr-"-';

vn. :v(.I'{a '•HIlA.. r»!;ii!/V ,) _^-f:,> -:-;' 'i;. ;_,/>!, i>'.',r.

)! ^!i ' -C y'-!'j(',

' I J , f.^.i ^ii; /•:( b'rr'A A\,r

U'-'Vx^'^ '/I.

pathway is present in most eubacteria and plants, but is absent in archaebacteria, fungi

and animals [Rodriguez-Concepcion and Boronat, (2002)]. Plants have two metabolic

sources to produce terpenes; the cytosolic MVA pathway provides the isoprene units for

the assembly of sesquiterpenes and triterpenes, while the plastidic MEP pathway provides

the isoprene precursors for the monoterpenes, diterpenes and the tetraterpenes. The MVA

pathway (fig. 1) is derived from acetyl-CoA with 2 units of acetyl-CoA condensing to

form acetylacetyl-CoA, and a 3'^^ acetyl-CoA condensing with acetylacetyl-CoA to form

3-hydroxy-3-methyl-glutaryl-CoA. This intermediate is then reduced to form mevalonic

acid, which is then condensed with three units of ATP to form isopentyl-pyrophosphate.

The MEP (fig. 1) is derived from glyceraldehyde 3-phosphate and pyruvate.

These two metabolites condense to form 1-deoxy-D-xylulose 5-phosphate. This

intermediate is reduced and isomerized to form 2-C-methly-D-erythritol-4-phosphate,

which is then condensed with CTP to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-

erythritol. This metabolite is then phosphorylated by ATP to form 2-phospho-4-(cytidine I 5'-diphospho)-2-C-methyl-D-erythritol, and the cytidine nucleotide is then removed to

form 2-C-methyl-D-eryrthritol-2,4-cyclodiphosphate. This metabolite is dehydrated and

then reduced to form the isopentyl-pyrophosphate. f e

"•<•'/ HB^M t>rtj "r' 'Hfe "t i'..

'>'>'', ?^•».d iMru;i*J .filJUdi

'. i; i. l>''.'-; ,/«.

('' 1

(' > 1-', ii«- . r-.i

l(l' ,»f?i! mi (it -jJ '^>»n'\(br. /

i'-:

-G bjlf^:; mro' i*f 'Tr::: sr.

i .'

C; bWltfT io!r!ri.-'f3- tt-i;

Jir' r Mevalonic Acid (MVA) Cytosol

3x SCoA OPP Acetyl-CoA Isopentyl-pyrophosphate (IPP)

Sesquiterpenes (015) Triterpenes (030)

Figure 1: Metabolic Pathway of Plant Derived Terpenes. Diagram illustrating the spatial separation and the source of the primary metabolites of the two metabolic pathways. r-

j?-^:::''"." ..;-- • .i,, ... £. v:x*r»'S- ,,»gif^fr

i A H HCG J

^ft'f.WBf-

t .i

<*- 8

The pathways for terpene biosynthesis converge at isopentyl-pyrophosphate (IPP).

The pathways are spatially separated as the MVA pathway is found within the cytosol,

while the MEP pathway is found within the plastid. It has been suggested that there is

cross-talk between the two pathways however it is estimated to be below 1% under physiological conditions and the rates of cross-talk seem to be species specific

[Eisenreich, et al, (2001)]. The synthesis of both pathways is highly regulated since the production of terpenes is crucial for normal growth and development. Over-expression of the PYSl, a gene involved in carotenoid biosynthesis, resulted in a dwarf phenotype since

the geranylgeranyl precursor was not available to produce giberillins, as it had been shuttled into phytophene production to produce carotenoids [Fray, et al, (1995)]. The

biosynthesis of terpenes is spatially and developmentally regulated to optimize the growth and normal development of the plant.

1.2.2- Early monoterpene biosynthesis within C. roseus

Early literature has suggested that the terpene moiety is derived from the

mevalonate (MVA) pathway [Coscia, et al. (1969)]. Feeding experiments of ['"Cj- mevalonate to freshly cut Catharanthus shoots yielded an incorporation rate of 0.05%

into ['"'Cj-vindoline amongst other radiolabeled alkaloids [Money, et al., (1965)].

However, since the discovery of the MEP pathway in plants the metabolic source of the terpene moiety has been re-established. The current literature suggests that the terpene

moiety is derived from the methylerythritol 4-phosphate (MEP) pathway [Contin, et al.,

(1998); Eichinger, et al., (1999)]. The conflicting data between the old and the new

literature suggests that there is cross-talk between the two pathways. However, transcript

analysis by both Veau et al. and Chahed et al. in 2000 clearly illustrated that expression '• ' -- .•.'^'"!:,'5 ij3i>ri.:f,; • '.i^4r,ji-

\ : i.ni KIT

n :n'KV Hth > .!.'•;>-:;,(: }';:>{{ ,*^ii J.iW.Jo M •i ailt

'' »t.»i',/'' 'j] '_\\

U5 .y, -^K /<}*- -.;' (;. f>

">:«, ;;' 'tx ''O^fi

iV.rj'j r-'iij !i ,J . lO. . .> ,,

' ' ,' ^v^^ >. ,.V« ;';g '^1 . ^' '>' j V. , V. i. ,?v'' '

' '' '» -. . tnms 3f'> " ; J .''ofj --aSl

->: !•,>' ,1*. 9

of the MEP pathway genes correlate better with the accumulation of the monoterpene

indole alkaloid ajmalicine rather than the expression the MVA pathway genes. Under

these assumptions we assume that the majority of the monoterpene precursor is derived

from the MEP pathway rather than the MVA pathway. ;-.:»h '*«:*> f ^"- I~'i«cx-' D-xyhdme

1.2.3-Cellular Localization of the MEP Pathway

In situ hybridization studies have shown that three genes from the MEP pathway (1- deoxy-D-xylulose 5-phosphate synsthase (dxs), 1 -deoxy-D-xylulose 5-phosphate

reductoisomerase (dxr), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (mecs))

are preferentially expressed in the internal phloem assisted parenchyma (IPAP) cells of

the vasculature [Burlat, et al, (2004)]. This data suggests that this cell-type is providing

the terpene metabolic precursor for the monoterpene indole alkaloids. The plastids of the

IPAP cells would provide the MEP precursors as well as geraniol to fuel the metabolic

synthesis of monoterpene indole alkaloids. However, additional studies by Oudin et al.

(2007) labeled hydroxymethylbutenyl diphosphate synthase (HDS), a marker enzyme for

the MEP pathway, with an inmiunogold antibody and localized the protein to the IPAP,

mesophyll, and epidermis cells. Their data suggested that these three cell-types are

capable of producing MEP-derived terpenes, however the epidermis had 100-250 fewer

HDS's (gold particles) as compared to the IPAP cells.

1.2.4- Gene Regulation with Respect to Monoterpenes ofC. roseus

Gene expression is crucial for the regulation of metabolic pathways. Synchronized

expression is required for optimal metabolite production so that the plant may respond to

its changing environment. Methyl jasmonate is a signalling molecule involved in

pathogen and herbivory defense. Methyl jasmonate is involved in the signal transduction , '

rUi \ >

„'"\ •! i/ ^' < .,;;... . vV.

'' '*> :'.''' (,;•' ' >'' ,, '::>h.-'i j'y't i' ,;,/ "T , 'U*.'.

?'/ -\ v- .'v. , ^v.tV>

. ' '' ',,':'_ - ' " V,, »;V,i\.. , • \';\' . \ ,-".,, VV-V^

'!C ;fl ,,..( f.

- •;;'. ; .- > • .: :h'i<.n ri}

'

. ll,':-.- 1« .i..;i;,' r 'I'll ';:.>, ' . , i». /\ I

" .- .' i ', f' . . i^ )( i;? .'A' i: ; ..,: ^.

!;'-/ ' >. »:-: ii-'*- J'' (i* »! r' • ^':i/- •;i: 1j *

• '''^"'- •>'!• • r'^f bfv'i •'U:"a,.f'Hrv. r' •.' -j -.-. 10

cascade which activates the octadecanoid-derivative responsive Catharanthus

APETALA-3 binding protein known as 0RCA3. Cell suspension cultures of C. roseus were transformed with Agrobacterium to yield cell lines which over-expressed the

0RCA3 transcription factor, suggesting that the MEP pathway gene I -deoxy-D-xylulose

5-phosphate synthase (dxs) was up-regulated [van der Fits and Memelink, (2000)].

However, gene profiling demonstrated that three additional genes were up-regulated in response to methyl jasmonate treatment, namely (1 -deoxy-D-xylulose 5-phosphate synsthase (dxs), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (mecs) hydroxymethylbutenyl diphosphate synthase (HDS) and geranyl pyrophosphate synthase

(gpps)) [Rischer, et al., (2006)]. This data suggests that the MEP pathway is up-regulated in response to methyl jasmonate, however this data does not determine whether the isopentyl-pyrophosphate will be channelled into MIA's or into other mono-, di-, or tetraterpenes.

1.3- The Iridoid Biosynthetlc Pathway

1.3.1- General Iridoids Biosynthesis

Iridoids are a special class of monoterpenes that are characteristically differentiated by their highly oxygenated pentacylic ring and the presence of a glucose moiety [Jensen and Schripsema (2002)]. Iridoids have been well studied at the

phytochemical [Damtoft, et al. (1983); Breinholt, et al., (1992); Damtoft, et al. (1997)]

and biochemical level [Uesato et al., (1983); Uesato et al., (1986); Frederiksen, et al.,

(1999)] and have been used for chemotaxonomic classification of plants [Weigend et al.,

(2000); Albach, et al., (2001)]. Iridoids have three putative biological functions. Iridoids have been implicated in the loading of the apoplast to create an osmotic potential within m

My i: f; .:V.

ikfi hlD)< fV}

'V ).>!' •^ J '* A.

K.. »'.. ;((' f( •r't:.*-!VJ <:J V ^*i;i^*iv -ijj'h ; jfH:''"s .A^ >»

''>r I >!«'nr'

.fvfKn I'i.if oir.'y \o c MM "iia ^-.flwinfc'':, -j'-l \iv^ hH^dt^^

':<

,-i»«,j«,..-r :(* . to >:'.tii\i I.U>s:]-'

f(j l>"jii> ;? ifs/; ,'v.'d H/iid ,'f>iot>rii

,A-> \- 11

the phloem [Voitsekhovskaja et al. (2006)]. Additionally a select set of iridoids participate in the biosynthesis of MIAs [Thomas, (1961)]. Lastly, some iridoids have anti-

feeding and anti-microbial properties due to their bitterness and their ability to crosslink-

proteins [Konno, et al, (1999)]. Both plants and insects have exploited the anti-feeding

properties of iridoids. Some Lepidoptera (butterfly) have co-evolved with iridoid

producing plants and have become specialists [Penuelas, et al, (2006)]. These specialists

lay their eggs on the leaves of the iridoid producing plants and in doing so they protect

their eggs from predation. The iridoids produced by the plant are bitter and toxic to the

egg predator. Alternatively, some insects (Chrysomelina) have evolved to produce their

own iridoids which are stored in their fat bodies [Burse, et al, (2(X)7)]. In either situation

iridoids are acting as an anti-feedant and illustrates the convergent evolution of iridoids.

1.3.2- Iridoid biosynthesis within C. roseus

The first committed step to iridoid biosynthesis involves the oxidation of geraniol to

form 10-hydroxygeraniol (Fig. 2). This intermediate is then oxidized to the dialdehyde

10-oxogeranial, which serves as substrate for the iridoid cyclase [Uesato, et al, (1986);

Uesato, et al, (1987); Sanchez-Iturbe, et al, (2005)]. The cyclized product is then further

oxidized to form 7-deoxyloganetinic acid [Uesato, et al, (1986)], which serves as the

substrate for the glucosyltransferase that yields 7-deoxyloganic acid [Yamamoto, et al,

(2002)]. There is a branching point in the metabolic pathway as illustrated in Fig. 2. One

pathway involves the oxidation of 7-deoxyloganic acid to form loganic acid [Kanato, et

al, (2001)], which is then methylated to form loganin [Madyastha, et al, (1973)] and

then further oxidixed to form secologanin [Yamamato, et al, (1999)]. Alternatively, the

loganic acid can be oxidixed to form secologanic acid [Yamamato, et al, (1999)] and rr

!;.' '' \'\<' '•.. ', ^!; .,« , , I /^ ( iV; 'M.-

.JV. [ i;t ^:j

,) • J ':it 'i. ,|.' .!i» ;.,i/, ''-.r'- - ,i.

• vxl; V i'- -If -y ,,[ • •'.,v"

'; ri: " •in A iQ

." '^i;'j)' .I't; ;i -iL)"

<' .i>«.

:• :>-/ , ' ,(V,, • ,., ^ i/x , .J'',] :

^Si'.' ;, .^;';m • 'v, T, >.. -id V ^

'j' .• .'-';- .-< 'v. I 'v .,• -ih

f..'- '/;.(•. ii

:•>:. : xo : :..'M .i

;' -"i • :.^ ;.tr ^ >/ ., ,i-

'^ -.<' .il.".:'', ir. vii:.-/-) c^;^ (

.' " '. i _ • '>» 1 . -~{ :t'^y- >

•"^ ..' ", '; , i:-i

h'; I /.; .-if

.' .'.'' •! «;'. .. : ''i' b"-: ! X! V i.

T*-: •! : f

V- />ii; ' w. rHj/ ','";>. 12

Geraniol OMe f^ OH

G10H

OH 10-Hydroxygeraniol OH

10HGO H O ,0H "V^ 10-Oxogeraniol ' 10-Hydoxygeranial .OH ^^ H 10HGO J H^.O 10-Oxogeranial O

Cyclase

O HO N>H o^k^H

3x Oxidation ^ q 7-deoxyloganlc acid Iridoidial 7-deoxyloganetinic acid

Figure 2: Iridoid Biosynthetic Pathway. Proposed pathway from iridoid producing plants: GIOH-Geraniol 10-hydroxylase, 10HGO-10-Hydroxygeraniol oxidoreductase, GT-Glucosyltransferase 7-DH- 7-Deoxyloganin hydroxylase, LAMT- Loganic acid methyltransferase, SLS- Secologanin synthase. sr

'S*:J

u^r. irsf}

^SSS'v' - « »*o

«;'t-\

H "'\>H •JjW' v;r'i y.-HOf ,

niroJ

s- ,; ra-

r».

C

'iK.ift J^f "rtiU' "V^ .>T.1> 13

further methylated to form secologanin [Madyastha, et al, (1973)]. The biosynthetic order of the late iridoid biosynthesis (post-cyclization) seems to be species specific.

1.3.3- Cellular Localization of the Iridoid Pathway

The spatial localization of the iridoid biosynthetic genes is appears to involve more than one leaf cell type. The gene geraniol 10-hydroxylase (GIOH) that commits geraniol to iridoid biosynthesis was located in two different cell-types [Burlat, et al, (2004); Murata and De Luca, (2005)]. In situ hybridization analysis showed that (glOh) was preferentially expressed in the internal phloem assisted parenchyma (IPAP) cells that also

preferentially expressed the MEP pathway genes [Burlat, et al., (2004)]. However, RT-

PCR analysis of cells harvested by laser capture microdissection, suggested that (glOh) transcripts were also found in leaf epidermis in addition to those of vasculature cells

[Murata and De Luca, (2005)]. The result coupled with the work by Oudin et al., in 2007 which demonstrated that the MEP pathway was expressed at low levels in both the epidermis compared to those of IPAP cells suggests that the latter compartment was more

likely to supply 1 0-hydroxygeraniol for MIA biosynthesis. The last step in iridoid

biosynthesis is the oxidation of the pentacylic ring by secologanin synthase (SLS). The transcript for this gene has been localized to the epidermis of C roseus [Irmler, et al,

(2000); and Murata and De Luca (2005)]. The problem that arises is that the first step in

the iridoid pathway is found within the IPAP cells and the last step is found in the epidermis, but the cellular localization of the other 6 putative genes in iridoid biosynthesis have yet to be localized. Therefore there is an unidentified transport form that must be transported from the IPAP cells to the epidermis. '

rt

I- I , I-.. X'- .>-{ ''/. Ml -»K 'Vi'.) O

Of

I ;' ViO . !i! } M;. ,rv ,-, .-j^i- ^ V

•>;• ;,:»( I' 1 ';. ( < ry ;.'! i;),.'C'-«' i .»> i;

'.': -• 'a-"-':. {' K'fh) ..< 'M i, jyy,I

I ^a

'i: "H' f!- ..

'' -.., ';.' -^f'!'.'"! '. -•'.*

'Hi'. "';(r' J ' 1 'M.-->.':j;ti >iiv.

:" K, I ;!i/.

'ij;

! i.v?'i -rJ,,. r;

;; .-:,>^ ;,;ii . ;•,{ 4>.' J,. ; ij^;- )K ^; , '^ifr hsy.:- J", ^i^ /. ; u M'C.i '^n

:; •-! q'i-. ! ,;x, • i'i !,-

'>'.,,. ! f, -V>-^'' "ij- ::Ki1

it,^'! in* Hi./i(, .;:; V'i "/ '•/ r;y t? r

1.3.4- Gene Regulation with Respect to the Iridoids ofC. roseus

The regulation of the iridoid pathway seems to be similar to the regulation of the early terpene pathway, however, there is some conflicting data within the literature. Early reports which were based upon over-expression of the 0RCA3 transcription factor demonstrated that there was no significant increase in the transcript of glOh (the only cloned iridoid gene at that time) [van der Fits and Memelink, (2000)]. However, later reports based on gene profiling suggested that two consecutive genes in the pathway

GIOH and lOHGO had a significant increase in their transcript levels when treated with methyl jasmonate (MeJA), while the transcript of the last gene in the pathway (SLS) was neither up- nor down-regulated [Rischer, et ai, (2006)]. This data suggests that the early steps within the iridoid pathway are regulated differently than the later steps, which may or may not be a consequence of their spatial separation.

J. 4- The Early MIA Biosynthetic Pathway

1.4.1 -General Early MIA Biosynthesis

Chemical structural diversity is imperative to the diversity of biological function and activity. The MIA's for example are all derived from a common precursor strictosidine. Secologanin (iridoid) condenses with tryptamine to produce the common intermediate strictosidine [Stockigt and Zenk (1977)]. This common intermediate can provide a vast amount of structural diversity (Fig. 3) and removal of the glucose moiety produces a reactive aldehyde, which produces the driving force for the structural diversity of MIA's found in nature. The diversity found between ring arrangements seems to be

pathway and species dependent [Zhu, et al, (1990); Szabo, (2008)]. It is also evident from Fig. 3 that the position of the methyl ester denoted by (*) is variable. The methyl •«r

'^^ >V ..; >. i.-.iH't\^ ' i) '. \.\

' 'yj . ii rv'j'i „ /ti'Wf ! y i: O

JiV. •

I. -^ ••(..', -/rv, iM- !r< /r- i,: :,id! !

,'i;,"., I

,:l r ' * ^:?0I b

I j!"- ''•V-i ir '

'«'-, " ^.< ..: 5^: b ion -«

')^i>.h

' "" ; , 1 .' 1^:^ iJ.SC. ..

V.iA^ \':

' 1. 1. : r ,. . ;y n'

>> I' .1 1 /!• H ;. : x'T

1^,'' :jl1f>- -JiWii

' ' •!' 1 '. .,i)t' 1 I,.

'••'.-rr .;i

;^ .-(..v -v ^o;i' .vH 15

ester can be used as a reference point to illustrate the spatial rearrangement and variation

of the terpene moiety. The vincosan-type is the simplest form. There is no rearrangement of the strictosidine backbone. The vallesiachotaman, corynanthean, strychnan and aspidospermatan skeletons differ in that they are derived from an isomerization of strictosidine. The other five classes differ, as they are a resultant of bond breakage, rearrangement and reformation of the terpene moiety. The ebuman and the plumeran skeletons are derived from a common intermediate, while those of the tacaman, capuronan and heynan skeletons are derived from another [Zhu, et al, (1990)]. The

structural diversity of these alkaloids is emphasized with their varying biological function. These compounds are produced in only a select set of plant families

(Apocynaceae, Loganiaceae and Rubiaceae) and each member only produces a subset of these structurally diverse compounds [Szabo, (2008)]. However, within Catharanthus roseus a member of the apocynaceae family, three major ring structures predominate, the corynanthean (ajmalicine), plumeran (vindoline, tabersonine) and heynean

(catharan thine).

1.4. 2-Biosynthetic Pathway of Early MIA 's

Early monoterpene indole alkaloid biosynthesis begins with the condensation of

tryptamine, which is derived from the decarboxylation of tryptophan by tryptophan

decarboxylase (TDC) [De Luca, et al., (1989)] with secologanin which is derived from the iridoid pathway described above. These two moieties are enzymatically condensed by strictosidine synthase (STR) [Treimer and Zenk (1979); de Waal, et al., (1995)]. The strictosidine then serves as a substrate for strictosidine P-glucosidase (SGD) [Geerlings,

et al., (2000)]. The terpene moiety undergoes ring rearrangement after the '

et

' 0''H,-^ri! 1^ . :!\:}.''.^'V'f. .u^i >ij!\-iic^ >( < '> .' iii'^i n) irfi--"^

i: . , '(!>

fli.fif'jV: ,'i ftt-r'.''* ., i'u. :;r>fr t^'>.:/ 'S.jiT 'Sff / l<

:•> ; h b"'-i< > 1 .' .-(. OTif

.

'./I >f 't(:(.t{ii -ylidv. •») £';>'i' 1, jli

fv';'!( ,j''^! 'i,,i;Vf •:=!,.^ >:^f^}!>j;' 'v. .. i-;i'i;/.i !./.'i.:;: •f-^ i^.•.nJ'yl hi

'L' J>>)h

b" H;< 'no:* T

"7- !i''^y .«".'^' u.. > ,.r'rJ:!i*j ,'r?^fiO'.') .t

:(• -.' Mn:}t'» :.:"Ti! :

":';(*» (,-.<.»•, ij.-"!yr!!j? ^ wisi'i.'Ti; .-; r'bK:;'';M'i,? \^ :.'.,liS'

. /'fA^j* tV'.ij.rv \ ^ n:,-^ i:\-y]

:' I ' i'"...i'?nT

*u,-.ur;:v;v, yr: nr-'iuuyiY: ' iv Mf */ ,^ ' .f'^-:-*:: ^4' mon! ^S-Mr

I 4,'.i !•: M. V

.fi L;;»5!'^'^^;'^^• v:1j 7iu;r': JH - J. J('

f ^'"^i • -jKif I ^ ,A>^ U'.'

,«..'! '^ "i,M"^''0| ;?.) *i.s .y-A' •V. !i < 5 iiH Jr^i Ai Hi

>f'i 7*Vij"'"'i '^rt/T'irji ,,J'^ .i 1€

deglucosylation to form cathenamine, geissoschizine and stemmadenine intermediates

[Qureshi and Scott, (1968)]. These metabolites are then modified to form stable intermediates such as tabersonine, catharanthine and ajmalicine.

1.4.3- Cellular Localization of Early MIA Pathway

Early monoterpene indole alkaloid biosynthesis requires the co-ordination of two metabolic pathways, the monoterpene pathway and the indole pathway. As stated above the last step in the monoterpene pathway (SLS) was localized to the epidermis.

Similarily, in situ hybridization demonstrated that both tryptophan decarboxylase (tdc) and strictosidine synthase (str) transcripts were localized to the epidermis of leaves, stem and flowers, in addition to the protoderm and cortical cells of the root tip [St. Pierre, et

al., (1999)]. RT-PCR of laser capture microdissected cells also demonstrated that both tdc and str were preferentially expressed in the epidermis of C. roseus in addition to strictosidine P-glucosidase (sgd) [Murata and De Luca, (2005)]. The transcript analysis

from the early MIA pathway is the first localization data presented in this report, where both the transcript data from in situ and RT-PCR of laser capture microdissected tissue correlate. This data strongly suggests that the epidermal cells are biosynthetically active in producing the early steps in monoterpene indole alkaloids.

1.4.4- Gene Regulation with respect to the Early Monoterpene Indole Alkaloids of C. roseus

The enzymes strictosidine synthase (STR) and tryptophan decarboxylase {TDC) are found in all organs of C roseus and the specific activity of these enzymes is developmentally regulated in young seedlings reaching the highest activity within 5 days

of growth [De Luca, et al. (1988)]. Later, it was reported that the expression of these . dr

.•«-:

n;;>i'

•: ii .!> .,1 ., -t ,1'Vi > >n' ),'.,;n;i.i

' aV vi^rH^:j

*j .' '" j'ii?.' ,.;:t Vij;''' lp;'.^>(>ir

' ; - :aa>' ) .Ut /i '« '»r;orn ;: l!r/

.I'^i !!.'ui'::.-[ rJJ

»V>.^. >u ,/'.

. .-i!,!!.'^ fll .

' ',h.'U- ,'i:tiV<- !";,.•:.; .!;> )

il'-H.-iy^j. 5^

!";<»' tl A i tr t. I '>';>„!;r, ,\J|)| ^h

.1,.•:,'.'> !^ '•' *« b

'^ /l!„-,J^J;;rv.-" '.u. "/.; •- '.,';.u.':;.t ;^,:j ^;.«t, -•;,-,.,.= :• ^^V'--.\ K.W^i.

''!_ ..•.(. 'j 5. a _j.r."fi' '•. -.i

*\^i,, ^J ; .\- vv

., '\v/.«.V. \'

'" i-'i'^ '.•(* J 'i> I'll*, if i >

• /' -' ' ' . 'f-M ij; > i 'i/ - :U'J

j,. r,, ;) •li. • ',' h^><. 1 i :«. J ;r-..; f 3< 17

Vailesiachotaman

Plumeran Vincosan

Capuronan Aspidospermatan

Heynean

Strychnan

Figure 3: Chemical structure of all the characterized classes of monoterpene indole alkaloids. Emphasizing the structural diversity of the terpene moiety and the location of the methyl ester (*). \ tr

) ; \t!. r- :.to fh>»ra0lJp')W i. y

w'~"*^ V-.

/'^ X \

/ —

! ^1 18

genes (tdc and str) was up-regulated when treated with a fungal elicitor (Pythium aphanidermatum) peaking between 8 and 28 hours post-treatment [Roewer, et ai,

(1992)]. Over-expression of the 0RCA3 transcription factor showed that the transcript for tdc, str and sgd accumulated [van der Fits and Memelink, (2000)], and similar results were obtained by the gene profiling of MeJA treated cells for tdc and sgd, however str

did not show any significant change in its expression [Rischer, et al., (2006)]. The lack of

change in the expression of str is interesting because it has been reported that transgenic

C roseus plants expressing a GUS reporter gene fused to the 577? promoter revealed an increase in the transcript levels of gus when induced with MeJA [Menke et al. (1999)].

This data suggests that MeJA is responsible for the regulation of a transcription factor

which binds to the STR promoter. Promoter analysis of 577? demonstrated that it contains a G-Box element which is responsible for binding CrMYCl, a known transcription factor

involved in the MeJA response [Chatel, et al., (2003)]. In addition, the transcription factors ZCTl, ZCT2, ZCT3 (Zinc Cys2/His2-type) bind to the promoter of both TDC and

577? and act as repressors of the methyl jasmonate (MeJA) response [Pauw, et al.,

(2004)]. The data presented here demonstrates that expression of the early genes involved in MIA biosynthesis (TDC, STR and SGD) is coordinated and responds to MeJA

treatment. These genes are directly related to strictosidine biosynthesis and it is the re- arrangement of the terpene moiety which provides the structural diversity of the monoterpene indole alkaloids and the variation in the biological activity. Treatment with

MeJA shows that the up-regualtion of the early MIA genes (tdc, str and sgd) is directly correlated with the accumulation of MIA's as tabersonine levels increased by 309%, vindoline by 111% and catharanthine by 203% respectively, as a result of MeJa treatment . ef

'} . an I"? jC ."u^ u\

t'iilfVij^,- .' Hhi ili- C'l J=^t .uHl'-h

?-:. "^ -livi ;v. vt;.-,t ;tvji,' i>i\:{J ^jls. , (>-;|i.' Win ti.

^'^ !'• '•^:-:ifi-ii ij:f^) Ir^n:)**!*)-! Ui^&sl /yd Ji v^'tjfi.. .'VJ ' i !V, ^3

.ff^Of'h !.'-• >!-. '( ,>-• V^^ \. ^isfitq /• ;

«'! ,.>•».;;:; i; jro D": ts^ i'^vSP: ^r .VVo. iO>.':!

i"ior5.-i*j ../FtlRit -u*

u- i"

'' Ol -fc

•1 -^ Ai n t-a > ' y: jtt. it>

:?!», V .' .d! ''^i/ /•,;'( a:;hf.v Vj^'OtH ^nmjVi)

'" »- .*<"- ;'' -,^ ( = ;S r.-:'-- 1»

[Aerts, et al, (1996)]. The accumulation of the transcripts increases the metabolic flux

• towards these MIA' s. •; -- ; ^. iiv -tvJrj;"

1.5- The Late Monoterpene Indole Alkaloid Biosynthetic Pathway • * tu" ;'.(.«;'

1.5.1- ; f General Late Monoterpene Indole Alkaloid Biosynthetic Pathway ! luw.J.s '

The late steps in MIA biosynthesis involve modifications to the stable intermediates produced by the re-arrangement of the terpene moiety, resulting in alkaloids such as tabersonine, catharanthine and ajmalicine. The most well studied alkaloids from C. roseus are derived from tabersonine. Tabersonine derived alkaloids are found in all organs of C. roseus, but the modifications to the tabersonine moiety are organ

specific. Tabersonine is a common intermediate which can either be converted to horhammercine, lochnericine, and 6,7-dehydrominovincinine in root tissues, or vindoline in leaf tissues and aerial organs [Laflamme, et al, (2001)]. Since the vindoline

(tabersonine derived) pathway is the best characterized pathway within the MIA's of C.

roseus, it will be used as a model to describe the spatial localization and regulation the

'- ^ : . ; v' .: i. , ^ . pathway. . T v

1.5.2- Late Monoterpene Indole Alkaloid Biosynthesis within C. roseus [The Vindoline Biosynthetic Pathway]

Vindoline is one of the monomers of the bisindole alkaloids vinblastine and vincristine which are known for their chemotherapeutic properties. Vindoline is a MIA derived from tabersonine through six enzymatic steps (Fig. 4). The first step involves a cytochrome P450 mediated hydroxylation of the indole ring, as catalyzed by tabersonine-

16-hydoxylase (T16H), to yield 16-hydroxytabersonine [St-Pierre and De Luca (1995)].

The hydroxy! group is further methylated by an 5-adenosyl-methionine (SAM) dependent m

i :f ^-'t/'iiir,; .' I'l '.'I' ', ' f'v.r.t

,.t-A^

rtr. . 'n v,<'*">\','.r. ' i

/.' . tt f'

^'1 ' ^' .,.ti?*- 'i . ;i^« iV'-fp.a J' -ir (^

>i I'.

.- '! , s; V' t/nal V . ->. ..,> ti

iU- J^ . .v

• ri>;: ri u' • TvjC"'-' f'AC*^;' ."'i.-K^ .r' 'IK

!. ,_j;;('- j'i,";:'t

- ' -i ,':i. , "V^ .;a) .'a>itT'(:-t'r; U

•;; •?:; » -i''-!; -V «)--\ . ..'! .'or.

, ';• '''' .1) ';, \j 'h'i' Oj 1 ,/!-»r"JT

-',>.',> ••,>''.i,.« • ,' •, ..'.VV..-; V -i''^- :',.iT,, ':-i'vV.;>» t.t'''':-,^-t 'V:\ . \ i."^

:,' ' '• '! . i- ..i'..r. »{ -. ' i^V-','-,]-\'\--' {U\ -^ . ..- :;(' ft" I', a'l • ':i

! ;<. ; ;- -I .-." fu.i:' > '5: !^- .>>•' ri.-vfv;:,-!

»-; /I V :- /r ":'*•' s^iti' ,i("'/:V;'>* ^' * :V.'''!..;l'»

-.iH-J'' ,^-;bvE{ ':'

,' t .,w •'; •• -li '.-.,;

(L l^/t'"- fs^u r :, ..K^M.' 20

O-methyltransferase (16-OMT) [Fahn, et al, (1985); Levac, et al, (2008)]. The 2, 3-

double bond is then hydrated by a yet uncharacterized mechanism. This intermediate is further A^-methylated via an A^-methyltransferase (NMT) that has been partially characterized and that has been localized to the membranes of chloroplast thylakoids [De

Luca and Cutler (1987); Dethier and De Luca, (1993)]. Hydroxylation at position 4, via an Fe"^ /a-ketoglutarate dependent dioxygenase, desacetoxyvindoline 4-hydroxylase

(D4H) yields deacetylvindoline [De Carolis et al (1990)]. The 4-OH is then acetylated by deacetylvindoline 4-0-aceyltransferase (DAT) to yield vindoline [St. Pierre, et al,

t: (1998)]. :: .. -fr. ii i

7.5. J- Cellular Localization of the Late MIA Pathway [The Vindoline Pathway] ,

The localization of tabersonine 16-hydroxylase and 16-C)-methyltransferase to leaf epidermis [Murata and De Luca, (2005)] suggests that this cell type is the primary site for the expression of MIA biosynthesis in the leaf. However, D4H and DAT, the last 2 steps in vindoline biosynthesis, were localized to the laticifer and idioblast cells, specialized cell-types within the leaves (Fig. 4) [St-Pierre, et al, (1999)]. The partially characterized

A^-methyltransferase has yet to be cloned but its activity has been localized within the thylakoid membranes of chloroplasts [De Luca and Cutler (1987); Dethier and De Luca,

(1993)]. The association of NMT with the chloroplast suggests that it is found within the mesophyll layer, a chloroplast-rich area within leaf tissue [Murata and De Luca (2(K)5)].

Murata and De Luca (2005) have proposed that either 16-0-methyltabersonine or the 2,

3-dihydro intermediate may be transported from the leaf epidermis to other leaf cells for jV-methylation. The transport metabolite has to exit the leaf epidermis and enter the second cell type, passing through two cell walls or via the plasmadesmata. .J j;vr--' ;f'*^*!t ,u. tH ,nuci"i; 'T!Wk5-dO o^i^i '^'vi.i.

-1 ij V

.;•'.: i"'y-\) .ili-tlr^ft- -i Hi n'v b:

•' ^ '-' ' '<'•-. •. . y.-VfJvM .r - vt. t

;- 'Mt .• ;.i\-

• I 't '.. ..fit .'1 Hl>-P ifi" i-ih^i^i ^v ^•J

J

J,'..f',\u' V \Vv \ nvuM'-o\ {JiU. V.u\ •^\\ ':i. i'-'. i^Uv .i^v:

" !i :'^:: • :,->i^ * ^tb'/; 1 ',

•lU-ur^ -' *i ' *tr;5 'b> ^ui^ 1.+., -'.^^ti' \it'^"^'^ ,jrHjJ 9Vi bfu. i-J^

''y-^u'- .iJ./i, i?- •i.f'r.tjJM tr'.: riJi'"-''s. -)U ii. *n. '(uroi ^t/^/ ./U'^;

i V- I -jni I': ^v.^'^ -/.,

' 't^'; : '< lij ) / l.'ixilfJ^iTii ,";i' y 'i < i'. JJO *V;'.;-4:> v'E '11 I.- dV.i,

' '( ;C I'f/. i") .nuj ii^

•' ui!'j.r bui; ^ -; V tf't*; v,';*^ :-'.:>, "!^i'l. :-'U •bfi''^^ l;Vfi>:'l:i :;/!'(

.;•! KH^^

:>•''!.

-.(' i ''•*•: ^: . . w*} " T* f,)r!i .f-^i;-

, •;! M »'." • '1 , .'•'rnit::rtr j!(;< . ilfi?V,!;;?i

IM i>i':cf, <;•. 21

Epidermis Secologanin Tryptamine

Strictosidine

1 1 Multiple Steps

16-OMT =< Cytosol O H3CO

Tabersonine 16-Hydroxytabersonine 16-Methoxytabersonim

Hydration? Unidentified' ^ Unidentifed Cell-Type

NMT H3CO Plastid H3C /O H3CO H .6 Desacetoxyvindoline 1 6-Methoxy-2,3-dihydro- 3-hydroxy-taberson irmy

D4H Idioblast/Laticifer

DAT

Cytosol H3CO 13V* y H3CO H,C /O Vindoline Deacetylvindoline

Figure 4: The Vindoline Biosynthetic Pathway. Diagram describing the six enzymatic steps from tabersonine to vindoline, illustrating the cellular localization of the pathway. T16H- Tabersonine 16-Hydroxylase; 16-OMT- 16-Hydroxytabersonine 0-Methyltransferase; NMT- W- Methyltransferase; D4H- Desacetoxyvindoline 4-Hydroxylase; DAT-Deacetylvindoline Acetyltransferase; ER- Endoplasmic reticulum. , . on

-'"^ .''*<,"!( '• i*;.,.H.» i'j .'iH-'':\ \ii t% .ftJr'i; ?T?vK?oi) ""srrJiv.rti'arf

" . '?•/ i-•.^^..r^^ : .( L

,'' '( »/"> Mbir ,Md) i''Ui-^.) -'>-i nt f.'

-,>--.;(: . i / .iH

4 : •?;

• i iV' V-.

:v. ' Vv .-.Mi-^! :c .y v? ; f".. ) t-

»* '\>y'. 1 >.' !. -vsVi t, V ^ .y, '.v

'^'-•0 .". '. / v'i ','..'1^ L ••. v5;..i -jU

i' '/jf!.':! '.f» ,>!l; ,r.^

-• ';! 4' .: i,f -, ^;frrit^;fj'J :<.';t

c* ^I'u;- U-tf ' ?-:( 21

Epidermis Secologanin Tryptamine "1~

Strictosldine

L I Multiple Steps

16-OMT =< Cytosol O H3CO H .6

6-Methoxytabersonin( Tabersonine 1 6-Hydroxytabersonine ^

Hydration? Unidentified' ^ Unidentifed Cell-Type

NIUTT

H3CO Plastid H3CO

Desacetoxyvindoline 1 6-Methoxy-2,3-dihydro- 3-hydroxy-taberson iru

D4H Idioblast/Laticifer

DAT

Cytosol H3CO H3CO H3C

Deacetylvindoline

Figure 4: The Vindoline Biosynthetic Pathway. Diagram describing the six enzymatic steps from tabersonine to vindoline, illustrating the cellular localization of the pathway. T16H-

Tabersonine 16-Hydroxylase; 16-OMT- 1 6-Hydroxytabersonine 0-Methyltransferase; NMT- N- Methyltransferase; D4H- Desacetoxyvindoline 4-Hydroxylase; DAT-Deacetylvindoline Acetyltransferase; ER- Endoplasmic reticulum. rs

{

>, TV;

? ti- i- O

r-^" >

O:^

tJiJit-on;

--. Jgh

"N.^ "*'**^«*«»«

1 -fh 22

1.5.4- Gene Regulation with Respect to the Late MIAs of C. roseus [The Vindoline

Pathway]

The vindoline pathway is regulated in two ways, by MeJA similar to earlier parts

of this pathway, but uniquely as it is regulated by light as well. Firstly, methyljasmonate has been shown to increase the transcript levels and expression levels of desacetoxyvindoline 4-hydroxylase (d4h) [Vazquez-Flota and De Luca (1998)], which was due to the methyljasmonate induction further complementing the environmental and developmental events that were already present. Over-expression of the 0RCA3 transcription factor showed that the d4h transcript was up-regulated [van der Fits and

Memelink, (2000)], however the transcript of d4h was not detected in MeJA induced gene profiling [Rischer, et al, (2006)]. Again, indicating that the results obtained are technique dependent.

Late vindoline biosynthesis is found in the aerial part of the plant and has shown to be regulated by light. T16H, the first committed step in vindoline biosynthesis catalyzes the hydroxylation of tabersonine to yield 16-hydroxytabersonine (Fig. 4), and

is induced by light and its level of mRNA increased between 22 and 28 hours post light induction [Schroder, et al, (1999)]. The last step in vindoline biosynthesis DAT hdiS, also been shown to be regulated by phytochrome, as etiolated seedlings exposed to light exhibit an increase in enzyme activity [Aerts and De Luca, (1992)]. Interestingly, the penultimate step in vindoline biosynthesis is the oxidation at the 4-position {D4H) (Fig.

4) and the level of enzyme activity is increased upon red light treatment and decreased upon far-red light treatment, which suggests that D4H is post-translationally modified by a phytochrome dependent mechanism [Vazquez-Flota, et al, (1998)]. D4H appears to be ;>C>

> 1 ^ ;j< ...-'^ ' M V.H ..• 'l'.'\

•i. •< 'iri j o.n..'.. -,i - i:- !•: i-;;i;iU!;yl (•-.wrf'tVj !., , -td'l

',"! I , **J ;. 'i'- -: f r'-<;-< . 7 n;,- M

'' -in'''^; .ii id' 'JJSjSrJi'i '' nv/'j.'ii, •'iO':

.'/ ' i< I- .u,.t : < i/.''(„ ^ ;" > >:»-ii m

Y,i .-(I ' I . --it! V\ ;n:>i'r'A ,r'i^ < : t;-. 'i urx/h;:! •jiim^T-Min: fi' ;, -n'' ol

Mc .') ;. ^ 'K-K^;' ./ ?- jti'v h f 'n-./',- li;

/ (-. .l;jtv-;-u,M ---^ •-.'V.vi '.>*, •>-- I?..:- ,>?'•;---* jJor]

'. '' '>' :.M ]. .j;::-ij Jf^r ;.- ' iV--U

\:. .^y/\ .(lA./]::; , 'ii ,i.j|,'

'"•' • - j' .i . : :> , t\^^'l.. : wMr : .'^:

.' i: '-'11 ',u>

'> •;, ^' 'iffsr^^ ^<,j "'i7'« uJi i;.'.!v f;/ vnir^j/.-i.;-;- I--

-' ': i;if:. '.''* li'i (' _ i-'A y.

" ,t! Zl:: ,i1' ]rf:' .'.11 V-

'>''•>_?!:< :>,, -•-jUj'ijJ-^, Oft Ci

)(' ': /U'.i . ^ \{ V

'Si-', si J I/O •'iri =(i ('.i'>ri ' r.' qajx

'»- '[' " ''J ! ii 4^ ni':

>.=' ..i'>i;.'-.r'f I(fgi5 b:>5-jR' r^

, -i-'. .'^'L!«. 1- ;o, .('"iMi 2t

the pivotal enzyme involved in vindoline biosynthesis, as it is regulated by MeJA

similarly to the MEP, iridoid and early alkaloid genes, but it is also influenced by light similar the first and last enzymes in vindoline biosynthesis.

" : '^; !/ 1.6- Conclusion and Perspectus -wwl k, . U/- iu . .c . d*; .:,y withtis ^ p ,

It is evident from this literature review on the monoterpenes indole alkaloids of C roseus that there are two factors which limit the accumulation of MIA's. Firstly, the pathway occurs in at least three different cell types, namely the internal phloem assisted parenchyma (IPAP), the epidermis and laticifer/idioblasts cells. This indicates that there are mobile metabolites, which need to be shuttled between cell types, however the

transport mechanisms and which intermediates are transported needs to be addressed. It is evident that the MEP pathway is the major biological source for the monoterpene moiety

and it is most probable that the major site of synthesis is within the IPAP cells. An

unidentified monoterpene is then transported to the epidermis where the last step in iridoid biosynthesis occurs. The early monoterpene alkaloid biosynthesis is then localized to the epidermis as well as the first two steps in vindoline biosynthesis. An unidentified

MIA is then transported to the laticifer/idioblasts where the last two steps in vindoline

biosynthesis occur. Secondly, it is apparent that the MIA pathway is induced by MeJA and regulated by the 0RCA3 transcription factors. Many genes from the MEP, iridoid, and MIA pathways are up-regulated in response to the MeJA pathway.

Future research should investigate the regulation of the monoterpene indole alkaloid pathway with respect to transcription factors and epigenetics. How does a cell become destined to be an IPAP, epidermal or an idioblast/laticifer cell, and could this cell fate be modified so that one cell type could produce the whole biosynthetic pathway for )

'(..I .-^l 1! A. ulii'ni' i"i !v»; r. ^ 'jm'xas ftjovio 7ff)

;. «-i,

;.i-(;v •;'!':' ;^'' n: i^^rn . -;i ti

:-'': ,>' '^-/.W: ":->S?V\V S^' • ^^ \

I- ''' ' '\-ur*r ."iT .ruMi.i j}tr u.^t^' . - f i'S

:'' J,~- / .-.;i lU'L! ' r'id': -'-:» ;..

.-' !»" '" - -^'^-' ^: v.i) 7i\,.' . rj

.^-i-T • '-1 , f, ^•. <->b)( ,

^'.' 'i: ..• .-.,'/: ' 'o/ ; :J^.' r , .-:;

(• ;<'- :• t ;/ ; ... uyi'M i-, •i-.-Tt r

*' ."," ,><( ' '1*^*1 ."; • !; i;' , ,"! i. I'!- . / / ., ^/

.'.I- f; .'i; •: I ":'>-.,'/ 'j: n -r V '!; f'-^afln V

,>.(•;: i<, :( ,-..-,i-

-'- - .V' - ". L"f'^.4i'

• '•.' : J ' i >:V 'A

>>' t tn-

< ,'ti-.'-/^ \uy'^r, f

ij. I" ; ^'1 y i\< •„) -\ ',,:' , !>3-

/; J ti

-< >- '1 :) "j. . 11

:-':l 'l ' ' I

i, -!i

•' i.»'jr!ih')i« 24

MIA's? Additionally, the transport form of the monoterpene moiety should be addressed, which iridoid genes are localized to the DPAP cells and which are localized to the epidermis. However, before these genes can be localized, they need to be cloned and functionally characterized. Moreover, the spatial localization of the MIA pathway within other MIA producing plant families such as Loganiaceae, Rubiaceae and other

Apocynaceae could be studied to determine if the patterns found in C. roseus are the same with respect to the IPAP and epidermis enrichment. '

.^s

,* '••^'^.'-liii •' ':l;,»t.'^ rl'j»t>cS »/;./'!(": lOfiO"! :jdJ Ut

i •?.

baa \--iiOi 'jf! >.A {-.)0. 'tmj JjrnCirm' :>1 (

vi^'."^} ii'i", ' 1 •,'.. i'jit>- 25

Chapter 2.

The Epidermome analysis of Catharanthus leaf reveals its biochemical specialization

This manuscript was published in The Plant Cell (2008) 20, 524-542 ."JS

I: 26

The Epidermome analysis of Catharanthus leaf reveals its biochemical specialization , . , , ,

Jun Murata^"^, Jonathon Roepke^ Heather Gordon^'' and Vincenzo De Luca^**

Centre for Biotechnology^.Departments of Biological Sciences'* and Chemistry', Brock

University 500 Glenridge Avenue, St. Catharines, Ontario L2S3A1 Canada; Present address: Nara Institute of Science and Technology, Graduate School of Biological

Sciences, 8916-5 Takayama Ikoma Nara 630-0192 Japan

2.1- Abstract

Catharanthus roseus (Madagascar periwinkle) is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine that are components of the commercially important anticancer dimers, vinblastine and vincristine.

Carborundum abrasion (CA) technique has been used to extract leaf epidermis enriched mRNA and random sequencing of the derived cDNA library has established 3,655 unique

ESTs, composed of 1,142 clusters and 2,513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only 4 known genes were found in the publicly available 12, 627 Catharanthus EST dataset. Several novel

MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid 0-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very long chain fatty acid biosynthesis were also located in this cell type. The results clearly illustrate the biochemical specialization -K 27

of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST dataset for biochemical and biological analysis of leaf epidermal cells is also discussed.

2.2- Introduction

Plant tissues are composed of various cell types with unique sizes, shapes and biological functions that play different roles in normal plant growth, development and reproduction. Among these cell types, the epidermis, composed of epidermal cells,

trichomes and guard cells, constitutes the surface layer of the plant that is directly exposed to the outside environment. The leaf epidermis usually constitutes a single layer

of cells that serves as a protective barrier to environmental factors (i.e. UV light, water loss, herbivory and pathogen attack). Similarly, the leaf epidermis also has specialized cells such as glandular trichomes that possess highly specialized systems for biosynthesis, secretion and/or accumulation of toxic phytochemicals to defend plants against insects and pathogens. Guard cells, in contrast, are the important physical openings that control gas exchange between the plant tissue and the atmosphere. In underground plant parts, the root epidermis is involved in water uptake from soil, where these cells also encounter a variety of microorganisms in the rhizosphere.

Recently, the developmental regulation of epidermal layer cell patterning has been extensively studied to identify various genes that trigger the formation of guard cells and trichomes from parental epidermal cells (Martin and Glover, 2007). Unfortunately, the mechanisms involved in biochemical differentiation and global gene expression of epidermal cells remain largely unknown, partly due to perceived difficulties associated with selective isolation of leaf epidermal cells which are intimately associated with s

d'r ,:,>!??, .4). ,:.i(ii i'\i(iim^-i:ti-qili'ni'< a; t'\ ": ju*j*y»f? mk v-Vl -mr^-A

.( ^i 'ii'> in.ji"i«';y .

_\,. /:;'•,?;.!' < ^^a , l'y> rM

' ->-w ri .ii

,^-.;'t -^;r,'i'-5V3 i'?

..)' . :: 'inj ; 'lO wl.'i.i dV^i-.V:'!^

;.-t ivli:'.' „:rlifiL V'.l ,3.1} I' Aii ri'JV!' • ^'-r!:

^; ?f'» .: *(: '"iiiv^i>»*id >>«rw>*^-i ...... 0 /jfi'^j.ri .ito».{i , .., . ..Kioi+'shj

I'JT?;')-. Ihrii .;^(iiii3f;.. .J-'Wif-lOi nr ,».il'xy h

/• i-,iri:t-:.or>'i O^dii rlh-j 5*,%fj 313riv, ,ii«>, .TK.tfl >*.7^»;^t< V.m'*' ft* Ii=?v!,/'/f;i

' i'-.'iSi V l('>.^ i/jii i»i.fnrtJ:,u::' 'lo

' !

»;''; 'o Ui»!. .

"''' . iiti '-Ki'i <^.'. 'ii;7,'>>i*.)') f'i. !: .' '\ P

,, ;« '^'>i,' ' >'.'fS*(i 'I^U. 'Ol. •'; ,^i(5-; 28

adjacent cells within the tissue. However, several protocols have been developed for the isolation of other leaf surface cells, like glandular trichomes which protrude on the leaf surface (Lange et al, 2000; Gang et al, 2002; Wagner et al, 2004).

Some biochemical features of epidermal cells have been well studied mainly by chemical analysis and microscopy in the last few decades. Two key characteristics of

epidermal cells include: (i) the ubiquitous biosynthesis of cuticular wax for producing the protective barrier of the surface (Kunst and Samuels, 2003; Shepherd and Griffiths,

2006), and (ii) with the exception of guard cells, the apparent lack of chlorophylls. The

cuticle is composed of wax containing a mixture of very long chain fatty acids (VLFAs), primary and secondary alcohols, aldehydes, ketones and esters, and cutin polymer esters that are primarily composed of Ci6 and Cig unsaturated hydroxy fatty acid monomers.

The extremely hydrophobic wax layer minimizes not only water loss from the plant body, but also the growth of fungi or bacteria on the surface of the plant. In contrast, the

chemistry of the cutin layer is slightly more hydrophilic than the wax layer, but its biological roles remain to be studied in detail. The chemical composition of wax and cutin layers, as well as the morphology of wax crystalloids are quite variable among different plant species, and therefore, have been used for taxonomical analysis (Barthlott et al, 1998). Apart from guard cells, other epidermal cells do not usually contain significant levels of chlorophyll, but they have non-green leucoplasts whose detailed biological functions have yet to be elucidated.

In most plant species the epidermis also plays specialized roles in the biosynthesis and accumulation of a wide range of secondary metabolites, including flavonoids, terpenes and alkaloids, as illustrated in recent localization studies (Dudareva S«U

•Si*! Kj'f ;)>; ^;'>.^. (wad ^

'• ' I«'^ .. -K

' .(fcOOi Av.v> Kiu'^!^ '''Aim

/(!

*0 •.»•! ^: (: -If;©!,* - )b ^'-r U'.

lot): . rJITi*

;n >

- ) gaol {-13 •' >!.

fofio 4;: ritibavoaa t^r:.-., .•ik

51! ,,?.iWti:y.S

'.. hyl .19V B' «4iW -afM i«t^ Jiiii!(-yli>v:ri ^Wtil V.

.tiA' !o i'H nh:

.*: nafjA -i.^V-'

'-i,;,',r*o ,tf^'

i

jf; .1- .-. r-X.: fw'ijf •ijc'!*:!;' fi/ ''f o-'(»» adi ^^' >3»"n«

ft; ';?"i;j'it.j->ii; v;j;bti' ,' 'o .^lit. ' '?t:>rW ij ^i

' '.ji.; ^::-i?.:i> :iolJi,M'r .' !•. > -n ?i '• 29

et al, 2005; Kutchan, 2005; Mahroug et ai, 2006; Murata and De Luca, 2005). In the case of Catharanthus roseus (Madagascar periwinkle), leaf epidermal cells appear to be specialized for monoterpenoid indole alkaloid (MIA) biosynthesis (Mahroug et ai, 2006;

Murata and De Luca, 2005; St-Pierre et ai, 1999). This medicinal plant is the only commercial source of the valuable dimeric MIAs with anticancer activities vinblastine and vincristine, which are derived from combining vindoline and catharanthine monomers. Remarkably, studies using in situ RNA hybridization and immunolocalization techniques showed that vindoline biosynthesis occurred in at least 3 cell types (epidermal cells, idioblast cells and laticifer cells) within the leaf (St-Pierre et ai, 1999) with early steps occurring in leaf epidermal cells and terminal steps occurring in idioblast and laticifer cells found in the mesophyll leaf layer. More recently, laser capture microdissection (LCM) and carborundum abrasion technique (CA) (Murata and De Luca,

2(X)5) were used to expand the understanding of the importance of the epidermal cells as

the primary site for biosynthesis of 1 6-methoxytabersonine from the secoiridoid,

secologanin, and tryptamine. Further studies using in situ RNA hybridization showed that three genes involved in the plastid localized 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway as well as geraniol-10-hydroxylase (GIOH) that commits this pathway to the production of secoiridoids were preferentially expressed in the internal phloem assisted

parenchyma (IPAP) of leaves. These latter studies implied that phloem parenchyma cells

participated in MIA biosynthesis by supplying an undefined isoprenoid pathway

intermediate that would be translocated to the leaf epidermis for elaboration into

secologanin and into MIAs (Burlat et al., 2004; Mahroug et ai, 2006). '- -' .V.

>'4 f!>i

"iil ,«jt-'.J iirtt R;.j1'. _ .:,. ;XJ :<*!.- ,,. .. isi;.» f !i«, (ii'/ J.

„fc: >f!:i!0*Ki<>! 'MM twuiX -m-rifV^StCiiJJ'/AOiL'tArr-dJ '{fi* ^J.PlMte/'iOid ^&>

(H;{l'/. j'«ifq^t»*i7-Mojrtf*?\ni3-&-lY,^,n;;.. >-'! t»SJiHr>J UjJ-*«iq %b n\

osMif-yfi n.Mjiffe':} JiitMslai ."jrl; m i>a'-isB-jA'3

vi^.'-t!!^;t

>.•' ..U- V. :;• ff'ictv; 30

To obtain novel genes involved in MIA biosynthesis, a useful approach for non- model systems like Catharanthus involves random sequencing and development of expressed sequence tags (EST) from cDNA libraries prepared from selected tissues.

Random sequencing of Catharanthus cDNA libraries prepared from whole organs

(Murata et al, 2006; Shukla et al, 2006) or from induced cell cultures (Rischer et al,

2006) have produced potentially valuable candidate genes for MIA biosynthesis, virtually no MIA pathway genes have been identified and functionally characterized using these approaches. These results suggest that the MIA pathway was poorly represented in the

RNA populations derived from leaf, root and induced cell cultures and that new approaches like laser capture microdissection and carborundum abrasion technique

(Murata et al, 2006, Murata and De Luca, 2005) were required to harvest cell-types specialized in MIA biosynthesis.

This report applies CA to produce a leaf epidermis-enriched cDNA library and shows that the moderate population of 3,655 ESTs (CROLFING dataset) produced by random sequencing contains essentially all the known MLA pathway genes that are

known to be preferentially expressed in Catharanthus leaf epidermis , including

tabersonine-16-hydroxylase {T16H) (Schroder et al, 1999), but not deacetylvindoline-4-

hydroxylase {D4H) or deacetylvindoline-4-O-acetyltransferase {DAT) that are known to

be preferentially expressed in Catharanthus idioblasts and laticifers (St. Pierre et al,

1999). Furthermore the EST dataset been used to identify, clone and functionally

characterize loganic acid 0-methyltransferase (LAMT) involved in secologanin

biosynthesis described here, as well as 16-hydroxytabersonine 16-0-methyltransferase

(160MT) that catalyzes the S"" to last step in the tabersonine to vindoline pathway (Levac "^

-ihj-i ^.:^ f. ' > * ^' .'r,..;-^at: l,«..'.*i/u «i ,^j>^,':w / ;;>( A l >:o i; io/ni

'0 ':r ' vt > fl.-^r^:

.< ("»<" ^ ..-;--.':; fjt^j.i^s'v tj;>-iM(^'-r. r ,r A- '.Ju «7' M^

-' '• yioT!

i; i"^^ Kvn.'"*:v i.5-' "i^oijii:)'^ .';•'. .

,'',1 ::>>,•:!

Vf .',*- ;•. • / 3;*,

/-•.-ri i.:/fi cm iiswian .'/it

"I'.i? -,., SA>j.i«.it'i^ (j:iJjjfiij';nrhs:, N; ! u i'»/t-if*amM ^^h^vv ,

' '?^ ,, .. .iJ»''-!»iji TttiV,

vi;) •^•a; :;!j,- : •! O :> 1 Tt^^l tj.

t''';v. >\-.t-yif U.\ ' :'.- ^Vi n* V) vii >v. .4'(i\^^

1 . : '\\~iv:

'^'.•j ':- ''I' ''<: ;* -'..v w./]Tuia;-;'; ..I :/»1' f»; . ;?; ;<; 31

et ai, 2008). The CROLFING dataset also contains genes involved in the mevalonic acid

(MVA) pathway, together with post-isopentenyl diphosphate (IPP) biosynthesis genes

and oleanane triterpene biosynthesis genes. The exclusive localization of the triterpenes, ursolic and oleanolic acid to the cuticular wax layer provided a basis for the leaf epidermis localized expression of this pathway. These data suggest that epidermal cells

are involved in the biosynthesis of triterpenes, as well as the monoterpene component of

MIAs. The identification the entire pathways for flavonoid and very long chain fatty acid

(VLFA) biosynthesis further demonstrated the high value of CA based leaf epidermis enriched cDNA libraries. The results clearly illustrate the biochemical specialization of

Catharanthus leaf epidermis for the production of multiple classes of metabolites (i.e.

MIAs, flavonoids, triterpenes and very long chain fatty acids) and predict the putative mechanisms for directed transport of various metabolites from the epidermal cells to proper destinations: some MIAs to laticifer/idioblast cells; the triterpenes and very long chain fatty acids to the leaf surface and the flavonoids to the plant vacuole. The value and versatility of this EST dataset for biochemical and biological analysis of leaf epidermal cells also highlights the versatility of CA as a unique tool to identify the complex biochemical pathways associated with leaf epidermal cells.

2.3- Methods

2.3.1- Plant material

The Catharanthus roseus (cv. Little Delicata) plants were grown in a greenhouse under long-day photoperiod at 30°C. Young leaves (1.5 cm in length) were harvested for cDNA library construction, and more mature leaves were also used for other experiments. iT. iO

'•(! ?i^:..i iB!tn'i?:,

-f |>^j-ji-

x'jinTft!^ >;?? y'Tijoshi U' j'voi onvtfiit » pib A'3 lUi

'• '', rii n

•.!.-ii ; ;v ., ';'i i,'^ ; -

32

2.3.2- Carborundum abrasion (CA)for cDNA library construction

Carborundum abrasion was performed primarily as described previously (Murata and De Luca, 2005), but with some modifications. The upper and lower epidermis from 5 g of young Catharanthus leaves were selectively abraded with carborundum (SiC)

(Fisher Scientific, Nepean, ON, Canada) using a cotton swab to apply even pressure. The epidermis was rubbed 8 times per side (upper and lower surface) and the leaf was then dipped in 4.0 mL of Trizol (Invitrogen, Burlington, ON, Canada) at 4°C, and gently agitated for 5 sec to release the epidermal cell content into the solution to obtain 2.5 mL of extract from 5 g of abraded leaves. The extract was then used for RNA isolation

according to the manufacturer's protocol. ^'

2.3.3- Construction and sequencing of leaf epidermis-enriched cDNA library

The Catharanthus leaf epidermis-specific cDNA library was constructed using

SMART cDNA library construction kit (Clontech laboratories. Mountain View, CA,

USA) according to the manufacturer's instruction. The cDNA was then amplified by PCR prior to the packaging to Gigapack III gold packaging extract (Stratagene, La Jolla, CA).

The primary library (1.0 xlO^ pfu) was directly converted into plasmids by in vivo

excision, and the E. coli colonies obtained were randomly picked for single path

sequencing using primers from the 5' end of the inserts. The sequencing reactions were

performed using Templiphi DNA sequencing template preparation kit (GE Healthcare,

Piscataway, NJ), and the resulting DNA templates were sequenced using ABI Prism Big

Dye terminator sequencing kits and an ABI 3730 genetic analyzer (Applied Biosystems,

Foster City, CA) - ^ • ,

-.. -^ -i >'>ni. 'y-'^'i: ..

'"» »' ^!iw?<;' rim' - * t- ,K0 V .in

' "'. f '-.It. o :;,'; 'A .-M t'l

If [!'x^;n ;; _ . 33

,, -. 2.3.4- Sequence analysis ;4 ; ^y

The sequence files in ABI format were analyzed using the BLASTX algorithm

(Altschul et al., 1997). Multiple clones with the overlapping areas of identical sequences were clustered and classified as 'Clustered', while sequences that appeared only once in the ESTs were classified as 'Singletons'. The threshold of the sequence similarity was set as E-values at 10"^ and lower, while sequences that did not show significant homology were named 'No hits'. The sequences were archived in FIESTA software package

(http://bioinfo.pbi. nrc.ca/napgen.beta/Zlogin.html) at the Plant Biotechnology Institute of the National Research Council of Canada (NRC/PBI) in Saskatoon, SK. The functional categorization was first done automatically to produce putative annotations, followed by the manual inspection to verify the reasons for the annotation. All the sequences will be available to public after certain suspension period of time according to the rules of the

Natural Products Genomic Resource (NAPGEN) consortium http://pbi-ibp.nrc- cnrc.gc.ca/en/CEHH/napgen.htm

2.3.5- Extraction and analysis of triterpenes in Catharanthus leaves

Fresh leaves from different developmental stages [120 mg of young 1.5 cm long

(Y), mid-aged 3.0 cm long (M) and older 4.5 cm long (O)] were harvested. To obtain surface extracts, leaves were dipped in 3 mL chloroform (Gniwotta et al, 2005), vortexed for two minutes and incubated for five minutes at room temperature. Surface stripped leaves were then rinsed with H2O to remove excess chloroform. To extract intact leaves or chloroform surface stripped leaves, they were separately frozen in liquid Nitrogen and homogenized with 3 mL chloroform in a mortar and pestle. The extracts were filtered through one layer of miracloth (VWR Canlab, ON, Canada) and the filtrates were «

r:^r<

'..', '' .yjCi''>-^*'> ^ '•..•'I'^tf iif m-^ii !.;e,i-i\t.,];y . .^jKv 'i>f5i:!:: ^'

)-».-. i-^t,': , i^.'.'l'iO '>l'-.:;.-'-'n Vdl .

U'l« *'

i<;( •r-'i^'OO';

' / -i

[!',- :>5^>:;W 'fH).. ..,.'., i/u.fr .yi'J^'-'V-i- i" -' ' ni ."V^\lti 'H'sW

']' •_)' :-(".'' ;> • ).' : ..Ti^iv . ;j '.'i-i-ii'; tf, /i.'i, ,'J

':...' :'i !.>;-'•' M ; -i' u . .-;vy-i ^ j .;u: •:!' -\y*i ''^[1

' -"> • :"- I'f' ;»^-, I r. • ' . "is' [ ;i _:.,,,..- '. 'Tr ' 'i. ,.,_i\L', > :(fl 34

transferred to 15 ml conical tubes together with 3 ml of water. The samples were mixed by vortex treatment (Genie 2 vortex set at 10, Fisher Scientific, ON, Canada) and then centrifuged at 5,000 x g for 10 min to separate the phases. The chloroform component was harvested and evaporated to dryness by vacuum centrifugation in the SPD SpeedVac

(Fisher Scientific, ON, Canada). The dry residues were resuspended in acetone (300 //I);

2 fi\ were spotted on Machery Nagel Silica Gel G thin layer chromatograms (Fisher

Scientific, ON, Canada) and submitted to TLC using ethyl ether:petroleum ether (9:1) as solvent (Wagner et al, 1984). Chromatograms were sprayed with Vanillin-phosphoric acid reagent (VPA: 500 mg Vanillin dissolved in 50 mL of 50 % phosphoric acid) and developed over a hot plate for 5 minutes until triterpenes could be visualized. Ursolic acid levels could be determined using an Ursolic acid standard curve.

2.3.6 Capillary HPLC MS analysis of triterpenes

Ursolic Acid Standard (Mr 456) (Sigma Aldrich, Toronto, ON, Canada) and

Catharanthus roseus triterpenoids (crude extracts or samples partially purified by TLC) were submitted to analysis using a Bruker HCT+ LC/MS System (Mode:ESI-Negative

Inlet Agilent 1 100 LC; Solvent System: AcetonitrileAVater; Column: 50 mm Cis with a pre-column guard). Both crude and partially TLC purified triterpenoid samples displayed similar retention times (RT 16.0-16.2 and 16.4-16.6 min) and identical masses (455.3 m/z) as authentic Ursolic acid standard. This verified that the major triterpene of

Catharanthus surface extracts was ursolic acid as suggested in previous studies with

whole plants (Usia et al., 2005). ;nr. < Li".'' 35

• - 2.3.7- Scanning electron microscopy ' .. ;^ • 1^

The images were obtained using an AMRAY 1600 Turbo scanning electron

"., .'.,».''« microscope as described previously (Murata and De Luca, 2005). > . t .

2.3.8- Laser capture microdissection for the isolation ofRNAfrom single cells !!<;.;

Catharanthus roseus leaf was fixed and embedded in paraffin as described previously (Murata and De Luca, 2005). LCM mRNA isolation and amplification were

'. performed as described previously (Murata and De Luca, 2005). !f , ,. - ; *>

2.3.9- LAMTfull-length cDNA cloning and protein expression in E. cell

The putative LAMT full-length cDNA sequence (Accession #EU057974) was assembled in silico from 12 different ESTs to produce CL57Contigl. PCR primers

(LAMr 5 MrG-5'-CACCATGGTTGCCACAATTGATTCC-3'; LAMT 3'UTR- 5'-

TTAATTTCCCTTGCGTTTCAAG-3') were designed to amplify the putative open

reading frame based on its similarity to known carboxylic acid methyltransferases. The

PCR Conditions used were as follows (20 |iL Rxn Volume containing Takara- Ex Taq

Polymerase and a Template composed of SMART cDNA from the leaf base of

Catharanthus roseus; Denature- 15 sec 94°C; Re-anneal- 10 sec 55 °C; Elongation- 40 sec 72 °C; Final Extension 5 min 72 °C). Since the coding sequence of L«Mr had 5

restriction sites (Eco RI at 320 bp, Eco RI at 549 bp. Sac I at 640 bp, Xmn I at 969 bp, and Bam HI at 1003 bp), the open reading frame was inserted in the expression vector

pET 30b (Novagen) using Nco I and Sac 1. For this reason the original PCR primers also

contained Nco I and Sac I sites that were used for the subcloning.

The amplified PCR product was ligated into pGEM-T Easy (Fisher Scientific,

ON, Canada) and the ligation mixture was used to transform heat shock competent DE3- •^

^Wv 3t.t«'.»iW »i

.ftrOO?: jvyj.J '^J hfit. rjnMih'

VX^i

' ' '•• \.'^ xJ -gii.:'S;T - ''>V a;»,K '

Oi

Kijij':/' T/^

>.) ;'j! •iV.-i

JCi.

'/ ,?:!)'^f' : , I -i "iO o ?(^' !"i' jt,^•ll ) - , . . T-v ti-;i;jXj;., n<5,.: 36

MRF' E. coli cells. Cells were grown at 37 °C for one hour and then streaked onto LB agar plates with ampicillin as the selectable marker. Plates were incubated overnight at 37

°C. Positive inserts were confirmed with colony PCR using single colonies as a template and the primers LAMT 5' ATG and LAMT 3'UTR. The MMr insert was excised from

the pGEM-T Easy vector using the restriction endonuclease Sal I and Nco I (Fisher

Scientific, ON, Canada). The protein expression vector was digested with the restriction

endonuclease Sal I, Eco RV and Nco I (Fisher Scientific, ON, Canada). The vector and

LAMT insert were ligated with Takara DNA Ligase (Fisher Scientific, ON, Canada). The ligation mixture was used to transform heat shock competent DE3-MRF' E. coli cells.

Cells were grown at 37 °C for one hour and then streaked onto LB agar plates with kanamycin as the selectable marker. Positive inserts were verified with colony PCR using individual colonies as a template and the primers LAMT 5' ATG and LAMT

3'UTR. The plasmids with positive inserts were sequenced (Robarts Research Institute,

London, Ontario, Canada). The original sequence from CL57Contigl contained a stop codon in the middle of the open reading frame that turned out to be derived from

sequencing error (data not shown). The cloned 1 11 6 bp LAMT coding sequence encoded a 371 amino acid ORE with calculated molecular weight of 42,018 Da.

2.3.10- Enzyme assays for recombinant LAMT (rLAMT):

The standard radioactive enzyme assay (100 \iL) contained 1 fig of crude desalted rLAMT protein, 2.5 nCi S-Adenosyl-L-['''C]-methionine (Specific Activity 58 mCi/mmol; GE Healthcare Canada), 0.5 mM Loganic acid, and LAMT enzyme assay buffer (100 mM Tris-HCl pH 7.5, 14 mM /3-mercaptoethanol and 25 mM KCl). Enzyme assays were incubated at 37 °C for 30 minutes. Assays were immediately frozen in liquid "> ,, . . , -. .,, hjij;- .'flit 'n"/? T' ji , „ .,:ii«i': ir>;l) i!i'v!

'^ TtVjAJ. tKJ.s OTA TMAJ i*'tii«!«

'v-'^. r. ir'jMiiai.;.'.! !»>}?Ko'>r' l3i!aoi'? =>?;(jrip'>"-' Ib-fiij^tr.,' »*ifr .!«!>;

;. . . I.i (^ :

•' '°; :-^ '.\<

nitrogen and lyophilized overnight. The dried material was resuspended into 15 (iL methanol and centrifuged at 10,400 rpm for 10 minutes in a ThermoIEC, Micromass RF microcentrifuge (Fisher Scientific, ON, Canada). The supernatant was directly added to a

0.20 mm Polygram® Sil G/UV254, Macherey-Nagel TLC plate (Fisher Scientific, ON,

Canada) and the reaction products were developed with Chloroform:Methanol (7:3). The radioactivity was detected by exposure of the TLC to a storage phosphor screen (GE

Healthcare Canada) for 16 hours and the emissions were detected using Phosphorimager

FLA-3000 (Fujifilm, Tokyo, Japan) with Multi Gauge ver. 3.0 software (Fujifilm, Tokyo,

Japan).

The standard non radioactive enzyme assay (100 jiL) contained 4 \ig of crude desalted bacterial protein with rLAMT, 7.5 mM AdoMet, 1.3 mM Loganic acid, and

LAMT enzyme assay buffer (100 mM Tris-HCl pH 7.5, 14 mM P-mercaptoethanol and

25 mM KCl). Enzyme assays were incubated at 37 °C for 30 minutes. Assays were immediately frozen in liquid nitrogen and lyophilized overnight. The dried material was resuspended into 200 ^L of 0.5 % Formic acid and 50 fiL of acetonitrile. Samples were centrifuged at 10,000 xg for 10 minutes. An aliquot of 2(X) |iL was then filtered through

(0.22 urn) PALL filter (VWR Canada).

Reaction products were analyzed with UPLC™" (Waters Canada). The analytes were separated using an Aquity UPLC^"^ BEH Cig with a particle size of 1.7 \im and column dimensions of 1.0 X 50 mm. The solvent systems containing A (0.5% Formic acid) and B 100% acetonitrile were used to form a linear gradient [Time 0-2.99 min (0.1-

30% B), 2.99-3.50 (30% B), 3.51-4.00 (30-50% B) and 4.01-5.00 (50-0.1% B)] at a flow 1

;..;';' '. ' v^l-y T: '"M ::[ < jnu J-;. J . , hw hat fij^i'ymn

V . -r-y l*i r:

;' ' ( '.,.1 -j&f. ';»(.,." SVw.IJj' ;/!, i i iV>l'. !

' " ;0\\,:--: , •- »v ''

•'*, ,•!'.••:•- ^' I;.' ^. i V. ., .! rvi

!•"' nu (1.; wv. i-fV ,i, '.' ,'<

-O'V ,rr^<) '>.>.'.>&! jU V-.v- -o-v-- i( ^,1- ./ :?

i'.i' M , YV-, fi '.,u; , l^'^:-fM;Ji;'

'li, a- ^:'»-:.f ; .!; <>-V! •TV v'. i' ...jd ,,P. '^f

» < I IV^j. :-: (•;• M r- y ,,

1 . •I i . .•: >':. ^(ttysn^

i*; s

»< > ). r;-(';;. ^r i ;t'';i.n3V' -i. \;;s---:'.y' .jr

I ' 'I a\',)'\ t\ 1; ' :;. VV'i

4A ';. -.». LHJJ.O)

' .« -'"v :^>('- .'-r r / J ;

'-' I- y ..fi;., -1' ./ , ;..; ; ^.^-:-:r

' I . '': ! '^'l 0"v!'^ 'li.c AJMU

it i '.':.

I 1 K, ;?nn( ^

/i.ii .'M I -J'' 38

rate of 0.150 rtiL/min. The reaction product detected at 240 nm eluted with an identical elution time and uv spectrum as authentic loganin (retention time 2.2 min).

This reaction product and the loganin standard were analysed using a Bruker

HCT+ LC/MS System (Bruker Canada) (Mode:ESI-Negative Inlet Agilent 1 100 LC;

Solvent System: AcetonitrileAVater (0.1 % formic acid); Column: 50 mm C\s with a pre- column guard). The reaction product displayed similar retention times (RT; 8.6-8.8 min) and identical masses (445.1 m/z) as authentic loganin standard. f

2.3.11- Computational Methods for LAMT sequence alignment and modeling of the active site

ClustalW (http://align.genome.jp) was used to produce an alignment of JAMT,

SAMT, LAMT and JAMT using a gap penalty of 10 and a gap extension of 0.1. The protein alignment was then submitted to SWISS-MODELLER using the alignment interface http://swissmodel.expasy.0rg//SWISS-MODEL.html (Schwede et al, 2003;

Guex, and Peitsch, 1997; Arnold et al, 2006). The software predicted the crystal structure of SAMT PDB# 1M6E.

Using QUANTA 2005, (Accelrys, Inc., San Diego) three-dimensional all-atom models of both S-adenosyl-L-methionine and loganic acid were built. Partial charges on the atoms were assigned using charge templates of QUANTA and the charge was

smoothed over all atoms to give a total charge of -i-l for SAM and -1 for loganic acid.

The initial conformation of SAM in the model binding site of LAMT was obtained by superimposing atoms onto the corresponding coordinates of the S-adenosylhomocysteine in the crystal structure of salicylic acid carboxyl methyltransferase (1M6E) (Zubieta et al,

2003). Two different orientations of loganic acid within the putative binding pocket were m

^>i'Vt:v%\M i -.A-f ""^ih^: iTflf! (>f ': tii L'.i yy h

• )V >-' i^Q ii ,1 ivn ..> . i

r<-,>r'>;»v :(d ! .'J^iao;^ .'!(;';!•

;im'!n'.»in"'t'.ivi •i>«vr>,.'.:-,i, \V>/',.' /'* 'V«-^A '. oV.;,\hi.\!"'.v,'>

i')3S)r.n,^Ir, m; '^mb ,'V-t>j sjG.i; ^;; Jw !'/ :Jrv ;.,>!•' ;J;ii?'/>'.'r}Ji! '/Vlsi^uiO

»(i^-ftfr;;tiK; aut jJiuKtr il^UJ^'.i;'^^ <*f^1!V/*-: :,». L^-jLu{.it- f.-ji-? .-•rA,' Jot;

''^';f. tf.j»'V.'i<> Mii (WJ3i&-".u< s->». *$>>« -J^f-f ,.ik. V--. -•:'0iT;/, .\'V'-:';

rni/fi.

/(*

V -^Jcn'! , ,.;< y. 'li.'- ^ip

.'^ >K c c:

' :'' n -i-a ^n;t(!Tri •>-'iu??!): v:! . . 'u^iu'jiWj-.l ^u ^m-^hi-y-'^ if\i 39

obtained by superposition of the carboxylate, Cq and Cp of loganic acid onto the J

coordinates of the substrate salicylic acid in the 1M6E structure. The hydrogen atoms on .

the LAMT protein were generated using the H-build algorithm of CHARMM 31.1. i

; . (Accelrys, Inc., San Diego; Brooks era/,. 1983) ^.j

The CHARMM 31.1 algorithm was also used for all computer simulations employed to locate low-energy conformations of loganic acid in the putative binding site of the model for LAMT. Non-bonded interactions were smoothed to zero beginning for

atoms whose centre-to-centre distance was greater than or equal to 1 1 .0 A, with a final

cut-off radius of 14.0 A. Non-bonded neighbor lists were employed, for which the radius was set to 15.0 A; the neighbor lists were updated using heuristic methods (Fincham, and

Ralston 1981). All energies were computed in vacuo. Covalent bonds to hydrogen atoms were constrained using the SHAKE algorithm. (Ryckaert, et ai, 1977) During molecular

dynamics simulations, a time-step of 1 fs was used.

An initial energy minimization was done of both AdoMet and loganic acid in the

binding site. The atoms of the protein were fixed and unfavorable interactions between the protein and the cofactor and substrate were relaxed by a sufficient number of steps of adopted-basis Newton-Raphson (ABNR) energy minimization so that the final root- mean-square-gradient (RMSG) of the potential fell below 0.5 kcal/(mol A).

Subsequently, constraints on the protein side chains were released and another round of

ABNR energy minimization was done until the RMSG was reduced to below

0.4 kcal/mol A. In order to allow the system to overcome energy barriers and explore nearby low energy conformations, quenched molecular dynamics runs were done starting from these energy-relaxed structures. The backbone atoms of the protein remained fixed ., ::.Pi>;,' ' ::.;.', ^••,;' ! :;/;'. it Ka/'Mi y^^jjii^ Iftilini r,'-

'{- i': ><. !)- " (- ^j . A^':. V\:\ A\\ii' . /'f Ij,,. i i : _,::t ^\a.' d.; ; 'jlC ;0/ v«^'K

r-^U :.,;:\ -fi j|„4i "' '!» ;;r :U'v^;f3'

'/, ''' ' '• '-.: >^^ i U d 4 :r\ 'rr ya< rh y/.i/V^ 40

during these simulations, amino acid side chains in the active site as well as AdoMet and loganic acid were allowed to move freely. During the initial 5 ps of computer simulation, the system was heated to 500 K by rescaling the velocities of unconstrained atoms and increasing the temperature by 5 K every 0.05 ps. Subsequently, the system was allowed to equilibrate at 500 K for 100 ps, during which time period the atomic velocities of unconstrained atoms were rescaled only if the average temperature fell outside a

temperature window of (500 ± 10) K. Conformers were saved every 1 ps. Subsequent energy minimization of these 100 conformations comprised the quenching stage - the

ABNR minimizations converged with all RMSG of 0.001 kcal/ mol A satisfied. This resulted in 100 low energy conformations that were analyzed for both interaction energies and hydrogen bonds between loganic acid with the protein.

2.4- Results

2.4.1- Construction of leaf epidermis-enriched Catharanthus cDNA library

In order to study the biochemical specialization of Catharanthus leaf epidermal cells, CA technique was used to extract mRNA (Murata and De Luca, 2005) for producing a leaf epidermis enriched cDNA library. Briefly, the upper and lower surface of young Catharanthus leaves (1.5 cm in length) were selectively abraded using a cotton swab coated with carborundum particles, and the leaf was dipped into Trizol reagent to release mRNA from leaf tissue. This RNA was then used to make a leaf epidermis enriched cDNA library and 8,527 colonies were randomly chosen for sequencing to produce 3,655 unique sequences composed of 1,142 clusters and 2,513 singletons. The

average length of unique sequences from this library was 43 1 .5 bp after removing low quality clones and vector sequences. Genes were annotated accord to their putative ,»••- :i;;. -vM:

' '5'' '; ; '•r-.tvi *: .; v/

•^ f..y

•' y^:. U'i

..^•'" 'iv'ti . V ,K V, •lA' toOL-M

'' ,' .;' - iV l>' J. "I'i ;• .V 5fluii;

';.•! d: "'i ••..•?'' u, --ii-,' f,( ,r,;

;iv: -;.* ( i;! • ^^K i:i:\;r'\i ;

•• ..'. -i. Jv.v. ' I ^. •ir>- ' ' !v^. vi:-,-iv >r< i 'Oi ,,i uit

tJlr?

<»; ", "'•< . \'i !• ^vr'' .'.Vi' -..' r*i.

- A«> ,. ^ '^i (,r:;-:.... i : / - : >*v 'yi-:;. ;>.! :$tno«J'>tnOi

'.. L .J ,in'_. ' j,,h..;

'*" '. -'->- !.' i 1 ii' r'i I: ,'}'-t,,'' ^.vA-'M

>. : • •;;,.>• ;, r( ! w :>:i.;;;i HT

;!.r-i' is^!-

i .- <"

'»/--0 . il .. '' rjt j!!;v-"^>

jiji .'• (•,. ^'}(loi^J> ''': ".f, by *sii* F.U:Sl'A jT^u H;vi,jy!tio« sysiea.

:/! y- . K M,.J- h'liKJ-

f^;*«•-

#1

functions based on their similarity to sequences in the GenBank database using the

BLASTX program coordinated by the FIESTA gene annotation system

(http://bioinfo.pbi. nrc.ca/napgen.beta/Zlogin.html) developed at the Plant Biotechnology

Institute in Saskatoon, SK, Canada. This dataset was named as CROLFING according to rules developed by Natural Products Genomic Resource (NAPGEN) consortium

(http://pbi-ibp.nrc-cnrc.gc.ca/en/CEHH/napgen.htm) at PBI. The validity of putative annotations was checked manually through the FIESTA system.

2.4.2- Data mining of the CROLFING dataset and categorizing the genes of interest

After annotations for each gene were verified manually, they were categorized

into genes involved in the biosynthesis of (i) MIAs, (ii) isoprenoid precursors, (iii) other terpenoids, (iv) flavonoids and (v) lipids. Since many MIA biosynthetic genes remain to be cloned, additional genes encoding certain classes of putative enzymes were also annotated, including (vi) methyltransferases, (vii) acyltransferases, (viii) cytochrome

P450-dependent monooxygenases (CYPs), (ix) glycosyltransferases, (x) dioxygenases

and (xi) transcription factors. ;

2.4.3- The CROLFING dataset contains virtually all known MIA biosynthetic genes.

Detailed analysis established that the CROLFING dataset was a particularly rich source of genes for MLA biosynthesis, based on the representation of most functionally characterized genes, from the pathway for secoiridoid biosynthesis to the early steps in the conversion of tabersonine into vindoline. This included 10- hydroxygeraniol oxidoreductase (lOHGO), secologanin synthase (SLS), cytochrome

P450 reductase (CPR), tryptophan decarboxylase (TDC), strictosidine synthase (577?), strictosidine Ji-glucosidase (SGD), tabersonine 16-hydroxylase (J16H), octadecanoid- "'* : A'' f- r J") b-

.'I '; :-'•

h;^>i-/: ':- ^,

' •. -;...... ^^"V. ^,. -..A , .[ ^ „ , v.v: , A. ^

•;;»•.<-. .',\>:VM . .rH'l'lf ,/ «s^,.-^ -\ iS. -''^v>t'.; 'v«iV»»V,» . 42

derivative responsive Catharanthus AP2-domain3 {0RCA3), box P-binding factor 1

(BPFJ) and zinc finger Catharanthus transcription factor 2 (ZCT2) (Fig. 1 and Table 1).

It was quite significant that SLS, TDC, SGD and TJ6H which have been shown by various localization studies, to be expressed exclusively in Catharanthus leaf epidermal cells (Irmler et al, 2000; Murata and De Luca, 2005; St-Pierre et al, 1999), were also identified several times in the CROLFING dataset. This is particularly striking with the

SLS contig (CL19Contig4) which is represented by 25 ESTs and is considerably enriched in the leaf epidermis. These results dramatically show the effectiveness and the value of

CA technique in constructing a leaf epidermis-enriched cDNA library and the specialization of this cell type for MIA biosynthesis.

The representation of functionally characterized MIA pathway genes in the

CROLFING dataset also strongly suggests that other novel and uncharacterized pathway genes are very likely to be represented. For example, there are four different clones that belong to the CYP72 family of cytochrome P450 monooxygenases (Table 1). Since these genes have sequences that are extremely similar to (over 95% identity at the amino acid level), but slightly different from SLS, they may encode 7-deoxyloganin hydroxylase that preceeds SLS (Irmler et al, 2000; Yamamoto et al, 1999; Yamamoto et al, 2000). The functional characterization of these candidate genes will help to complete our understanding of the terminal two steps in secologanin biosynthesis in Catharanthus, as well as in other species of plants that accumulate these secoiridoid compounds.lt is interesting that a single EST for geraniol 10-hydroxylase {GIOH) (CAC80883; Collu et al, 2(X)1) was represented in the CROLFING dataset as well as two additional CYP

ESTs with significant similarity to a gene annotated as GIOH (CAC27827; Meijer et al. 1

'}-; . / . )V, t

\'i ' ', '< , >vs-'^\ ;rtv; k'rt ^'^i

1' r; -,- 'J :• 'A I

«<:. !!,•%! /S-.-:- .'•:.'}• \>-

V; ;'t,.^ '».'..

( i,: . :/l •I"-. './3> ;.-M.'5»r:

>f' '; ' !'"'! i I •I' '-*';

1.'. in;'.'/

-d..-7j /

-•' ..,>;'(. i ' Hi ( J»j

'jf? .,., -W

' • • et: .r, ^' M-'

! ... f!'

' t . ;i lo :^i;"..

•'^ . ft^'i.i

f' -,i-i

., - 1 i ,

••'' , i I. 43

1993), but whose biochemical function has yet to be described. However, this clone

(CAC27827) is not likely to catalyze the GIOH reaction since it was more similar in sequence to other CYPs, including T16H, than to the functionally characterized GIOH clone (CAC80883). In general, the ESTs that encoded MIA biosynthesis enzymes were represented several times compared to those of the three transcription factors {0RCA3,

BPFl and ZCT2) (van der Fits and Memelink, 2000; van der Fits et ai, 2000; Pauw et al,

2004), that were only represented once. These relative differences implied that transcription factors were expressed at lower levels than those of the MIA pathway enzymes that they regulate (Table 1) and that the overall profile of mRNA expression in the leaf epidermal cells of Catharanthus was largely maintained in the CROLFING dataset.

2.4.4- The CROLFING dataset contains the genes for the mevalonic acid and triterpenoid biosynthesis pathways.

It is well known that plants possess two different biosynthetic pathways for making isopentenyl pyrophosphate (IPP), the key building block of all isoprenoids including pigments (chlorophylls and carotenoids), phytohormones (gibberellins), sterols

and other terpenes (Lichtenthaler, 1999; Rohmer et al, 1993). It has been suggested that the cytosolic (MVA) pathway produces precursors for sterols, triterpenes and polyterpenes, while the plastidic MEP pathway supplies IPP precursors for chlorophyll,

carotenoid, phytol, monoterpene and gibberellin (Lichtenthaler, 1999) biosynthesis. It is remarkable that one MEP pathway EST (4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; MCT), four MVA pathway ESTs {HMGR,AACT1, AACT2, HMGS) and three - '"', > ;> •!. z^'" !"i<'>'' ; \.H-i'

:i; !.,,<«;(;• ."I ' .. " ;v .i"v

' ;'- J ,:>; d .'v''^'* '/ ^.

Ivr'

I n H .• . : ? 'M i; -!. >i ,• ..(•X(L^ 44

Putative numbers of Enzymatic steps

Glyceraldehyde y-pnosphate

DXS 1-Deoxy-D- xylulose -5-phosphate m *DXR •0 2-C-methyl-D-erythrilol-4-phosphate(MeP) Vascular cells I DIphosphocytidyl -2-C-methyl- and/or D-erythrltol-2-phosphate 9 Epidermal cells IMECS 2-C-methyl-D-erythritol- 2,4-cyclodiphophate (MEC)

Isopentenyl diphosphate (IPP)

i T Geraniol idOH 10- hydroxy geraniol 4-10HGO Loganic acid ILAMT Loganin Tryptophan ISLS Itdc Secologanin Tryptamine

I I STR -15 Epidermal cells Strlctosldine ISGD Cath^namine

Tabersonine IT16H 16-hydroxy tabersonine

I 160MT " "TBHTTetboxyla be? son ihe" Mesophyll cells? 1 6-methoxy 2,3 -dihydro tabersonine V Desacetoxyvindoline idioblast/Laticifer t D4H Deacetylvindoline cells i DAT Vindollne

Figure 1 : Vindoline biosynthesis and its proposed localization within the leaf in

Catharanthus roseus. Among -30 enzymatic steps involved in vindoline biosynthesis, 17 genes encoding known MIA biosynthesis enzymes and transcription factors as well as the recently cloned and characterized 160MT (Levac et al., 2008) and M/WT described in this paper are represented in the CROLF1NG dataset. The leaf epidermal localization of 160MT and LAMT (highlighted in gray) expands the list of genes and enzymes expressed in this tissue and highlights it as the major site of MIA biosynthesis up to 1 6-methoxytabersonine. Putative numbers of the enzymatic reactions in each cell type are shown. Cytochrome P450 reductase (CPR) is believed to be involved in all the reactions catalyzed by cytochrome P450 monooxygenases. DXS, 1 -deoxy-D- xylulose 5-phosphate synthase; DXR, 1 -deoxy-D- xylulose 5- phosphate reductoisomerase; MECS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; G10H, geraniol 10- hydroxylase; CPR, cytochrome P450 reductase; lOHGO, 10-hydroxygeraniol oxidoreductase; LAMT, loganic acid methyltransferase; SLS, secologanin synthase; TDC, tryptophan decarboxylase; STR, strictosidine synthase; SGD, strictosidine beta-glucosidase; T16H, tabersonine-16-hydroxylase; 160MT, 16-hydroxytabersonine-O-methyltransferase; D4H, desacetoxyvindoline-4-hydroxylase; DAT, deacetylvindoline-4-O-acetyltransferase; ORCA3, octadecanoid-derivative responsive Catharanthus AP2-domain3; BPF1, box P-binding factor 1 ; ZCT2, zinc finger Catharanthus transcription factor 2. frr

W i , ! oil 9a '6 !«•»»<>»< 'H

I 1

a!li>o

1 45

late isoprenoid pathway ESTs (GPS, GPPS, IPPT) (Supplemental Table 1) were represented in the CROLFING dataset.

The enrichment in MVA pathway genes (13 ESTs in total) compared to the single

MEP pathway EST identified, strongly suggests that the leaf epidermis of Catharanthus expressed higher levels of the cytosolic MVA pathway. Since Catharanthus accumulates considerable amounts of the oleanane-type triterpenes, oleanolic and ursolic acid (Usia et

ai, 2(X)5), it is possible that the MVA pathway in leaf epidermis is involved in their biosynthesis. The CROLFING dataset also contained squalene monooxygenase (5 ESTs) a key enzyme for sterol and triterpene biosynthesis and Xl fi-amyrin synthase-\]kt ESTs that may commit oxidosqualene to the formation of oleanane-type triterpenes

(Supplemental, Table 2). The representation of these transcripts strongly implies that the leaf epidermis of Catharanthus may also be specialized for triterpene biosynthesis and accumulation.

2.4.5- The CROLFING dataset contains the genes for flavonoid biosynthesis-related genes

Many genes involved in flavonoid biosynthesis were also found in the

CROLFING dataset (Supplemental Table 3). Among the ESTs reported only cinnamate

4-hydroxylase {C4H)

Table 1: CROLFING genes with significant similarity to l

Gene o3ifoiii;."?c> nrj 3rt

H'^y-

Ui-

y^A'

h —

I

^ 1*1 ; ;

m

>•• i,v(.;t'^,'^3 I f^rtnr/ MJU'> Tf'i J 46 ' d>'

,.-.4';.

I'? Si;.-*; «,•

«. ( »^.^lt1^r; M; >

I ~N(.-«;K'>'l.t

t tfl." l.ll )^l > y.

iSi»(io'JV'A-^j >

'^' >«*£(;!•< '•l«-^«H;

i

^iv.w'.'^vtf.o a.-iT

1 ^ .

I , , ;i I ^.-• ' f.». »!. invvi f ,yo

*<.'- • • ^ 'i; * J' , (ii >«»A .v.',' , •.>efl-

I

'.i.j'|-£.-. I

•in .>

I :• ; yy.i>--'Ui

I

•-t."*!' ,:»

V .T.i

i-«V . cli

'.: ^t ' «• IJ-: • 47

had complete sequence identity to a functionally characterized C4H previously reported in Catharanthus (Hotze et al, 1995), whereas the other ESTs are novel with considerable similarity to known flavonoid pathway genes described in other plant species that include: phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone 3- hydroxylase (F3H) and 2 '-hydroxy isoflavone/dihydroflavonol reductase (DFR). These data were not surprising, since flavonoids appear to play vital roles in the control of epidermal cell fate (reviewed in Broun, 2005), have long been proposed to protect plants against UV-B radiation (Schmitz-Hoemer and Weissenbock, 2003), and have been shown

to accumulate within the leaf epidermis of Catharanthus by microscopy (Mahroug et al.,

2006). Together these data suggest that Catharanthus epidermis is a primary site for

'': flavonoid biosynthesis. •;;,i-0!i\>^'it'fi?' -^rsr \r irifi

It is also interesting that another EST (CL314Contigl) is represented 4 times in the CROLFING dataset and is annotated as a putative flavonoid 0-methyltransferase

(OMT) with 77% similarity to a previously functionally characterized flavonoid 4'-OMT

(AAR02420; Schroder et al., 2004). In contrast this EST was not represented in the

CrUniGene set established from sequencing of cDNA libraries prepared from the base part of the leaf and from the root tip (Murata et al., 2006). The public database did contain one EST sequence (EG555799) from a Catharanthus roseus flower bud cDNA library identical to CL314Contigl, whereas the root tip library was only represented with

0MT2 and 0MT4 that had been previously cloned and functionally characterized as flavonoid OMTs. This novel OMT contig turned out to encode 160MT (Levac et al,

2008). ^>- <• -%p':-A.:.i .. \-.c .;;,k;;' •f^MV.li

.. /:.'! t^»'. -• :,'"; . ,:: 1 ,,^U V. ,:5A-f:,.iI X'

v. 'M*^ V, i'(ti

'- "»V>;V S'.v,,vi r,tiv>.rtu-»u v

' i.OTi'V V t.'ili;

^.': - » < I ;.<; v :)'; ,r: uwi

., "yty- ;i<-t '1';

::, -.%, 7" :::.>!. ;. .Httithc' -Mij-rihsit ?}-'/U

'> -' 'Ifi'n ' HiM

'»' 1'; ". i^;ii''; •li '• •yaj'»T

>'<: :'•, >! -< •! -'i i i

t: • i hsH,

•' /' ;•>{, /!!.>„. /li .iii'T-.f I 'j'-vij-^ n ^ t, oi

'' 1 ^ ': - ::.'^ ; '! r r,.iM ')' . ;.•:' u' '

] K j^ "'

•'.•:'^-^-.-",./''. *. r; ..j^u'c

''I- ;^,l •' ' ' V"' ; ; ^ .i:^l• . !U»'^ i-a L 48

2.4.6- The CROLFING dataset contains many genes for lipid biosynthesis.

Leaf epidermal cells are important sites of biosynthesis of (VLFA) lipids that are secreted into cell walls to form the impermeable cuticular wax layer. Many fatty acid biosynthesis-related genes were represented in the CROLFING dataset including: /affy acyl-ACP thioesterase {FAT), fatty acyl-CoA synthases, fatty acid desaturase (FAD), carboxylic acid ester hydrolase (CXE), phospholipase D (PLD) as well as lipid transfer protein (LTP) (Supplemental Table 4). Among these, the number of ESTs encoding LTP was quite high (121 ESTs), suggesting that this type of protein might be very abundant in the epidermal cells of young leaves. LTPs are composed of two subfamilies, LTPl and

LTP2, which are remotely related to each other by sequence similarity. LTPs bind to different types of fatty acids and hydrophobic substances in their hydrophobic groove in a

non-covalent manner. Although there is much to be studied in order to assign in vivo functions of LTPs, this class of proteins is implicated in various biological processes

including cuticle biosynthesis. It has been claimed that some LTPs are localized preferentially to the cell wall of epidermal cells or in cuticular wax layer of various plants

(Pyee et al, 1994; Sterk et al, 1991; Thoma et al, 1994). Moreover, barley seedlings accumulate cuticular wax and express LTPs in response to the abiotic stress caused by heavy metals (Hollenbach et al, 1997). These previous observations support our result that LTPs are abundantly expressed in the leaf epidermis of Catharanthus. In addition, the putative orthologue (92% identity at the amino acid level) to Arabidopsis 3-ketoacyl

CoA synthase (CUTl) was represented with 7 ESTs in CL135Contigl. Previous studies

showed that CUTl was involved in VLFA biosynthesis and that it was expressed

specifically in leaf epidermal cells (Millar et al., 1999). '

',\' V.V'.V;

4tir. .I: >„ i" :/^yi ic,

» V

<'' .1 ', < >i/' ^«f rs',,: «, ii ,,:., 'S-i i,v ^' «?'v,i5 bitJi,.!.'>i'J5f <-ijt!i..-,','. .'^t

?i;.\-.

' l'''Ui /; lb.' , : 'i'l f':^ i> 'W;i><'\\V r '.^W :) ^i.l>l. *ii\7-..o*i'

(' .1 t /),:"?>'',•: : i ,(?!'' ^ t;* ! * ; .i'j,ui 'yii'r

' >;'t .' ' ' - /h^- 'Xur-- i:.

•' I ^'f i ;,.-j(' :tTii ..(» f'itfi'.v

.•'; •;' '. '' t.--. iw l-^ y i;;i 1 Ji. -Vi /., v;*l-i i-'

',. ;!i i/n ..' '--.H: ;K,.;v;!^

<,,;>> I I j mi

' ( tO- '!>( 1; .','< a' n- '

'\ /'

.'-* .>\

' 4- '^^ >' ' ' r > y- „f' , _,.;i ^v ;. i''-3i\\i'i

s9'. \i\

', I '..!,' . .-i •!>, .'f:^t:f><;>«tJi; -yu-

: I'V ft ..' ..''>

'' ',> ' •• .'•'.>! > I- >U='

" ^ : It-

^*i "tK'iii''^''/ 'i rrt Jih^.-rriO' 2.4.7- The CROLFING dataset contains genes for S-adenosyl-L-methionine-dependent

' methyltransferases h ,

Several classes of genes were also identified that could be potential candidates for identifying novel MIA genes that remain to be characterized. In addition to 160MT

(CL314Contigl) described above, 20 putative OMTs were annotated, including three

ESTs with identical sequences to known Catharanthus genes; methionine synthase

(CL69Contigl) (Eichel et al, 1995), S-methyltransferase (169-E03) (Coiner et al, 2006) and cajfeic acid OMT (101-B09) (Schroder et al, 2002). It is well known that the MIA pathway leading to vindoline biosynthesis involves 3 separate methyltransferases: loganic acid OMT {LAMT; Madyastha et al, 1973); 16-hydroxytabersonine-16-OMT {160MT;

Fahn et al, 1985; Cacace et al, 2003); 16-methoxy-2,3-dihydro-3-hydroxytabersonine A^- methyltransferase (NMT) (De Luca et al, 1987) that remain to be cloned and functionally characterized. Previous studies have suggested that LAMT might be expressed in the leaf epidermis, since SLS converts the LAMT reaction product into secologanin (Irmler et al,

2000; Murata and De Luca, 2005). Inspection of the CROLFING dataset revealed a full length cDNA (Supplemental Table 5, CL57Contigl composed of 12 ESTs) encoding a putative AdoMet dependent carboxyl methyltransferase such as LAMT. However, the

CROLFING dataset is not expected to produce candidate genes for NMT, an activity associated with chloroplast thylakoid membranes within leaf mesophyl cells, rather than with leaf epidermal cells (De Luca and Cutler, 1987; Murata and De Luca, 2005).

2.4.8- The CROLFING dataset contains acyltransferases, Cytochrome P450 monooxygenases, glycosyltransferases, dioxygenases and transcription factors «^

fvj ciut: ^ -. ... .vji -HI \it

->"Vi.i\.fVV »' Vi ., . . .. *'*')! ,, , ,„ ,..' ,\i,sV!. ^>/:mi'A ti?

'^'-^ A^?A -4^!; fafft fflwor ' -f »f ,(£^)tiC .^vi va (i9b<"m;-i?!'t (<*',

:\-A»Y"*\( TMO-^4 - - - '»; J^'TV )v

3i0ifi3ik.

Vh

}f;:?bo^f73l> »»l

i.^fli ?rid.Un , . . ..,-M m!l:i''f , r^v

'.I'f-t 'S»\'.S-Si^,i«>'.7 A ,v\.UV;\ .ij-!,',', .'IV. /.n.AlT>f> • <.a*VjS,'S35ij

:.:'. -f:\t.\i.. 50

The database contains a significant number of ESTs encoding acyltransferases

(Supplemental Table 6), Cytochrome P450 monooxygenases (Supplemental Table 7),

glycosyltransferases (Supplemental Table 8), dioxygenases (Supplemental Table 9) and transcription factors (Supplemental Table 10). This rich source of genes promises to hold a number of MIA pathway and regulatory genes that need to be functionally characterized in future research.

2.4.9- Functional characterization of a novel LAMT obtained from the CROLFING dataset.

The last step in loganin biosynthesis is catalyzed by LAMT, which has only been partially purified and characterized (Madyastha et ai, 1973) (Fig. 2). In order to demonstrate the value of the CROLFING dataset, a full length candidate gene, obtained by overlapping 12 separate ESTs (C57Contigl, Supplemental Table 5), was cloned and functionally characterized. This unique sequence was not found in the EST datasets obtained from cDNA libraries produced from young leaves or from root tips (CrUniGene

database; Murata et al., 2006) or from other Catharanthus tissues described in the public database. The clone was 1,396 bp long and exhibited moderate similarities (44% amino acid sequence identity) to a putative carboxyl methyltransferase from Medicago truncatula (ABE92230) (Table 5). Since LAMT may catalyze a reaction preceding SLS shown to be expressed in the leaf epidermis (Irmler et ai, 2(XX); Murata and De Luca,

2005), it was possible that C57Contigl (Fig. 3A) encoded LAMT. The putative open reading frame of LAMT (Fig. 3B) aligns to three (35, 36 and 36% amino acid sequence identity) previously characterized carboxyl methyltransferases from the SABATH family

(Salicylic Acid methyltransferase (SAMT) (Ross, J., et ai, 1999) from Clarkia breweri. ^r

ri-Kid^ari 1.

> V>

''" ^ _" »r»' '•'ij , , 51

Geraniol —

G10H T 10-hydroxygeraniol 10HGO

10-oxogeraniol

MTC: T 7-deoxyloganetic acid dlgt:

7-deoxyloganic acid

J.AMT \7DLH OGIc COOH COOCH3 7-deoxyloganin loganic acid

7DLH"'--. LAMT COOCH3 '— loganin SLS CHO COOCH3

secologanin

OGIc

Figure 2: The proposed pathway for secologanin biosynthesis in Catharanthus roseus. Enzymes whose corresponding genes have been cloned are shown in bold. LAMT, which is cloned and functionally characterized in this study, is shown in a gray box. Dotted arrows represent uncharacterized enzymatic steps, respectively. G10H: geraniol- 10-hydroxylase; 10HGO: 10-hydroxygeraniol oxidoreductase; MTC: monoterpene cyclase; DLGT: deoxyloganetic acid-0-glucosyltransferase; LAMT: loganic acid-0-methyltransferase; 7DLH: 7-deoxyloganic acid hydroxylase; SLS: secologanin synthase , .

v M<"

r •i>j 'jr

. *" . J

^' •. .(

HO

v.^

• H. T .

ti'^

""" Tit. I. O k ' ^iKi^ 4 >

1 52

Jasmonic Acid methyltransferase {JAMT) (Seo, H., et al., 2001) and Indole-3-

acetic Acid Methyltransferase {JAMT) (Qin et al., 2005) from Arabidopsis thaliana.

Based on the crystal structure of SAMT (Zubieta et al, 2003) amino acid residues

composing the putative hydrophobic active site of LAMT include Y159, W163, 1224,

P227, L244, H245, 1320 and D359 (Fig. 3B). Within these active site residues LAMT has

two notable substitutions where the non-polar aromatic residues W226 and F347 of

SAMT are replaced with the polar charged residues H245 and D359 (Fig. 3B).

In order to test the activity of the C57Contigl gene product, the corresponding cDNA clone was isolated by PCR amplification using specific primers based on the

C57Contigl sequence, using the Catharanthus leaf base cDNA library as a template. The product was sequenced to obtain a 1,113 bp product identical to C57Contigl (Fig. 3A) encoding a putative 371 amino acid ORF (Fig. 3B). Recombinant LAMT protein was expressed in E. coli cells to perform functional studies. Enzyme assays with crude recombinant protein, 5-adenosyl-L-[mer/i>'/-''*C] -methionine and loganic acid produced a

radioactive product corresponding to loganic acid (data not shown). These results were corroborated by repeating these assays at a larger scale and by isolating reaction products by UPLC (Fig. 4). Loganin production only occurred in the presence of recombinant enzyme, AdoMet and Loganic acid (Fig. 4 profile A), but not in reactions with boiled

recombinant enzyme (Fig. 4 profile B), in reactions lacking loganic acid (Fig. 4 profile C)

or in reactions with E. coli cell free extracts expressing the empty vector (Fig. 4 profile

D). Mass spectra analysis (Fig. 4, Table insert) illustrated that the reaction product (Fig.

4A) is loganin based on its base peak at 435.1 (m/z), consistent with the mass obtained

with the loganin standard. «e

' '.'" Vii,-i bn rt^rW ";ii»>j:''sl xr.?n'.r. ,r,|-)-, ii .;' •:i' -j, i:i u :34i^.fOIi O

;- ;^!i' Tf^/,

^'*^ ' •iJ*. J -i .? ')!U r,.v r J at

....; :i ^1, >J t

/'•''.'Li >-f.d li.

'.i ^" ).: !.. ., ,w•^:^,. ^s. !:

] {:><,> !,- <,^ 'y-: M' 'i

.:(!'. :i .; I .Tj

t • / r

-1 JSirt^fv-* :j

ViM

'•. .1 ^ .ihw ii ,*(<. or

'«}! .• i' 'Jf'i) Df.'JXJiq iifj'-t^'.Vji It;:-: ).,j!i- :. !i; i'^'s' ,. ,r.' -; ., «-i3eifj/

I ,: ilj iS;i i <:i'<'.

;i.^ ':i;iii?i''ii ;>!. EL

53

(A) 0.5k! l.OK LAMTCDNA 139 B02 130 F06 I hi 035 G02 028 H04 128_G11 *" 037 EOS M 004 C04i 056_E03 055 B07i iSQ^CIIjw. 126_D11 • (B) 142 C03 ' JJUCr MEVMRVL HMNKGNGETS YAKNSTAQSN IISLGRRVMD EALKKLMMSN SAMT MDVRQVL HMKGGAGENS YAMNSFIQRQ VISITKPITE AAITALYSGD

LAMT M„Ari i.^IKK rA_t-rA .-bAl. F" :K'.: : L .:H;% i ::,:, ;ijKa v; LA/iKAv r,- LAVt;i;K:-:,i.j: -MGSKGDNVA VCNMKUSRJLL SMKGGKGODS YANNSQAQAM HARSMLHLLE BTLENVHLNS ?i 120 3E ISS IGIADLGCSS GPNSLLSISN IVDTIHNLCP DLDRP-VPEL RVSLNDLPSN TV TTR LAIADLGCSS GPNALFAVTE LIKTVEELRK KMGRENSPEV QIFLNDLPGN

K;' ivt:.:VETr.vK .:Lg;;'i i'i.v ;;•, t !-kL';;vk;; SAS PPP FTAVDLGCSS GANTVHIIDF IVKHISKRFD AAGID-PPEF TAFFSDLPSN

180 DFNYICASLP EFYDRVNNNK EGLGFGRGGG ESCFVSAVPG SFYGRLFPRR SLHFVHSSSS DFNAIFRSLP lENDVDG -VCFIHGVPG SFYGR1.FPRN TLHFIHSSYS DFNTLFQLLP PLVSNTCMEE CLAAD GN RSYFVAGVPG SFYRRLFPAR TIDFFHSAFS

240 LHWLSQVPCR EAEKEDRTIT ADLENMGKIY ISKTSPKSAH KAYALQFQTD FLVFLRSRSE LMWLSQVPIG lES NKGNXY MANTCPQSVL NAYYKQFQED HALFLRCRAQ ! 'vf .'Ki./ LHWLSQVPES VTDRR- SAAYNRGRVF IHGAGEKTTT AYKRQFQAD LAEFLRARAA

xi 300 ELVPGGRMVL SFLGRRS — DPTTEESCYO WELLAQALMS MAKEGIIEEE KIDAFNAPYY E\A/PGGRMVL TILGRRS — DRASTECCLI WQLLAMALNQ MVSEGLIEEE KMDKFNIPQY .;^i t,r,/-. -..M-, I ,ip:;::.:-; -:..:/• Lc^ VA ;LL Hr_... vi.'LMK L .N^ ;. J . t.KK ;;i ,: f N-i . y EVKRGGAMFL VCLGRTSVDP TDQGGAGLLF GTHFQDAWDD LVREGLVAAE KRDGFNIPVY a g 111 ~360 AASSEELKMV lEKEGSFSID RLEISPIDWE GGSISEESYD LVIRSKPEAL ASGRRVSNTI TPSPTEVEAE ILKEGSFLID HIEASEIYWS SCTKDGDGGG SVEEE — GYNVARCM :..; a-. APSLQDFKEV VDANGSFAID KLWYKGGSP LWNEPDDAS EVG RAFASSC SB o 410 rawepmlep TFGENVMDEL FERYAKIVGE YFYVSS P RYAIVILSLV RAG- ravaepllld HFGEAIIEDV FHRYKLLIIE RMSKEK T KFINVIVSLI RKSD raimecilte: FEIVTKNLfiiE JviJ'HVFDKElB K0ADL3X,VLK RKC.N rsvagvlvea HIGEELSNKL FSRVESRATS HAKDVLVN-L OFFHIVASLS FT- o §»n(O r- ^

Figure 3: Identification and functional characterization of loganic acid methyltransferase from Catharanthus. (A) Representation of CL57Contig1 composed of 13 ESTs that encode the full length LAMT obtained from the CR0LF1NG dataset. The consensus sequence contains a putative open reading frame with significant similarity to known carboxyl methyltransferases. (B) Protein alignment of Jasmonic Acid Methyltransferase from Arabidopsis thaliana {JAMT) {Q9AR07), Saliclyic Acid Mettiyltransferase from Clarlda breweri SAMT (Q9SPV4), Loganic Acid Methyltransferase from Catharanthus roseus (LAMT; highlighted in grey) (EU057974) and lndole-3-acetic Acid Methyltransferase from Arabidopsis thaliana (lAMT) (NP20036). Highlighted in red are important active site residues which form the hydrophobic core, based on previously reported active site residues of the SAMT crystal structure (Zubieta, 2003). Interesting differences compared to JAMT, SAMT and lAMT occurring in LAMT are denoted by an acidic (aspartic acid) and basic (histidine) amino acid substitution in amino acids 359 and 245, respectively. vv«... . »•/

f I. 54

2.4.10- Recombinant LAMT is a highly specific 0-methyltransferase

The rLAMT showed striking specificity for loganic acid since it was not active with other similar iridoids including deoxyloganic, dehydrologanic, epiloganic, loganetic

acid or with the reaction product, loganin (Fig. 5). Various benzoic acids (salicylic, benzoic, p-hydroxybenzoic and anthranillic acids), phenylpropanoids (caffeic, ferulic, sinapic, gallic, chlorogenic, p-coumaric, and trans-cxmrnxmc acids), dicarboxylic acids

(malic and tartaric) and hormones (indole acetic and abscisic acids) were not accepted as

substrates by rLAMT nor was it active with MIAs (16-OH tabersonine and 3-OH tabersonine) (data not shown). Together these results confirm that the 12 ESTs used to assemble C57Contigl encoded authentic LAMT, that Catharanthus leaf epidermis is specialized for MIA biosynthesis and that the CROLFING dataset will be a valuable resource for cloning many more unknown and uncharacterized genes in this pathway.

Biochemical and Kinetic properties of rLAMT:

The pH optimum of rLAMT was 7.5 with loganic acid as a substrate and co- factors such as Mg^"^ or K"^ were not required for activity nor did they enhance enzyme activity (data not shown). The Km for loganic acid of rLAMT was 14.76 ± 1.7 mM similar to the results obtained for the partially purified enzyme isolated from cell

suspension cultures of C. roseus (Km = 12.5 mM) (Madyastha, K., et al., 1973). While the Km value for the co-substrate AdoMet was not previously reported for LAMT,

rLAMT showed a remarkably high Km (742. 1 ± 37 ^M) compared to (J AMT) (Seo et al,

2001) and (SAMT) {Ross et al, 1999) whose Km's were 6.3 and 9 ^M, respectively. A \y. >.•

>: ten ^

' 'I .1 I. t .' I. :•-,.,'//( .. -f' Hj :ji.'j^-' riiK. J, (fr 7At dlr.v y; i.>t

it

I, (! / h ;. , •'r/u,io(>j 'a:' .f'^ 'i , •ih:

'.•(' ">.*' 5(5> :;i i - >r ,,1 vv;

::'ii,si-; •'• I. •d. -r'-: icj '>' i' J^ ?-LL

'»« 'If' t .

fv- ^ ,.:. . ''OK-;' !i ^. ,ti.\*u*v .w.;,.! ;*},i, \ ;1 I "»;)•; . y^i^^j

' ' :'?<<;;:'> ' } '„[: l^'j'V ;iic;j '> . i

'.'10 '' ./.• 'i •>.:. ";!!'. :>':. " 'i'^-^A'i' , r»i\

.1 -'.;- --v-v -A :

I n-' . '.1 'i

'.. '•-. : -.^"ntUiyr .•'t

»:, ': v: ^ -^ . I v:"' v:>; ":"!'f .i'fi'','.'

• rttfi- f.^f M ;;/' 1 ,, nMf.

' , 'I- >

-.' ;!/ .i • '^^•. ;./; i I: 1, 'f'A ^jM^;;. ;;.--;.- 'i;!' -j-

••^ II ^j

V .! -iv 'i ,^. >^ i.y 'H'i , t,:-^; 55

-£* Logan ic Acid

;' )'l iv;- |-4ao'. s>M T

Loganin \ I

D

B MLAl .w'.-r'rasi ;f v 2.0 3.0 4.0 Time (min)

Substrate ntjn/y'^

A 0.^ o.r

?4 * r.^-f-?' ?>•'"{ s

«».. p

'.C •*

- « . » l'* ti-'j -.o-*3i -^

Pf"yii ',)f-.lfi :-"i:»'»' 56

The high Km values for both substrate and co-substrate suggest that the catalytic efficiency of rLAMT appears to be very low (Table 2) compared to other plant OMTs.

The turnover number of rLAMT for Loganic acid was quite low (0.327 s') compared to

non-histidine tagged (2.8 s'') and histidine tagged (14 s'') rSAMT (Ross et ai, 1999) and to JAMT (25 s') (Seo et al, 2001). Remarkably, inhibition studies with the reaction products revealed that loganin is a strong inhibitor for rLAMT with a Ki of 215 ± 30 ^M,

since it has 50 times greater affinity for loganin compared to loganic acid. In contrast the

Ki (400 ± 47 nM) for S-adenosyl-L-homocysteine (AdoHys) was only 2 fold higher than that of AdoMet. Since the AdoHys inhibition constant was about 2-fold higher then that of AdoMet, inhibition of LAMT by AdoHys would depend on the ability of

Catharanthus cells to recycle AdoHys into AdoMet through the AdoMet cycle.

2.4.11- LAMT enzyme activity is preferentially expressed in Catharanthus leaf epidermis.

In order to localize LAMT in Catharanthus leaf, the enzyme activity of LAMT was measured using extracts either from whole leaves in various developmental stages or epidermis extracts of corresponding leaves obtained by CA. Previous studies with

160MT (Levac et al, 2008) showed that this enzyme activity was highly enriched in the epidermis of young Catharanthus leaves whereas the activity of the chloroplast thylakoid associated NMT (De Luca and Cutler, 1987; Murata and De Luca, 2005) is found within cells inside the leaf. In good accordance with these findings, NMT activity was only detected in whole leaves but not in epidermis enriched extracts in all developmental stages tested (Fig. 6A).) In contrast 160MT specific enzyme activity was 10 times higher in leaf epidermis enriched extracts compared to the activity found in whole young leaves

(Fig. 6A). The 10-fold higher specific activity of LAMT in the leaf epidermis iudi't •:. I [Mi

:• . i ?<' "'-^ ^r< i':0 'at:* i vmAkj b.t-^jjs^Boo V:. , ,Miv -ji -n: -niqti TT/

((.''« Jt fuit g>Tj«,i '. .. i '^) ' '^ TV ;;^'.:! TM>^.2 ,1 b. -ix^AAPui UmX'7 8 in;

-^li'. i.3^ i- ' r*,Ji'v.' •

iul^: rtit^' -",'

vr:jj.. -ah 'h; A yd TMA.] V> /i^

i

so ,^;i.- , ;p-,^

oci. o ^-^jifv;;") 'trig: ' --nv' /t;vi?>, 'Ai. '\j^ 57

:Hy U:-~'^i-.ji~:l

)^ ''• ViMc-, U'^.-.'i m

Substrate Relative Activity %

(. ,-<» ! 100*

CM H !> - ^••^fiy' OH Loganic acid \ .'tr.'-.',

OQIO ,v »*:.v.s 10 .vv

OH '.:'.'.. . Secologanic acid i..: r.>W.

Gl«

OH '' '' 7-Deoxyloganic acid -I^K':''.' S i' ''V:Vi-

GK

OH 7-Dehydrologanic Acid ;,'f-»« ).•'::'( (V

HQ^^ H OGIO

H 0-< H OH 7-Epiloganic acid <*"i 'hW: .•;V!y 1, hq^Jhph

o=< h oh Loganetic acid

*Specific activity of 1 00 % = 310 ± 30 pmol Iridoid/mg protein/hour

Figure 5: Substrate specificity of rLAMT. The LAMT only accepted loganic and Secologanic acids as possible substrates, but was not active with other similar compounds shown. * The 100 % value is related to the specific activity of the enzyme for loganic acid (310 pmol loganin/mg protein/hour).

•^>

; t he .i<-' yt'/l"^ ^/

fl" A"

'>''P-.» . ^.n

•3; T " .i„iv(,/ .,

t>ir*« rjiu- '.frii'-^-'i-?.'

» ,' ,'.i- , .

wa-i''- .-(

-y f- [ »-,.»• • - - -" "V

I'' ''I' '''' '< 'j . ;?," ill' ,;

I ^ 'V;-; •>'..' ^!". 58

than in whole leaves strongly suggests that this enzyme is also preferentially localized to the leaf epidermis (Fig. 6A). However the activity of 160MT was 35 times lower in epidermis enriched leaf extracts of L2 compared to those of LI leaves, whereas the activity of LAMT only decreased by about 50% in epidermis enriched leaf extracts of L2,

3 and 4 leaves compared to those of LI leaves. These data suggest that unlike highly regulated 160MT, significant LAMT activity is maintained as Catharanthus leaves age.

The reasons for this difference in regulation remain to be determined. Leaf, hairy root, stem, silique and flower extracts contained both 160MT and LAMT activities in all these tissues (Fig. 6B). However, the levels of LAMT (Fig. 6B) in hairy roots were 4 to 8 times lower than the activities found in the other plant organs, respectively.

2.4.12- Biosynthesis of pentacyclic triterpenes with an oleanane skeleton may be exclusively localized to the epidermal cells of Catharanthus leaf

The identification in leaf epidermis of putative jS-amyrm synthase (CL50Contigl;

13 ESTs) and squalene monooxygenase (CL247Contigl; 5 ESTs) genes (Supplemental

Table 2) suggested epidermal specialization for biosynthesis of pentacyclic triterpenes having an oleanane skeleton in Catharanthus (Fig. 7a), since none of these genes were represented in the CrUniGene set (Murata and De Luca, 2005) or in the over 10,000

Catharanthus ESTs found in the public database. Furthermore, cuticular wax components were selectively extracted by briefly dipping the leaf into chloroform (Fig. 7b). The triterpene component of the extract was analyzed by thin layer chromatography (TLC) to show that triterpene alcohols, composed primarily of ursolic acid and small amounts of oleanolic acid (Usia et ai, 2005), are exclusively localized to the cuticular wax layer

(Fig. 7C), suggesting that the leaf epidermal cells are the primary location of their biosynthesis. Together ursolic and oleanolic acid compose approximately 2.5 % of the dry weight of the young Catharanthus leaf (Usia et ai, 2005) illustrating the importance ' ' 1 I

^:'"." '-i-''- . i' ' . . : :U^ .-'Hi' : K.i;.,i.' i-' .^•i'':^t^ 1 59

Table 2: CR0LF1 NG is enriched in ESTs for enzymes in tine MIA biosynthetic pathway T 60

1234 1234 1234 1234 1234 1234

Leaf Developmental Stage (LI, 2, 3, 4) # -^ # i^ ^ # Whole Leaf Epidermis Enriched Extracts Leaf extract 2.0 16 B 2 c O flj

CO i A Ml

^'

Figure 6: Differential distribution of LAMT enzyme activity in Catharanthus leaf epidermis.

(A) Catharanthus roseus leaves of different ages (Leaf 1 , 2, 3 or 4) were extracted in triplicate either as whole leaves or by CA treatment to obtain epidermis enriched leaf extracts as described in Methods. The two sets of desalted extracts from each stage of leaf development were assayed for NMT, 160MT or LAMT to identify the differential distribution of these enzyme activities. (B) Whole Catharanthus roseus leaves, hairy roots, flowers, siliques and stems were extracted in triplicate as described in Methods. Desalted extracts were assayed for LAMT and 160MT to compare the distribution of these enzyme activities within different plant organs. Each point represents the mean of three assays ± standard deviation.

61

of this pathway in the leaf epidermis. Triterpene alcohols within the extract (Fig. 7D) analyzed by capillary HPLC MS identified ursolic acid was a major triterpene component based on its elution with the same retention time (16 to 16.2 min) and the same molecular

! mass (455.3 m/z) as authentic Ursolic acid standard.

2.5- Discussion Cy^"/^*^'^ »

2.5.1- Towards a complete understanding ofMIA biosynthesis in Catharanthus roseus

Significant efforts by various research groups have yielded more than 25,000

Catharanthus ESTs with over 12,000 unique sequences that have partly been submitted

to the GenBank database (Murata et al, 2006; Rischer et ai, 2006; Shukla et al., 2006).

While these efforts identified only four known MIA biosynthesis pathway genes (Table

2), the present study using a cDNA library prepared from leaf epidermis enriched mRNA,

identified ESTs for virtually all known MIA biosynthesis and regulatory genes up to and

including the T16H (Fig. 1) and 160MT (Levac et al., 2008). These results strongly support the biochemical specialization of young leaf epidermis as the primary site for expression of most of the pathway in vindoline biosynthesis from primary iridoid precursors and tryptamine to 16-methoxytabersonine (Murata and De Luca, 2005). Most

significantly, this dataset is very likely to contain the remaining uncharacterized biosynthesis and regulatory genes for the MIA pathway as shown by the CROLFING

(3,655 unique sequences) guided identification and functional characterization of LAMT

(Figs. 3-6) and 160MT (Levac et al, 2008). In this respect, it is relevant that the LAMT and 160MT sequences were represented 12 and four times, respectively, in the

CROLFING dataset, while only one EST for each sequence was found in published

N f^

,»<"'<««"'* ,i..^i,.;'ik»i' '.'bit ;jitf;-K

a

I'C'i'^JS , .V(. V-!

'• r-iovi^ ^?Nw:r, 'V-»iii .'^'X)! ,,.^. \-> :^:, -.J, vA:;;f.\ iii.y ,j .

^y(?f'! i<' ,M oj .

.-!: .- . V

t , 1, * - ti'i,xh:.fM,y-vi • >.^.. '..;. )!'! v.!..j^y^-':: rA' '>'Hii' '.. y,^-. 62

r'.r->

Cycloartenol cynthaco ,

(A)

rbj';.i.

Squalene monooxygenase

Squalene 2,3-Oxidosqualene m^: V"i?<'-^*^r

P-amyrin

synthiase „, (B)

Leaf surface in dipped CHQI |J-amyrin lis .U' (C) .^^ ^^, L-- .^-

V- >;k. >\\

• .h. i'-'}:,: :_i

(a>

>r>>; i\ 63

Catharanthus EST datasets (Table 3). These results highlight the remarkable value of random sequencing of leaf epidermis-enriched cDNA libraries for investigating the specialized chemistry of this cell type. The results also suggest that selection of candidate

genes based on their relative abundance in this library is a useful approach to identify

MIA biosynthesis enzyme candidates. In this context, further sequencing of this epidermis-enriched cDNA library should be considered in order to obtain more reliable

data on the redundancies of the ESTs (Table 1) and to reach saturation with respect to the numbers of ESTs that encode enzymes that are related to MIA biosynthesis. In addition, gene families with particular biochemical functions could be selected for expression analysis by RT-PCR using RNA harvested from different organs or cell types (Murata and De Luca, 2005) in order to select for candidates to be screened directly for biochemical function.

2.5.2- Use of the CROLFING dataset to elucidate the pathway for secologanin biosynthesis

Iridoids are cyclic monoterpenes, found in various plant species, where they play important defensive roles in plant-herbivore interactions. For example, aucubin and catalpol found in Plantago lanceolata deter and retard the growth rate of generalist insect herbivores (Harvey et ah, 2005), whereas insect specialists are attracted to and consume host plants to sequester iridoids for defensive purposes or to use them as oviposition cues

(Peiiuelas et al., 2006; Pereyra and Bowers, 1988). In the case of the MIA biosynthesis,

the iridoid secologanin is important as the source for the terpene moiety of all the MIAs.

While iridoids have also been used in chemotaxonomy because of their structural Vi j(i.lfc-< j:!((f l>.ini,V' -st^i !(i^(hbr''

":': :jJf*iij«b,u-;olv' aunootv. i«'' ay.l. <>it vn sU'f -•x*',!;.} fiiiy }fi.rfl )u rit^lm-:>r'

ci'h fr V 7as(l'.?,i j/".i*vo -•>;.«; 'tl !M ;u.

,;•.}-.-"! 3f'* 01 5 dti,v ^r ^-'Tii! : -jiiiX' ->

- r 1 - . »- .'jfj ijl ^• .^ ;:;vi'>:' 'M -^'^jV^

'A 1

ij(U; ;i>''HJ:j»;i) .J-mmr:'j ^m'^ ^^PfHim^i^^^u :; '''S-hs^ii^fc; m •^};'>i 3yj.-fi^'!jf; »,

" * ^;fi>^fr'>> -jrrft It jiO^';tc.,ns it'- n . .y^i. ..^iM^f ^-n-ad^ .i^:i)' 'uj

/<) /-*; irr.'U r,^; S ,

• ">' ; f •. . ^;;.'M'}'-v5-f bfi:^ 'iiv.-iyi ;^Jl):

-'* '^';- ) * '/ i'.i vir,!.)jit :. '. U 'lO'y; ' \t xi h':.- -iVu n,

)/ • !in. i«-.>/il U) w.nnj':i v/^fr- , ; -,lvi^ .r f:>..'i' cj^o :j;'h v '^.H .tbtobhi 64

diversity in various plant species (Lopes et al, 2004), their localization within the plant as well as their biochemistry and molecular biology remains largely unknown.

Recent studies in Catharanthus have shown that while GIOH is preferentially expressed in IPAP cells (Burlat et al, 2004); SLS (Fig. 2) is preferentially expressed in leaf epidermal cells (Burlat et al, 2004; Ixmler et al, 2000; Murata and De Luca, 2005) and in root cortical cells. This preferential expression of GIOH as well as several steps in the methyl erythritol phosphate (MEP) pathway has led to the suggestion that the precursors for secoridoid biosynthesis may be produced in IPAP cells and that an

unknown intermediate is transported to the leaf epidermis for elaboration into secologanin (Burlat et al, 2004). Further investigations into this question have been limited, since other enzymes in this pathway remain to be cloned. The present report has identified, and functionally characterized LAMT which catalyzes 0-methylation of

loganic acid (Fig. 3) and is strongly expressed in (Fig. 6), but not restricted to leaf epidermal cells (data not shown). In addition, the representation of a single MEP pathway gene {4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 3 ESTs; Supplemental table

1) and the GIOH (1 EST; Table 1) gene in the CROLFING dataset, together with the abundance of 10-hydroxygeraniol oxidoreductase like transcripts and SLS transcripts

(Table 1) in epidermal cells may mean that the complete secologanin biosynthesis

pathway is expressed in the leaf epidermis (Murata and De Luca, 2005). These results remain ambiguous and further detailed studies are required to elucidate if an isoprenoid

intermediate produced in IPAP cells is required for the biosynthesis of secologanin in

C(3//?ara7?//?M5 leaf epidermal cells. laW- i iri,. - ^,1,1*. 'Xyi

J:. ^ .? ^ ;if: m

i, \:y

f'Vv-";'iift" ^i*?-!**)! r?ui»..'^n-., V'f'''.ti-'i tr,'-fo.>i't?n om vr?-

..'- ^f J.i'i^.'^v^iA-j /li'^MfTSH'^rj •if f'" ,:sr' --.'J. .ft.TK)C

: !;-!;?, (.. ' /nv. . ,^ .iti fb* o- W (U'l m.

an !<»;) p>at. tHi^j fj Zi<,

o;;-* ...!;. 'i)

;i .'t,r'

to

a.-^l Li, .;:«. K./-^ ,^(; ,S!'l il ^•.»/;.^^«1«';i"J V .'t^^'nlH ?i >'':iti

-' ' <,'.';, ; ;i..z.:{r;?|i.v^ ,'i?i:j ,s?.»iH.M<- U.\ v<.! A^i\;'7U^-'3-^ V*\>.^i.

.?ii

.,".Ui>'';ut V-,AV. ^I'.'r.t .'tv|r-.J«i;fll':!:} ...,;; •jy.V.Viis'd'^-'.i^.^i': V'4ri,>j'\«si;^VK.CVvfe',f:V-^\. k>

.«':' .i"t*<.Xi',. 'f^fj; .^ji.-.i r'.i r.-i li-.tk'- Si^n-ilViqs 'li:-*! -yA

'*,""' ifi:T«:KJ?ov.(/ i-> PV^ib'i'/'>''-'i !:' -i L'-'r;':'^^ ! .nl^v 65

The epidermal localization of secologanin biosynthesis in Catharanthus leaf

fits their possible anti-herbivory role since the leaf surface is the first contact point

between herbivores and the plant. It would be interesting to know if other iridoid producing plants, including Plantago and Lonicera species (Harvey et ai, 2005; Peiiuelas et ai, 2006; Pereyra and Bowers, 1988), also preferentially produce their iridoids in leaf

^ '^ , * epidermal cells. vt . m

Two additional candidate ESTs in the CROLFING dataset could encode 7- deoxyloganetic acid 1-0-glucosyltransferase (Yamamoto et ai, 2002) (Supplemental

Table 8) and 7-deoxyloganin 7-hydroxylase (Yamamoto et ai, 1999) (CYP72B and

CYP72C in Supplemental Table 9) involved in earlier steps in the secologanin pathway

(Fig. 2). While these two enzymes were partially purified and characterized from

Lonicera japonica cell cultures, their corresponding genes remain to be cloned. Further experiments with these 2 candidate genes will be performed to identify their possible role in these biochemical functions.

2.5.3- Features of the active site of LAMT based on the crystal structure ofSAMT

The SABATH family of carboxyl methyltransferases has distinct characteristics which separates them from other 0-methyltransferases. These enzymes do not require any catalytic residues but rather provide a hydrophobic pocket or reaction centre where the desolvated carboxylate moiety can attack the AdoMet (Zubieta et al, 2(X)3). SABATH family 0-methyltransferases such as JAMT, FAMT, and SAMT (Seo, 2(X)1; Yang, 2006;

Ross, 1999) that 0-methylate fairly hydrophobic substrates require that the carboxyl

moiety be devoid of water in order to increase its reactivity. In contrast, loganic acid with d»

:.r''i>i'--'^. :.!''HJ:: :-'\ .••' 'i» i'=s v.j:-.i.'i,:i; ,:::»•:': -.i^-rvA tf^j;; Csjf.winV-Vv J<'i!'^i''f:-"fi ..Z^s^^iji

•*'. •i- /VZ':'. 'j.j-;':«'h: si-»

j^*\' /•'::^ •«:' lii^n^--'' [tAi^'i!^. .». \ > ^ .i: r % \ vV-»';:

EC (y *

v,>-, <,| ,..,. ,

, .'A- '. \ } •)» J'u'-'V.-

t> ,, : - !'' ?lv; Ji,: n ;j ifT'i^lf* .4if^ ^>:(s:,!'!:-r!„ ^!\;'4; .'^J^y/. m"';; K/ ?l:f;'; 1 AS.

-j/i// .>> s. >i «."»»;>>.:.'> r< ': ,:« V, i. 7t .' rii} .>r'i,-i 'u-i r-.rw£a8i»'

;I0;,'-: ri. 'K)l.^Ti;>' >-:ji, I f'/'J .'*;,;. < ...»v'i1S,'f'iUi;'!v 'j'Xf

XiKMi, '^U.V' Vii',

•-.;::.- '^ '>?.•'• ) :.':'I .Kr.ihVK- xi] -I. ! , ,; 66

its glucose moiety and its higher water solubility may require special conditions for eliminating the glucose associated water as loganic acid enters the active site of LAMT.

The protein alignment of LAMT to lAMT, SAMT and JAMT in Fig. 3B reveals that active site residues required for the hydrophobic core (Y159, W163, 1224, P227, L244 and 1320) are conserved in LAMT when compared to those based on the crystal structure of SAMT (Zubieta (2003). Two important differences involve the substitution of SAMT amino acids W226 and F347 with H245 and D359, respectively, in LAMT.

Preliminary modeling studies of LAMT with loganic acid superimposed onto the salicylic acid binding site of SAMT (Fig. 8) showed that two different orientations of loganic acid had favourable van der Waals and electrostatic interactions with the protein and could be accommodated comfortably. The average CHARMM interaction energy, over 100 minimum energy poses, for the first conformation within the LAMT comparative model was -100.57 (5.2 kcal/mol with an overall range of -113. 32 kcal/mol

to -86.78 kcal/mol. For the second conformation it was -87.98 (14 kcal/mol with an overall range of -139.29 kcal/mol to -70.37 kcal/mol. Given that both conformers have similar interaction energies with the protein, neither model can be excluded from consideration without further experimental evidence. We examined five amino acids in the putative binding site of LAMT, HI 62, H245, Q316, N305 and D359, which are distinct from those of the SAMT for putative hydrogen bonding with loganic acid. The

100 minimum energy poses of conformer 1 (Fig. 8, Conformer 1), consistently made

'good' hydrogen bonds (i.e. those having lengths less than 3.0 A) with both D359 and

HI 62. The carboxylate of D359 forms '

,»",

'l,'\'

»MA.^ ': !-.Wi Vd> ti'}^ ,,!"; ."j .>'^ TON:'// {,'j}.i{>v^^;vS/»ry>''' rtmiSo

I '\''

'.,.'0 ' , .itl. v'l'' :i ^ M !;

^/ -'^ it ;'Oit»!7i ':](;« -jV'-) fu iV.i'.- to Old) r'aDf'xj ./ii.-j,''! .( tCKjS.^ i.'SJ',;^^; i;

' *'' <• fd) ;uno ! .«:i).M bo« jint.^o! dr ''t/AJ

V'-^i;,

'(i^- -1':?.

'il-/. -. !""nj

, „('»\' ;.,>'« ,^^0 fij: "HA'

;v' i n-

>/.;'; *''.1»'>'>' -iiranity'r. ji liJOfJ 'U^i' n\y'\i) ; foAh:^^ '^.>}^i.ls.;.

/-' '. ii.-:-: jwi^'ip'i \i\\ ' ^ir!'!' nfiiO'i s>i I'lAA^': ^&i to

!'.< ' \ii''U 1 >n')'i j J ,\> -.n. ;'. •_: .i.o_ij

<>-;-?; .,.' S-,^ ilVii i,j,;/ ,,->,? ii ^j. d':r',:>\-i0\v&ii'jvj:' 67

H^ H Conformation One Conformation Two

Figure 8: Computational model for accommodating two conformations of loganic acid in the active site of LAMT. The upper panel shows the two orientations of loganic acid with respect to salicylic acid that were used to initially position the substrate in the binding site of the comparative model of LAMT. The bottom panel shows an image of the lowest energy pose obtained for conformers 1 and 2 from the 100 minimum energy conformations obtained from the modeling of loganic acid and AdoMet within the active site of the LAMT model. The LAMT peptide backbone is described by a ribbon structure and the polar/charged amino acids putatively involved in substrate binding are labeled. H245 is colored in grey to differentiate it from the sugar moiety of loganic acid. /X^! 68

hydrogen bonds to the C3 and C^ hydroxyls of the glucose moiety and the 7-hydroxyl group of the five-membered ring of loganic acid displays hydrogen bonding to the N(e) of

,;. ,

Analysis of the 100 minimum energy poses of the second conformer of loganic

acid in the putative binding site (Fig. 8, Conformer 2) revealed that in this orientation, the carboxylate sidechain of D359 consistently forms a hydrogen bond with the C2 hydroxyl

of the glucose moiety and occasionally with the 7-hydroxyl substituent of the five- ; u r membered ring. Concomitantly, the N305 amide side chain was sufficiently close to the oxygen of the 7-hydroxyl group to make 'good' hydrogen bonds in 65% of the minimum energy conformations. The N(e) of H245 also showed putative hydrogen bonds with the

C3 hydroxyl of the glucose moiety in just under a quarter of the 100 minimum energy poses of conformer 2.

The putative association of the 7-hydroxyl group of loganic acid with H162

(conformer 1) or N305/D359 (conformer 2) may explain why 7-deoxyloganic acid and 7- epiloganic acid are not accepted as substrates, since hydrogen bonding may be required for substrate binding and specificity. However, without a crystal structure for LAMT this

remains a hypothesis. Regardless, it is evident that the high Km (Table 2) value for loganic acid may be due to the tendency of water to associate with the glucose moiety

and this could affect its binding affinity to LAMT. Although beyond the immediate scope of this work, the above modeling study suggests that appropriate mutational studies of

LAMT might support conformer 1 versus 2 as the actual binding site orientation of

! loganic acid. wys a v ., 8ft

'\'>'-(/'f' ''> •" to/.j)ki mU "? -;:>'!>' !tt ni oix»Ji%«;! |f)ri i:.'-<'i'jrfj!ft'>r; "' to quoia

>it' ; :. -'<--\ A'^rviT^ .'ivk:'/

'1 ... 'Jf !

'Vt^'^-t .<' ! K

JI--J o! ->;'; >r: A

ry^VU:, -li: :. •l-.'y.vV'

'};. f-l'f vnK. ' "['.-iVj. ! rf^f\•:. r Sr ill! ,<', «!.;>-•:,?;

' •': ,t'iyp-»' yi '/Hru :a i / v i^i .'bvd "-J.!- -VOji:"**;**!:.' k-jr^j^ :>-.>. lOfl Bdu f

1^^' -I'iif./ il 5M;''' ' f'4 ff'-!', 0:' :.•! 'f?yt"^o-') '; ,--?'ii!>!.' j'if* ,^'»<=>it:

- ''^1/ "t j*i,*L jjari:' ;r-J ijr. '!>/ ^K j'''''^n('. / J ',-•! ylif-sVi^; ^f :.^^u.i ^ii !':

jj'- ' !• 'li - '.'.v fu :, I. - _, t. 69

2.5.4- Kinetic analyses and substrate specificity studies with rLAMT suggest a preferred route of secologanin biosynthesis in Catharanthus roseus.

It has been shown that LAMT from crude Catharanthus leaf extracts O- methylated loganic acid but not 7-deoxyloganic acid, suggesting that hydroxylation preceeds 0-methylation (Madyastha et ai, 1973). Studies with Lonicera japonica cell cultures suggested that 0-methylation could precede hydroxylation since 7-deoxyloganin

could be converted into loganin (Fig. 2), (Yamamoto et ai, 1999). The substrate specificity studies (Fig. 5) with cloned rLAMT show that the preferred iridoid substrate is loganic acid and that 0-methylation does appear to proceed after hydroxylation of 7-

deoxyloganic acid (Fig. 2) (Madyastha et ai, 1973) in Catharanthus rather than before it

(Yamamoto et ai, 1999). Further studies with the LAMTs of other secologanin producing species may also show that the substrate specificity of this reaction rather than that of the hydroxylase could define the order of secologanin biosynthesis.

The Km for loganic acid (12.5 mM) found for partially purified C. roseus

LAMT (Madyastha, K., et ai, 1973) was very similar to that determined for the rLAMT

(14.76 ± 1.7 mM) (Table 2). Remarkably rLAMT had a relatively high Km for AdoMet

(742.1 ± 37 \iM) compared to other carboxyl methyltransferases from the SABATH family (JAMT, Km 6.3 ^M; SAMT, 9 \iM) (Seo, H., et ai, 2001; Ross, J., et ai, 1999).

The high Km values for both loganic acid and AdoMet suggests that the catalytic activity of LAMT may be limiting the production of terpenoid component of ML\ biosynthesis in

C. roseus (Peebles, C, et ai, 2006).

Inhibition studies with the reaction products revealed that loganin was a very

good inhibitor (Ki of 215 ± 30 |xM) for LAMT, with its 50 x greater affinity for loganin %9

.'.!«:>'••;<'.'- .^r^^a.t:^.ss'l\VJ'.> vV> , it^Ur

fi^.-i'i't h-^

?.« >?.;»•%

"^ i .441

HfA^Ao 3fi: f- V ^ ...Ji.,^ 'Y'"

(f

<0; i'U i^-; /;i fi> 70

than for loganic acid. These results suggest that for LAMT to be active, loganin concentrations would need to be kept very low in the cell, either by rapid turnover of loganin into secologanin or by subcellular compartmentation. Substrate specificity studies have suggested that only loganin can serve as a substrate for secologanin synthase

(Yamamoto, H., et al., 2000). The association of this cytochrome P450 with endoplasmic reticulum membranes or related vesicles could rapidly convert loganin into secologanin

followed by its transfer to the vacuole (Contin et al, 1999) together with tryptamine for conversion into strictosidine by vacuole associated strictosidine synthase (Stevens, L., et al, 1993). In this way the secologanin synthase-mediated conversion of loganin into secologanin, together with its subcellular compartmentalization for MIA biosynthesis could prevent loganin from inhibiting LAMT.

2.5.5- Catharanthus leaf epidermis as the exclusive site of oleanane triterpene biosynthesis.

The plant kingdom produces many thousands of triterpenes exhibiting a broad range of pharmacological activities based on over 80 different carbon skeletons produced

from (J5)-oxidosqualene (Ebizuka et al., 2003). While the accumulation of high levels of

ursolic acid in Catharanthus roseus has been documented (Usia et al., 2005), the sites of accumulation of these products have not been elucidated. Studies with other plants

showed that ursolic and oleanolic acid accumulate on the surfaces of apple (Bringe et al.,

2005) and of grape berry exocarp (Grncarevic and Radler, 1971) as well as in the epidermis of grape leaves. In other studies with Avena strigosa, the expression of ^- amyrin synthase was localized to epidermal cells of root tips that accumulate triterpene avenacins (Qi et al, 2006). The CROLFING dataset suggests that the mevalonic acid 6X

>'< lESiO^^^viJ '--!:( V'i t^bl^' fli'j':' m'u f •-'i^ 7:'i7 r.Ym '>ii O! t ' .-JtiaDtJ

-! ' ^f^»*' It; -!€ t61^ k)l>Hl^gUP

< 1

/«*

Ui-.-A> V> '^\ii

r*?f '•JvJbfr? •vf^'.>"Vi i^» iiwct^in J'«i:rtaft:'> -tavu t'.iT lidv!;*1 ifj-'i^.x.- J -,»'tliv(j'j4,

k* ,^aJ!-; ';«J ,(<'t>01 ,;.k. v^i.ii'**'.?'! jj.'Ufi'jvR-Vrift ft4>-^i>i/r,lf t^ssi^io,'' f»i(Mi1rt«"'.,VM^:v»3

C -t):

'»'! «. yjniitr} -j-ffj^Tu'io f3->Ui!(v ;.'(!: :>i^- •.«»:..•_,- 'iU

-''i t'/ H

.t''~ ;-f\'j': ' J fi 'ill 71

pathway (Supplemental Table 1) as well as two key triterpene biosynthesis genes

[squalene monooxygenase (5 ESTs) and Ji-amyrin synthase (14 ESTs)] (Supplemental

Table 2) were preferentially expressed in Catharanthus leaf epidermis. The selective and quantitative extraction of surface leaf triterpenes with chloroform (Fig. 7B, C), suggests that once they are made inside Catharanthus leaf epidermal cells, they are secreted where

they accumulate on the surface of leaf epidermal cells (Fig. 9).

It is interesting that Catharanthus leaf epidermal cells are the site of biosynthesis of triterpenes that are secreted into epicuticular wax as well as of

secologanin that is then incorporated into MIAs. The more frequent representation of 5

MVA pathway specific genes compared to the single representation of a MEP pathway

gene (Supplemental Table 1, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase) does suggest that MVA pathway transcripts are selectively expressed in leaf epidermal cells compared to those of the MEP pathway. These differences might be attributed to the greater demand for biosynthetic precursors for triterpene biosynthesis, since triterpenes accumulate at much higher levels in Catharanthus than do MIAs (Usia et al, 2(X)5).

Further biochemical and chemo-ecological analyses will be required to elucidate the mechanism of triterpene accumulate in the leaf epicuticular waxes, their in vivo roles and the involvement of the MEP and MVA pathways in Catharanthus leaf epidermal cells in relation to MIA and triterpene biosynthesis.

2.5.6- The possible roles of the plastidic MEP and cytosolic MVA pathways in secologanin biosynthesis.

Previous extensive analyses of the MEP pathway in MIA-producing plants

have suggested that the terpenoid moiety of MIAs is predominantly, but not exclusively :

\[ J-'lv

' ! -, (

" ' .'!, ) :>>':.< V. n

,< >5 -f 'I -:rMU

t'^i /

M ')

V 'it.?' l..M...Ci«i(j[tnj'

.-.', I - " 'A , .: I ^ I' ;-">fJ! CW ?*-j1

^'u : t; ! (<;'', !T' is ii>-

if i' '.'^^ -J .'

' 'it.i.^' ^U '. , 'i. >i!> vii^.;:; k) r

' 1- •: 'i 'vsr;,^ ;;:».;: I I,

I,.: i,

-j^;. vM, \. 72

Surface wax layer VLFA8. tmerpenes

Figure 9: Catharanthus leaf epidermal cells express the MIA, mevalonic acid/triterpene, very long chain fatty acid and flavonoid biosynthetic pathways. The EST sequencing analysis described in this study suggests that the complete the mevalonic acid/triterpene pathway is expressed in leaf epidermal cells and that the triterpenes being produced are exported outside the cell into the surface of the leaves. Similarly the entire pathway for very long chain fatty acid (VLFA) biosynthesis is also expressed in this cell type and is exported to produce the surface wax layer of Catharanthus leaves. Flavonoid biosynthesis also appears within these same epidermal cells where flavonoid products accumulate within epidermal cell vacuoles as suggested by Mahroug ef al. (2006). The model also proposes that most of the Vindoline pathway up to and including 16-methoxytabersonine is expressed in the leaf epidermis and an undetermined

intermediate is then transported to adjacent mesophyll cells, specialized idioblasts and/or laticifers, for final elaboration into vindoline. In order to permit this complex transport, strict control mechanisms appear to be operating and these have yet to be characterized. In contrast to leaf epidermal cells that are enriched in the MVA pathway that supplies IPP for the biosynthesis of

triterpenes, there is strong evidence that the internal phloem assisted parenchyma cells (I PAP) preferentially expresses the MEP pathway as well as G10H. This raises questions about the roles played by these two cell types in supplying the necessary IPP for the biosynthesis of secologanin within the leaf epidermis.

73

derived from MEP pathway in cell suspension and hairy root culture systems of

Catharanthus roseus, and Rauvolfia serpentina cell cultures (Contin et ah, 1998; it i ',

Eichinger et al, 1999; Hong et al, 2003). These results are supported by studies in

Arabidopsis that monoterpenes are produced principally via the plastidic MEP pathway

(Lichtenthaler, 1999). In the case of Catharanthus roseus, recent in situ RNA hybridization studies showed that the MEP pathway genes, DXS, DXR, MECS as well as

GIOH, the first committed enzyme in iridoid biosynthesis, were expressed predominantly in IPAP cells (Burlat et al, 2004) of the leaf vasculature. Recent immunocytolabelling studies provided more quantitative information to corroborate the high enrichment of the

MEP pathway in IPAP cells, but it was also found at low levels in other cell types including epidermal and mesophyll cells (Oudin et al; 2007). The expression of GIOH was used to further suggest that MEP pathway-derived isopentenyl pyrophosphate was converted to 10-hydroxygeraniol or another derivative for mobilization to the leaf epidermis for further elaboration into secologanin and MIAs. While the baseline representation of MEP pathway genes compared to those of the MVA pathway in the

CROFLING dataset (Supplemental Table 1) provides support for the MEP pathway

enrichment of phloem parenchyma (Burlat et al, 2004), it also establishes the differential enrichment of the MEP and MVA pathways in each cell type.

Since only a single GIOH EST was detected in the CROLFING dataset, it does support in situ hybridization studies that show preferential expression of GIOH in phloem

parenchyma (Buriat, et al, 2004). Remarkably, it has been implied that the biosynthesis

of the similar iridoids in leaf beetles belonging to the Chrysomelidae family is divided 'iW'lu> JCiC . . ri' ft: /tr' ' il Liivii^l)

\y: {"{ |i-. -

:.'?' ui;!,' .;!l r^^iiy:jr^i'- w ?Jm > s^ J ^(K':: Av''v» -i^KiH ,9«^i ,

Jr..>;?) .,,u^^'-'

J -rrr^? .H.^'if t:

t ?'li •"),;<' / . i .

>.'.'. ':' ..*<, 'i:'*!'' < y^ChY \ < ^^K*! i^f itu•Jl"^'Vi^i ty-i'jiii

:\i' i".J; '. M '-1

!f; ..'v-ti. 1. ^ A'/?/ b;/. MSMiM;

I. ,.' >» > " -^UK,! '•- >r ill o;

^ > >''•' :,'.- fi ; ^v.V\-t:» t .%;.( ,-:'>)!

;fi, *v ''.>'.-.!'"'>' '••' '

: ;i . I^ ':':':. ;, : -J (!, \\ ,/ :i,vrr!' .!' »> '^t ;'. fjiobru 74

between the fat body and a specialized glandular reservoir that also accumulates these defensive secretory compounds (Burse et al, 2007). The fat body expresses the MVA pathway as well as a geraniol hydroxylase and glucosyltransferase to produce 10-

hydroxygeraniol-10-(9-p-glucoside. This glucoside is then transported to the glandular reservoir where a P-glucosidase releases 10-hydroxygeraniol for enzyme-mediated

oxidation and cyclization/isomerization for the formation of toxic iridoids (Burse et al,

2007). In the case of Catharanthus MIA biosynthesis, the identification of a similar pathway step or intermediate in the IPAP cells could be very helpful to completely prove that MEP pathway derived precursors are transported to the leaf epidermis from the internal phloem parenchyma for the biosynthesis of secologanin (Burlat, et al, 2004).

Similarly the hypothesis that leaf epidermal cells alone are competent to supply sufficient

MEP pathway intermediates for secologanin biosynthesis and MIA accumulation in

Catharanthus leaves also needs to be fully tested (Murata and De Luca 2005).

The unique biochemical properties of distinct cells (i.e. epidermal cells, mesophyll cells and phloem parenchyma cells) with respect to IPP biosynthesis are clearly related to their complex roles to produce different metabolites. For example, neither leaf epidermal cells nor IPAP cells, in contrast to those of the palisade and spongy mesophyll, require IPP for biosynthesis of chlorophyll and yet these 2 cell types require increased expression of the MVA/MEP pathways for their unique metabolic needs. The data presented here highlight the complexity involved and present novel approaches to study these questions. ."A

->'• '"'>>i '!> ,'•; *.

> /:-•;' % ' :j-- i'-| = - \^ : * 'iinii_.l!*j3 .f.v

f; • j'' .^^" ' '.''' •' .rj . . •''|/;ri!,;J .. ri^r. .,,,i^ ^f(4|- .^ M

•».. '•I.. Ji ' r' ••/; tr

Mi!.;L.ii'>i!t'^i>- .^ir- o)- ;,f! A -ltd Ai(/ tH'. .ivr.-nV'i 1 V' r.d< ni

;i.,., • •ir, . "y'l'i £/. ' Ij I)'.

'^'f'i^.

(«,'--jt^: f jrv.-> "(J

ii'iO fy.h j-K-;,p 'f:'.'-; „,

^n.i'

•'(','- ...... J/-) •' ur^l.^-i; ./i'tri -y of ^jr ;'.' o-^t:- ^eo

ji ' • «.•* h: I , . >.! ; I'v. 'ts '•!)>

-l-rv/si.-^ fif:?-}

>' i' i.^;M

^bfi. :it.ij 3

:Ui .' ' •:' 1'

-iJ 7 /*-. 'iinvi'j jih j-bi^if ff ^'.d 75

2.5. 7- The versatility of CA technique and its possible use for epidermome analysis.

Unlike LCM, CA can be used to harvest epidermis enriched materials from

intact leaves, making the analysis of mRNA, active protein and metabolite analyses

possible without significant technical difficulties. The technical simplicity of CA

technique makes it valuable for: (i) localizing enzyme activities to the leaf epidermis

(Murata and De Luca, 2005), (ii) large scale purification of leaf epidermal proteins (D.

Levac et ah, 2008), and (iii) targeted random sequencing of cDNA libraries produced

from leaf epidermis enriched mRNA, as shown in this study.

Our effort to establish leaf epidermis-specific ESTs not only revealed the

capability of the epidermis to manufacture monoterpenes and triterpenes, but indicated

the value of the CROLFING dataset as a gene discovery tool with respect to various

pathways in the leaf epidermis. For example, numerous putative candidate genes involved

in fatty acid biosynthesis were identified (Supplemental Table 4) presumably since fatty

acid biosynthesis is elevated for the production of cuticular waxes that are secreted to the

surface of the epidermis. Similarly, Catharanthus leaf epidermis may express the entire

flavonoid pathway (Fig. 9), as suggested previously (Mahroug et al, 2006), since the

majority of the genes encoding the putative pathway for flavonoid biosynthesis, from

PAL to DFR, were also represented (Supplemental Table 5). It is noteworthy that the

whole pathway for flavonoid biosynthesis appears within the same cell type, in clear

contrast to the vindoline pathway that appears to require the involvement of 3 or more

leaf cell types (St. Pierre et al, 1999; Burlat et al, 2004; Murata and De Luca, 2005).

VLFA and triterpene alcohols, on the other hand, are synthesized in the leaf epidermis

and are secreted out to the cuticular wax layer (Fig. 9). The wide range of biochemical ' '

:' .-A. ( V-' -W M .1 '.I'

-' '!'(»') -*..'«'<. ;:'-,; t- ,r'- f^wj /'-. , -,r ) . ,M'.;j ^)

.•i.; / •; .:,:vr In. J 'i. 1 ^ .;,c/':V, *:>:!.!

ji

>•>,'»;! »' *•;. n>, j< r>i J a. '

ii'f )^ ^.>>j":\:.i 'iv. lii-^ .'fti ^ :^ ..k^

'" 'I..';; I

ij" .)'i: /•«'> ,; 'K -irilJld/ t"?!'' i'^iC'-» ) -lli'i '\Q 'jy

'\ i' -V-r , :h.!.vff.n '=f,'

fii^.i ' 'an'^ • s'-y^/ ,.). iri:'

a!; '»!:; ii*;- J . ;;vH;'.»!5

' ") ^'>:i>(\ : tr! i>K'. :<. '; /; :C" . ''UJ i^«t4:i--.Tv -•'nil •)i~'':

; ' / ' r I o^'=

."•, >i 'i . jf:'!:- ^;i' )!.

'. y. -'i. l^.-.!), 'iui'

l.!',>f' I * :i:ii. . .V' , 'l", yi

•v'' n(> ,,';('.

•i I , il •') , h" ii' ':i J'.. / r ry.M' 76

pathways described here require the movement of different end products within the cell, outside to leaf epidermis or towards the mesophyll and suggests that the leaf epidermis also expresses the complex transport mechanisms and controls that permit the proper distribution of metabolites to appropriate destinations.

The discovery of entire pathways for the biosynthesis of triterpenes, VLFAs and flavonoids also promote the usefulness of CA technique as a very promising tool for global profiling of gene expression, enzyme activities and metabolites within the leaf epidermal cells. CA technique should in fact be considered to carry out analyses for any

plant where the specialized biological role of the leaf epidermis is of interest.

2.6- Acknowledgements

We would like to thank Dr. Larry Pelcher and his team in the DNA Technologies Unit for

DNA sequencing, and Jacek Nowak and Dustin Cram in the Bioinformatics Unit

(NAPGEN at the Plant Biotechnology Institute, National Research Council of Canada,

Saskatoon, SK) for their help in sequencing and in annotating the ESTs. We also thank

Glenda Hooper (Brock University) for technical assistance in scanning electron microscopy and Tim Jones (Brock University) for mass specirometric analysis V.D.L. holds a Canada Research Chair in Plant Biotechnology. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to V.D.L and by a joint NSERC Strategic grant held together with Dr. Peter Facchini, University of

Calgary, AB) ';i\

:'' ;t>t1? II |'ii-'>;i'<. >ry' i>j.?.t'^p' ; :\(; /i!-,.;'> jfn 9

?'?C'if'-<.1..':rU .U;

;i''l /\'> '.:; ;.>: ••.f i^

)i':. ' r:

t..''U'i.''~i ''^ -r*i-i--'^ ^o ri'Ji. -js'^tii^nJ otuMtifboJo: 'i^ Uyjj''!

'j)'.- %:\ii-'m-j- '\'' ^^'/'-•n h-'.'Hifi'i < vA (f%.--i')'n(ili :•

> '^<-i\-:- •i>'^ tyytf -*.'iW i J, >(<><> ^-^hui! j^a^uH u \ii^iO ({^\iVt

^" ^i ! ^-\.V \., t:'>tn^' irwrM'j _ ,/

>^;<'.. .' .:K*r: ti ^(JV* a1 i='. )i V.':'4 'n. i'-, ',)('jHr.'»ft? '>,'i?3^K int' 77

2.7- Accession Numbers

The EST sequences in Tables 1 and 2 as well as in Supplemental tables 1 to 10 were submitted to the GenBank (dbEST) database (accession numbers FD660726 to

FD661300).

2.8- Supplemental Data

The following materials are available in the online version of this article

Supplemental Table 1: CROLFING genes related to isoprenoid biosynthesis. Supplemental Table 2: CROLFING genes related to downstream terpenoid biosynthesis. Supplemental Table 3: CROLFING genes related to flavonoid biosynthesis. Supplemental Table 4: CROLFING genes related to lipid biosynthesis. Supplemental Table 5: CROLFING genes related to methyltransferases. Supplemental Table 6: CROLFING genes related to acyltransferases. Supplemental Table 7: CROLFING genes related Cytochrome P450-dependent monooxygenases (CYPs). Supplemental Table 8: CROLFING genes related to glycosyltransferases Supplemental Table 9: CROLFING genes related to 2-oxoglutarate dependent dioxygenases. Supplemental Table 10: CROLFING genes related to transcription factors. '

X\

•t I •:•' 'ii-. pi \i: ;i.?ft,> 'ti;t. ! Y^;^ !>}('{

,yiv.

-:!((' •>l. : a; " <.

..•s.^a 78

Chapter 3.

Spatial Separation of Early and Late Iridoid Biosynthesis within C. roseus Leaves

Jonathon Roepke -ih- '> ll iiil< •,'..«,-f- 79

3.1- Abstract

Spatial localization rather than substrate availabilty seems to be the limiting factor in

monoterpene indole alkaloid biosynthesis. As enzyme assays for the first committed step in iridoid biosynthesis geraniol 10-hydroxylase (GIOH), demonstrated a specific activity

ratio of 90: 1 (Whole Leaf: Epidermis) suggesting that its enzyme activity is preferentially localized to the specialized internal phloem associated parenchyma cells of C. roseus, while loganic acid methyltransferase (LAMT) a later step within the pathway is enriched

; ic' in the leaf epidermis. However, metabolite analysis of epidermis enriched extracts from

C. roseus contain only 2-5% of the total secologanin found within the whole leaf,

suggesting that once secologanin is produced within the leaf epidermis, some of it is exported from the site of synthesis to other cell-types within the leaf body. Metabolite extracts from leaf, flower, hairy roots, stem and siliques indicate that secologanin

accumulates and is 10-fold higher than the major monoterpene indole alkloids vindoline and catharanthine. The organ analysis revealed that the leaves, flowers and siliques are more biosynthetically active in producing iridoids and monoterpene indole alkloids than the roots and stems of C. roseus.

3.2- Introduction

Secologanin is an iridoid glucoside which is found in several plant orders

including the Oleaceae, Gentianales, and Comales [Soler-Rivas, et al., (2000)]. Within plants that make this metabolite, secologanin can be stored directly in vacuoles [Contin,

et al., (1999)], it can be converted into sweroside or into different dimers [Jensen and

Schripsema, (2002)], it can condense with polyphenols to form terpenphenolics [Soler-

Rivas, et al, (2000)] or with primary amines to form monoterpenoid indole alkaloids '' "Ijj; .

ii -.r.^ ' I

, '''K::

u,-yf:^ >' j :!b'k fcfif/f*")i;:?!j,i! h';]:, i'i--. »; KjUl'; fiBu;. ., ., . rn';:,;i -^fl:! oi b^

r ijii

!u.';^ .'ivK-i'-ii' 5ujl;'rmi» an^tt^jl^-ij lo • -k' . ^i^rVt:?!^ ;K»yjwr;lt .^>n;T*i-

;. *> !/ ;:><•./ xirnjuta:v 'i;^.! »(!! (-j: T.;?!0 r

; .:5(*'-!f.w:j*» ifjfit j;i;j,t';''i .'^Wj.iiii^ b/ir, rr^-^

.. ti-v- J« -^^m •/.- (yim fVino.'Si"'' > >'^ n«i.

"'j -•:--".:f! : Cj ctlr« 1

;-• -r"-.n' \')./ti;'. i^ic. ,>'-|-;:;^ ri; 'p ,. Muifxn ytiiiCiKj iiJi^tf 10 {(4j> 80

(MIA) [Thomas, (1961)]. An important class of secologanin derived MIAs include the commercially important anticancer agents, vinblastine and vincristine, that are produced and extracted from Catharanthus roseus. Within this plant the biosynthesis of secologanin may involve at least two cell types, the internal phloem associated

parenchyma cells (IPAP) and the leaf epidermis [Burlat, et al., (2004); Irmler, et al.,

(2000)]. The IPAP cells appear to preferentially express the methylerythritol phosphate pathway (MEP) as well as geraniol 10 hydroxylase (GIOH), the first committed step in secologanin biosynthesis, whereas the leaf epidermis appears to express loganic acid O-

methyltransferase (LAMT) [chapter 2; Murata, et al., (2008)] and secologanin synthase

(SLS) [Irmler, et al., (2000) and see chapter 2] involved in the last 2 steps in secologanin biosynthesis. Within the leaf epidermis secologanin can be converted into MIAs such as

16 methoxytabersonine by the vacuole localized enzyme strictosidine synthase (STR)

[Stevens, et al., (1993), McKnight, et al., (1991)] together with other pathway enzymes that include as tabersonine 16-hydroxylase (T16H) and 16-hydroxytabersonine-16-0- methyltransferase (160MT).

However little is known about the levels of secologanin found within

Catharanthus leaves or if it is entirely incorporated into MIAs. Studies with C. roseus cell suspension cultures have suggested that all of the secologanin accumulates within vacuoles (15 \ig/ 1.54x 10^ vacuoles) [Contin, et al., (1999)] and other studies with seeds

show that 0.8% of their dry weight is composed of loganic acid [Guarnaccia, et al.,

(1974)]. This pool of loganic acid decreased to 0.2% of the seedling dry weight after 8 days of germination and to 0.4% of the dry weight in mature plants [Guarnaccia, et al.,

(1974)]. The detection of secologanin within cell cultures together with the significant \\' t f :/iv. 4) :)t! ,' ir

•!' •: ,' ''"Jm;'''1"I '-'A' < l'.Ktj;/ 'l- , L •):>4i'';--)'' «(. riiHOo

'i! , :l' ^-Vf! '^ ' J'' - -'v » ORfc

', '.>-«ij •/!<;-i '''j?;m ir:"?';! T/':i -. V .Vx .4 ,((

•' If.;-. u .:'.. \~:i: >:. 'j 1, I'u;^..!":, ij'-'(

•.!j. '., /•q •; •!:i:^^^'.» 'iM;j.«r •>;!? -yu.x'i .:,.;jf 'T .-fii'y

:j: i> - 'i'i'Tuu .,-•*

O m::.- -JiUvviAi <.'•!, ,-[/:3 I ' f^-':: K) ;r. :; f. .yy.i tr^:.i -j.ij

l-(.^f:jf ' ):>" 7<«''i i>'i: .. >^ > .l-'US'l*^ S lUi<;,^:VJ\

./'?• -i! ' »:.: •,'''=. ..-•/>? - •jvh:;/.:-;i 'jfi /'If . Sii^\ «»[» u;

ii\ /

:'i - :'«•': ."'i; it ' -,. ' / v.<^r' .M; './ rj:\<-:Xi. .; i >•> ,< ,_U\ ,;. 154.1 u-I:jM ,1 W

,' •; %.-!o "v'iSf: j; i/. u-'Vi' _v ._ '>i ^>n»;; n;-"',

.( Ftv*

. -^j-i =''/ ^^"ni ^tv^li•>.-: 1*. .. ..-»f*r n- Kh;d ts

!:•'.•:(. "*! ,,,/ ' ib'l':(' yAif-^ ...i-ii >^r'»n< /l''W!(: .' -•, fi !u r;

" '.' /'' >•• ,',.:, ;,,- ;»--_) . ^i^.:..i.ri! i!f.:..i «d , ;|)

':\ ' j-- i 1 \'iU'-f 'Aily-r^: !;-.i' , lifir •' I- -fi,',|-C''.)! (>->1 ;''

J' «i('r^*j' .S i'".'i. Jf -''ifV" 'J -I !'*?!fV'-

' •.: tH •!' :t J' ^iii r : y.

i'' J'J':' .1 IV,'. J !;;• :•' , V, ,/'l •'(-. hn,; a

{' . ' ' ;->.>/ ' "H J .i'.'i' .tiii;."^' • i:'>< 1- 81

amounts of loganic acid detected in mature plants have raised questions about where in plant leaves such metabolites will accumulate. Despite the high levels of loganic acid in mature plants, the terpene moiety has repeatedly been reported to be the limiting factor

MIA biosynthesis [Morgan and Shanks (2000); Peebles et al., (2006)]. Feeding experiments with terpenoid precursors have increased the level of MIA's within cell suspension and hairy root cultures, but this depended on on the metabolic precursor used

and the growth phase of the culture [Moreno, et al., (1993); Morgan and Shanks (2000)].

Other studies with transgenic C. roseus cell lines over-expressing strictosidine synthase

(STR) demonstrated that both the indole moiety and the iridoid moiety could limit the

accumulation of MIAs [Whitmer, et al., (1998)]. Studies with developing seedlings demonstrated that the enzyme activity of tryptohan decarboxylase (TDC) was optimal in five day old seedlings and then decreased to -50% of the activity in 6 day old seedlings.

In contrast, the STR activity peak was broad, between 5 and 7 days and dropped by -50

% after 9 days [De Luca, et al., (1988)] of growth. The developmental regulation of TDC suggests that the tryptamine levels may change transiently for MIA biosynthesis;

however there is limited information on the iridoid pools within C. roseus throughout development. This study decribes the metabolite profiles of iridoids in relation to the major MIAs, vindoline and catharanthine, within Catharanthus leaves of different ages.

3.3- Materials and Methods

3.3.1- Metabolite Extractions

Individual leaves of C. roseus (Brock University Greenhouse) were harvested and their weight was recorded. Single leaves were frozen in liquid nitrogen and pulverized in a mortar and pestle. Methanol (5 mL) (Fisher Scientific, Nepean, ON, oi «>r'fi MiV'''-r. '.'V M rtgi^} a^'f M'i-.-i-' .:>lr^Juu:ij •>« lirvj -r. )rtSliI

•><]{;?: . i'/ix^';;; i-'y-v? -i ^ }f; !u .-.ff.-'? ^.kbtif:

t . U. i.

"•>>/ Uu j':;^ .\;'ii^r; ».'..:V of-" 5fii i»4U ^.^

' vi V i 1 Xi'jm'- i/i;:: -ky- '"tfi ?: 'i'>3-.'' ,Di:'n j «a* ^li^q ^tKi;:)B V\''

.i' S ," i 'i

<'-%'!

/fcortl';.

' ?.'^--' ':>> L; ji -"/"ii- .-^'M'-iifj /.V.^/.-: ^ O O

i' . i* jrfwv: a r.-t; -I' .A /.;>/-:,! jl'in:^

'/f ' ' I'.Ku,;; 1/ >,^•y^ U.ti ^iirtotfi t ^i l»\i', 82

Canada) was added to each mortar and pestle and the leaves were homogenized. The

slurry was then added to a 15 mL tube (SARSTEDT, PQ, CANADA,) and was placed on

a shaker for 30 minutes. Alternatively, epidermis extracts were prepared by harvesting

individual leaves and placing them into 15 mL tube (SARSTEDT, PQ, CANADA,).

Carborundum (Fisher Scientific, Nepean, ON, Canada) (0.1 g) and 3 mL of mQ water

was added. Samples were vortexed (Genie 2, Fisher Scientific, ON, Canada) at speed 4

for 1, 5, and 10 minutes. The leaf was removed from the 15 mL tube and the aqueous

solution was removed and lyophilized. The leaf was then frozen with liquid nitrogen, pulverized in a mortar and pestle and extracted into 5 mL of methanol (Fisher Scientific,

Nepean, ON, Canada). The samples were stored at -20 °C until further analysis.

Metabolites were analyzed with UPLC-MS-SQD^^ (Waters, Canada). The analytes were

separated using an Acquity UPLC^*^ BEH Ci8 with a particle size of 1.7 ^m and the

dimensions of 1 .0 X 50 mm. Analytes were detected by both photodiode array and mass

spectroscopy.

3.3.2- Iridoid Analysis by Ultra Performance Liquid Chromatography-Mass Spectroscopy Detection

Solvent A: 0.5% Formic acid (Water) Solvent B: 0.5% Formic acid (Acetonitrile) Wavelength: 240 nm

Table 1: UPLC Elution Gradient -Iridoids

Time (';. ;''.- M 4 ;,-i. , .litv >:J.,

A'-', -^'-i, irr f VTru;

'- - 'i'.

'' i'" ' .>'v^/ -'i : t,^ ' -. . v- ,. ii'j I

>". iVfi. ;;^ i I'M < <^' "' ,/','

r [M a: > t-'O .aiH;) •.»<.? , Sl

111! :V!; '".' f ;.. --vi^o

•' ^-.-i !fiv . : n-f '!^V'j"'t v'.^'Oi;

•':' !r^v^ ,; >''«;vS>;M'f . xr r :i.i'."'ii

i^y '• 'j- v J* iv U .

r.f ,T TV, >.'.;, //,. y!.:-;>i'(' •..:*?, J'T

; '-'.I.'. ,:

•*• ^i:: ;^^r ^ ;!.;,- > 0- *- , .J<"' -f' If/ -jrvkfw' -.tii,- (K X '

:'.>A>iS» ^;"t*'

J~. ; If •:.',,, 11

-fi '

'^X

.-0'.i 83

3.3.3- Alkaloid Analysis by Ultra Performance Liquid Chromatography-Photodiode Array Detector (305; 280 nm)

Solvent A: (MeOH: MeCN: 5mM Ammonium acetate) (6:14:80) Solvent B: (MeOH: MeCN: 5mM Ammonium acetate) (25:65:10)

Table 2: UPLC Elution Gradient- Alkaloids

Time / 'J -' r\:^'

>^ \ Ol'v

I-:. 11!

-'*-* v..

): 1 if.", w

i©T-A;c°'':>fK?^ !, 9^'-tCiQ nS'ti,'

i>: 1

(»r.

A n- \V..A v,^

'< •"...{?-,}(

. li: 1'; »; '/(

i. I -.l'.^ Vi.:::-. .. :^t J" Vf M ;i 84

New Jersey, USA) until they reached an O.D. 6oo of 0.5 (UVA'IS Spectrophotomer,

Ultrospec 3\00pro, Fisher Scientific, ON, Canada). The cuhure was then induced with 3 mM ITPG (Bioshop, Burlington, ON, Canada) and grown for 16 hours at room

^^ temperature on a rotary shaker (120 rpm) (Innova 2000, New Brunswick Scientific,

New Jersey, USA). The cultures were harvested by centrifugation (5000x g) (21000R,

Thermo International Equipment Company, Fisher Scientific, ON, Canada), the supernatant was removed and the bacterial pellets were stored at -20 °C until needed.

3.3.6- Isolation ofLAMTfrom E. coli cells harbouring pET30b-LAMT

Frozen E. coli pellets were resuspended in 4 mL of 100 mM Tris-HCl pH 7.5, 14 mM p-mercaptoethanol. The bacterial slurry was sonicated (Setting 4, Model 100, Fisher

Scientific, ON, Canada) on ice, 45 sec intermittent pulse followed by 45 sec rest each cycle was repeated 3 times. The sonicated material was centrifuged at 5000x g (21000R,

Thermo International Equipment Company, Fisher Scientific, ON, Canada). The supernatant was added to a PD-10 desalting column (GE Health Care, Uppsala, Sweden).

The eluent was directly used for enzyme assays.

3.3.7-Enzyme assays rlAMT

The standard enzyme assay 100 ^L containing 4 (xg of crude desalted protein, 7.5 mM S-adenosyl-L-methionine (Sigma, Oakville, ON, Canada) 1.3 mM Loganic acid

(Chromadex, California, USA) and LAMT enzyme assay buffer (100 mM Tris-HCl

(Sigma, Oakville, ON, Canada) pH 7.5, 14 mM p-mercaptoethanol (EMD, New Jersey,

USA) and 25 mM KCl (Caledon, Georgetown, ON, Canada). Varying concentrations of inhibitor secologanin (Gift in Kind Jochim Schroeder, Germany) and sucrose (Sigma,

Oakville, ON, Canada) were pre-incubated with the crude desalted protein for 15 minutes '- ..">-:' ,i •«) :'r. . i f,; ).'''' ,•'- ;i (> J. V i, ( ; ..''i'il< 4 ly^il^

..* ^> /"t "/ 1-. . :.'< >wr ' :

'• ' )• .*» ' ;» : /. ,i ".--'li :v'.-'< Ti^-:?-! •.v''' Vr >] , (Oi-, ; t'.'ri.r^JJK^''

••fi. I i

't itiU :"v'-'i.i ',;(,;

,^ f! ii :;.: y iK' tu o^i,

•( -^ > . i--:.y

< . i.f "' H.| nr Mr 'X>: .t'i ' ;; n ,,^ O-^wn-l

;>'; '!'

>'/ 1'^ ' '!''.- ^>'.',l!^ -

1' ' !v: I 't;

' ./ .. j • ) / tvCC':'/"' ?-iy>'i; !ii; .{ \:y

;.>'. I .::';i., J I'.' .

'a.-'^'h :.vv^vw)ot h-sf'.ii /(*'3rfr:t?>

-.. •>! CT ": '<- ; .,;;-' .' .4-D

W J.!'., -I :i.':;*.,:)

>-,-. '^ •> ^i . .A r!V: .! TKr., ;/.^-J ai.:-"!titfc

•• .' a-ij f' V i . Hf, :.^'>/iDi.

iir I (^ !!'.^\- .!><;' •^-. ')

-' I !- ; ni:'f,-.p;oc»>' V

>'f'i :^i'j*^' 85

before adding substrates and cofactors. Enzyme assays were incubation at 37 °C (Hot water bath, IsoTemp, Fisher Scientific, ON, Canada) for 30 minutes. Assays were immediately frozen in liquid nitrogen and lyophilized overnight. The dried material was resuspended into 200 |xL of 0.5 % formic acid (EMD, New Jersey, USA) and 50 [iL of acetonitrile (EMD, New Jersey, USA). Samples were centrifuged at 10,0(X)x g

(Micromase RF, Thermo International Equipment Company, Fisher Scientific, ON,

Canada) for 10 minutes. An aliquot of 200 ^iL was then filtered though (0.22 [im) filter

(PALL, Mississauga, ON, Canada). Enzyme assays were analyzed by UPLC Iridoids

Program.

3.3.8- Geraniol 10-Hydroxylase (GIOH)/ loganic acid methyltransferase (LAMT), 16- hydroxytabersonine 0-methyltransferase (16-OMT), N-methyltransferase (NMT) and deacetylvindoline acetyltransferase (DAT) Enzyme Preparation and Assays

Whole Leaf Enzyme Preparation: Fresh C. roseus leaves (Brock University Greenhouse) were harvested (6.0 g) and frozen in liquid nitrogen. The leaf material was pulverized and homogenized in 12 mL of extraction buffer [(100 mM Tris-HCl (Sigma, Oakville, ON, Canada) pH 7.5, 14 mM P- mercaptoethanol (EMD, New Jersey, USA) and 25 mM KCl (Caledon, Georgetown, ON,

Canada)]. The homogenate was filtered through 2 layers of nylon and centrifuged at 500x

g (2 1 OOOR, Thermo International Equipment Company, Fisher Scientific, ON, Canada) for 10 minutes, the supernatant was added directly to a PD-10 (GE Health Care, Uppsala,

Sweden) column for desalting. The desalted protein was used for enzyme assays and the protein concentration was determined with Bradford assays.

Epidermis Enriched Enzyme Preparation:

Fresh C. roseus leaves (Brock University Greenhouse) were harvested (6.0 g). The leaf material was added batch-wise to an equal mass of carborundum and a total of 60 mL of * . > !«»! 1 '•/' ? (i,; >. ::v':i :

ij' • ^-if.;- . •! !'. 'I --iyHj! 0' 1, } '-'C , >'>-h' :'-

.iiK,. 1 ).ii! ^ !«(. HlJlTiS

'^ ; ,' !. , hiu A ; .vr;.'!^ . ,.;

'] .' •!( • , ;''i!i.-! u"; Iff.!- ','.:•]' (•:. ' ,f I .

( ' -- ^.» . \ 0' :t v.c i-j «*. 'Hjr,,. n 'ti 1

ij-'iT:' . > t,ii, ^^:»'! r Tiifi, Mi/

'T'f".'''!!}'',

> ^ * ... .n-r ,-': v-lD /i.f^C

".!*- 'X 1 : -l^i<.v<{^>fTl• 'ii J: J ; '.?.'. i/lt^i /iitv/

' . I ,* -»H. ?v, ,i. .,*:.;'.;. 'r- yi:/- .^^ci^ti) s :iii i!i'i i.\ii,iy.n)\ fjiUir^

'" "- > <^"' i ';. {l ^:3.f^. • :A

'<', • rt i • .cj'Mi , , u;'»i /ii i. .: •'a4! ;. ,:*fl' ?.l,-*l 'H.'-

1- 'i:i-. /'f...' . !;.'>'iU

"-' ">' ; ' M'. 'lUa ( : 0?^i u '... f- '.4J

*,. >;.t; iV

^ f/ ', -;1.'1( .-.'•' Il!',i !>' I"! - .

>/-ia?j:rt { -'h^K if^V' ;...! !'!, 86

extraction buffer [(100 mM Tris-HCl (Sigma, Oakville, ON, Canada) pH 7.5, 14 mM P-

mercaptoethanol (EMD, New Jersey, USA) and 25 mM KCl (Caledon, Georgetown, ON,

Canada)]. The leaves, carborundum and buffer were separated equally into two 50 mL

tubes (SARSTEDT, PQ, CANADA) and vortexed (Genie 2, Fisher Scientific, ON,

Canada) at speed 4, for 3 minutes. The leaf material was removed from the buffer and the

samples were then centrifuged at 4500x g (21000R, Thermo International Equipment

Company, Fisher Scientific, ON, Canada) to separate the carborundum. The buffer (25

mL each) was then filtered via vacuum filtration through a 0.45 |J.m membrane (PALL,

Mississauga, ON, Canada). The other 5 mL of buffer was used to resuspend the carborundum. The filtered material was then concentrated (Millipore, Etobicoke, ON,

Canada) to a final volume of 5 mL. The concentrate was then desalted on a PD-10 column (GE Health Care, Uppsala, Sweden). The eluent was used directly for enzyme assays and the protein concentration was determined by Bradford assays.

GJ OH Assay Conditions: The complete assay for the epidermis and whole leaf assays were slightly different, each

assays conditions is described below:

Epidermis:

Geraniol (1 mM) (Sigma, Oakville, ON, Canada) was incubated at 37 °C (Hot water bath,

IsoTemp, Fisher Scientific, ON, Canada) for 60 mintutes in the presence of 1 mL of epidermis enriched protein (resuspended carborundum). NADPH (Sigma, Oakville, ON,

Canada) (1 mM) was added at 10 minute intervals over the course of the 60 minute assay.

Additional control assays included (without geraniol; without NADPH; Boiled Protein).

'(•< m

"' ; • ' iv '/. .'/Xj 'U'-'K^ r- >i ,,, \^ . t :. ;-^i-y f-"

.) J .

5 • 'r,. :.i-K^

'/•i'.v ' :H.(Ehim'

!iV

•:/-i;;Km '

f>''- ' »i..' liiioq!:'; !>«* lU-".^ 'AO \^'

*? .^'-^ 111 K f.j'*a>:>b A=)fu s^/ ^!y'i."^v*:/;iu' • .r" ,.If^^ ?; :C fiwoio'

' Jl-', ' ;

•Vt: -. :j 01-- 87

Whole Leaf: Geraniol (Sigma, Oakville, ON, Canada) (1 mM) was incubated at 37 °C (Hot water

bath, IsoTemp, Fisher Scientific, ON, Canada) for 60 mintutes in the presence of 1 mL of epidermis enriched protein (resuspended carborundum). NADPH (Sigma, Oakville, ON,

Canada)(l mM) was added at 10 minute intervals over the course of the 60 minute assay.Additional control assays included (without geraniol; without NADPH; Boiled

Protein)

Sample Preparation: After the 60 minute enzyme assay, 2x 500 |iL of ethyl acetate (Caldon, Georgetown, ON,

Canada) was added to the reaction mixture. The samples were vortexed (Genie 2, Fisher

Scientific, ON, Canada) speed 10 and centrifuged at 10 OOOx g (Micromase RF, Thermo

International Equipment Company, Fisher Scientific, ON, Canada). The organic layer was pooled and dried to dryness (Speed Vacuum, Thermo Savant, Fisher Scientific, ON,

Canada). The dried material was resuspended into methanol and analyzed by high performance liquid chromatography. The geraniol and 10-hydroxygeraniol were monitored by photodiode array detection at 210 nm.

Radiolabeled Enzyme Assays [LAMT, 16-OMT, NMT, DAT]

Epidermis:

Loganic acid (1.3 mM) (Chromadex, California, USA), 16-hydroxytabersonine (60 |iM),

2,3-dihydro-3-OH-N-methyl-tabersonine (60 |iM) was incubated with 220 000 dpm of

['''CJ-S-adenosyl-L-methionine (GE Healthcare, Buckinghampshire, UK), in the presence of 100 |iL of epidermis enriched protein. The enzyme assays were incubated at 37 °C

(Hot water bath, IsoTemp, Fisher Scientific, ON, Canada) for 60 minutes.

Deacetylvindoline (60 )iM) was incubated with 220 000 dpm of ['''C]-Acetyl-Co-enzyme .

* ' .-y/ iCi !• : a'ci-fif'^.Ti jy

.: ';!. tA x : ' ''

•/ ir..

-•-; msm.' /: >iv--n. V ,; .>(-!•-

•i-Si

(>>./.* '}' "J' 0' ] 1^

:Mt...; -'>!-" -;. < <: r ;.f»/M?o- ; •;': - yji,. ...i„^y:iin i''-^'-'- -:/'U ]:'A -.iif (

('•' ;'\ ' ^-r: ) 'uiii l>'j.v; fei,. P..

' .' i'.i-.k-i .<.:'M'

i'ijji/; ' - /;/-ii> '• I. ,.: ('.i^r- .;.. ,. . .*!"> >.,. -i U' ') yfl .!<

-i '-' ; J ?r- i

'' i .! V 'u .; 'ill . J-

('// (. -!;,' . ...

;(! i, 88

A (GE Healthcare, Buckinghampshire, UK) in the presence of 100 |xL of epidermis enriched protein. The enzyme assays were incubated at 37 °C for 60 minutes (Hot water

* • bath, IsoTemp, Fisher Scientific, ON, Canada). - r.i i -:

'i- - . Whole Leaf: , i-, v t ;'.v>. ;ii ^:^

Loganic acid (1.3 mM) (Chromadex, California, USA), 16-hydroxytabersonine (60 |xM),

2,3-dihydro-3-OH-N-methyl-tabersonine (60 |iM) was incubated with 220 OCX) dpm of

['"^CJ-S-adenosyl-L-methionine (GE Healthcare, Buckinghampshire, UK), in the presence of 100 |iL of epidermis enriched protein. The enzyme assays were incubated at 37 °C

(Hot water bath, IsoTemp, Fisher Scientific, ON, Canada) for 60 minutes.

Deacetylvindoline (60 ^iM) was incubated with 220 000 dpm of ['"CJ-Acetyl-Co-enzyme

A (GE Healthcare, Buckinghampshire, UK) in the presence of 100 |i,L of whole leaf protein. The enzyme assays were incubated at 37 °C for 60 minutes (Hot water bath,

IsoTemp, Fisher Scientific, ON, Canada).

Sample Preparation of [''^CJ -labelled Alkaloids (16-OMT, NMT, DAT Assays):

Enzyme assays were stopped by the addition of 20 |4L of 10 M NaOH. The alkaloids were extracted into 2x 500 |xL of ethyl acetate and dried. (Speed Vacuum, Thermo

Savant, Fisher Scientific, ON, Canada). The residue was resuspended into 10 |iL of methanol and spotted on to a TLC (Macherey-Nagel, Pennsylvania, USA). The reaction products were resolved in (9:1) (ethyl acetate: methanol.). The radioactive products were stored on a phosphor screen (GE Healthcare, Canada) for 16 hours an the emissions were detected using phosphor imager FLA-30(X) (Fujifilm, Tokyo, Japan) with multigauge ver.

3.0 software (Fujifilm, Tokyo, Japan) the radioactive spots was scraped from the TLC for

in.; quantification by a scintillation detector (Beckman Coulter, Mississauga, ON, Canada). -r A

\,l-i V-

•>;.l; 'i^ »

'•' !i/w . :

•;',) (... .«. .' X- t -'.-J! ^/ !f!;:f).ii'*'f ; ''/. !'f^h-;

•' ^--'f "•- d

'' ,A, ," . ;-.«:f' Ci ,:< M'. i i

'-',••' 'I, i"'.; •../!> H ,i..;r ".':,, 0(

.' , C'' y - :i\

' '• ^. --'... .' '•! -''Hi , ..4:.,; : .' ": ^,lii :-{r>

---^ < •fn'-

* y- : M\i»ir<,S<^

r( ,,,< u, ,_, ,-v / , ; ,v rVr;^'

'•! ',. "i.- > : T' f.h.,'

.•"<' h':.-- ; • :\ii<\ r:'^ -rirf

• < .-'(. •i'. .t ' -1 M !ir ^ ;3-

>5'j;\ i--:vi -i'K

>.l :':.. n>t> v '!

to ;jn(- 'I {^

' ".r ' I •. Ki.

<5i.i;]n! iU.-

Sample Preparation of [ C] -labelled Iridoids (LAMT Assays): .«, i;'

Enzyme assays were stopped by quick freezing the assays in liquid nitrogen.

Samples were then lyophilized and the dried material was resuspended into 15 (xL of methanol and spotted on to a TLC. The reaction products were resolved in (7:3)

(chloroform: methanol.). The radioactive products were stored on a phosphor screen (GE

Healthcare, Canada) for 16 hours an the emissions were detected using phosphor imager

FLA-3000 (Fujifilm, Tokyo, Japan) with multigauge ver. 3.0 software (Fujifilm, Tokyo,

Japan) the radioactive spots was scraped from the TLC for quantification by a

scintillation detector (Beckman Coulter, Mississauga, ON, Canada). -

3.4- Results

3.4.1- There are important pools of secologanin in different organs ofC. roseus

Since glucoside and methyl ester containing iridoids are acid and base labile, respectively, various plant organs were extracted in methanol without further processing.

To investigate the accumulation of these iridoids in planta, a reproducable and robust

UPLC-MS detection method was developed that clearly separates and identifies 7- deoxyloganin, loganin and secologanin by their elution and molecular ion profiles in a short 5 minute run. While each iridoid standard could be detected by UPLC at 240 nm, their poor UVA^IS chromatophore makes them difficult to detect with a photodiode array detector in crude plant extracts that are contaminated with other analytes. Instead MS

analysis was used to detect and quantify each iridoid based on the intensity of its m/z +1 ion (Fig. IC, D). The m/z +1 ion data representative of a young Catharanthus leaf extract

(Fig. IC) shows that secologanin (m/z 389) accumulates in this organ but not 7- deoxyloganin or loganin. The same results were also obtained with stems, flowers and , ,

, H

^ \\ y -"^

i< 1 I'' !l

• 't , -i^

'i.\ '/ i I 'Ji

• ^, - , ^

li" ? THt •VlV/,'. 'h' --, ^':

*ii;} :» •- ,.. Mil ). ',.'.- i-',' *ty)' '-/I. ..;'..':::

' >'*' v.>v V-- .•^ : • ,;c ;»•.,.. "'- '.>^''- .i'%1. , 'it-v> - ;, v' '•^n''*^--*'''' 'I'^s^ V

''f;.' 1;=";. -<" '! '/"-''' .Jli; ( if ;:..i .• 1;

,'-iA :-'. ?it;';ii'i

" !( ^«'i,<-(.^ \ :>i.- '^r'^t,, iisiv'; t^. "'/U

U 1 • >

r I: ',.fV

,ll..- f.i, ; ..i , ,f:ih

' )' 'iV ! , 90

siliques. The mJz +1 ion data also suggested that young leaves contain small amounts of

an uncharacterized metabolite with a mass-to-charge ratio of 375 mJz but it does not co-

migrate with the 7-deoxyloganin standard, nor does it posess its absorption spectrum.

All organs analysed accumulated secologanin with high levels found in leaves (3.5 mg/gfw), flowers (2.9 mg/gfw) and siliques (4.5 mg/gfw) compared to the low levels found in stems (0.75 mg/gfw). Remarkably, secologanin levels in each organ were significantly higher that those of the major MIAs, vindoline and catharanthine that occur in stems, leaves, flowers and siliques (Fig. IB) with the highest ratios of MIAs to secologanin occurring in leaves (0.59) and the lowest in flowers (0.0087).

3.4.2- Large secologanin pools are found in leaves of different ages and in hairy roots compared to those of vindoline and catharanthine.

Leaves of different ages were harvested as individual leaf pairs, where leaf pair one was the youngest set of leaves observed next to the leaf apical meristem and leaf pair four was the oldest leaf pair tested. Individual leaf pairs were extracted and analysed in

triplicate (Fig. 2, /-///) for their content of secologanin, vindoline and catharanthine. Since there was considerable variation in the levels of these metabolites between replicates,

each replicate experiment is presented separately (Fig. 2, /-///) to show that in spite of the variation found between datasets, the secologanin content per leaf increased with leaf age and was significantly higher than the alkaloid content in leaves of all ages. While the levels of secologanin, vindoline and catharanthine varied between datasets, secologanin u.f f,

^'"' :' >,'; : . 1' 'i- jJj'w U i! 'Vu *V^ itl'm ;- .; •'..jjii: t; 'f^'i

.K'»'ij--y .: h«.;. ;; '^L-vjf ,:v-!i »h-

;.l-

i-„- ;i

i^'-' i' a." _,. U/O:!?,! ;i'.i.

.' « .n. V M» tfi^1^>ji 5 ^^IWJ . . , . ! \.l ?>•«<>

'; >-..,;-' i '" 'i;--' b?i;. , JiKji: h vr'.rjtj: i/ T' 'yvo-j^iS ^a'/i.*}} lo^ ilNS Jri;>"

.': T.-«»v".-wt; 'iii-: 91

A)

GlcO/,,/0

H3CO

Catharanthine

B)

^m. (A. Onin

L?

00'.?*

'»^ '>0'

'*- % *-lA. ^M, •at. 92

(800 |ig) levels were up to 2.8- and 8-fold higher than those of catharanthine (280 p.g) and vindoline (100 jig), respectively (Fig. 2Aj & An). Depending on the replicate (Fig. lAiii), higher levels of secologanin, vindoline and catharanthine could be found in leaves but in all cases (Fig 2Ai-iii) high levels of secologanin accumulated compared to those of catharanthine and vindoline. When metabolite levels were compared on a per gram fresh weight of leaves, secologanin levels decreased (Fig. 2, Bj & Biii) or remained relatively similar (Fig. 2, Bii) with leaf age. However secologanin levels remained high compared with those of catharanthine and vindoline, as shown with the measurements for total metabolite content within individual leaves (Fig. 2A).

The hairy roots of C. roseus produce different MLAs than those above ground plant parts, such as tabersonine (5-10 ng/gfw) and the tabersonine derivatives, lochnericine (40-160 [ig/gfw) and horhammericine (5-50 jig/gfw) (Fig. 3A & B). In spite of the different MIAs that accumulate, hairy roots, like those of above ground plant parts

(Fig. 2), also contained high levels of secologanin compared to MIAs (Fig. 3B Replicates

/,//, & ///). When hairy roots were harvested into sections of different developmental stages (Fig 3C), each stage contained higher levels of secologanin, compared to MIAs, but levels of each metabolite/gram fresh weight decreased with the age of the tissue analyzed. '. •'.,/ 'i. [' . !.; 'I .-.' 'HI JTiAW ^Is-iT-'Sl d^l] 'V

^' \ i ' •, '.,.:) ;:. :; T 'ib. . ,( Ij 'M5 \i"^-K(f,'>(

v;r-'/ ,i '^r.

t:.r,.,ir -';.'.':T.

< ;T •<:'> r ', ' r''''-^.-' V\n']i j.- ;ii j{iu .1*' r '^.t

r ' • > ' '-' Jj1(' cm I 'i '>. '» ;.. 1 J

• ^>' ,;:' ' rhu. >.';'rri^i .t-f i 'y.i .' li, -y ; n

:^' , ,()-', , ftj ,- if ;> ,^' ,u'.r,:i,-' .;t > ;, ^ r-.

' / ,iiS>[i!i-'^>' .MKirrv i(!

i - I : ' . i .' -'l" Lm

*/' ' ''.'' !)'' ': ('.V; ( ii t ,ii, :V'ii. ri:: < V,

\ T' f . ;;-!;; "i''' •.}:: il " i' x ''!'-. " /y* ''l*.'l

^t. - >.' «);:•,. ^JM .'iS'; I iMft

' . [; A; K< i . i './ I' J .) ,! NV-,

.i')' 'i': 'ii;'.;' I'iVi)-: ii-,»..'

-' I i ''[•'nr-in ^

93

A) B)

|60oa

|40(»

o> n. J" 2000

I JM yfl •s 1£1 1234 1 Leaf Pair Leaf Pair

2 3 Leaf Pair

Vindoline

Catharanthine

Secologanin

Figure 2: Quantitation of secologanin, vindoline and catharanthine in C. roseus leaves. The results are expressed as A) micrograms of secologanin, vindoline and catharanthine/leaf or as micrograms of secologanin, vindoline and catharanthine/gram fresh weight of leaf The analysis was performed in 3 replicates (;, // and ///) with using individual leaf pairs from the youngest (leaf pair 1) to the oldest (Leaf pair 4) .0

« ^

(

;;» # i I "1 r 94

3.4.3- The terminal two steps in secologanin biosynthesis occur in the leaf epidermis

'. "^ but secologanin accumulates elsewhere in the leaf. _/ -

Previous reports have suggested that the early stages of iridoid biosynthesis leading to the production of 10-hydroxygeraniol are expressed in internal phloem associated parenchyma cells [Burlat, et al., (2004)], whereas LAMT [Murata et at.,

(2008)] and SLS [Irmler, et al., (2000)] that catalyze the last two steps in secologanin biosynthesis are associated with the leaf epidermis. CA technique has been used to differentially extract metabolites from the leaf epidermis [Murata and De Luca, (2005)], but care must be taken not to over abrade the leaf in order to obtain representative results.

To control for these limitations, metabolites were extracted and analyzed respectively,

after 1, 5 and 10 min of CA (Fig. 3A) by using vindoline as a metabolic marker for over

abrasion since it accumulates within cells inside the leaf (idioblasts and laticifers)

[Murata and De Luca, (2005)], (Fig. 3A). Since vindoline first appeared after 5 minutes

of CA, a minimum of 2% (Fig 3A, 1 min CA) to a maximum of 5 % (Fig 3A, 5 min) of the total secologanin appears to be associated with the leaf epidermis. These results suggest that while biosynthesis is completed within the epidermis, most of the

secologanin must be exported to undetermined cells within the leaf where it accumulates. •.. •t (J 'V,-,',. , ,

>i '. ' l»ft,! •••.,<".; "I'vKti •f^'i^jfp-;

• ' -y t- 1.1''.- , -I 'i

' >' ". ';.v '.•?i,' 'I--. ' ':>. (,

'<:'.--. •'"' ^^ ,(.;'>-; i .. :, :ii. SI'S' : i

- -., >.•-.. ,1,. ,f. .<.i(< "•- :• ;:0 . '''U. i i'vr^AX. . 'r, T;..

'"' I ii -. ,::''''•. 1 , i;>;*- -V. f;( 'r ?;„ 'n^'T Vii |A£ ,'* 'V.> fa;;.

-).; ij"i .i'i- .!•( :, ;! \v\ - i-tn--'

^•' .>,••'?»»'( i-jii-ior } \\ - ("'\'. ^;.-i; so'rj :ia I

-» ',- '^1-' C ' t

• ': - I ':,-->'i I: : / . (! .' ih i,:,(rVo J O:

-.,-J> • :j,V: i?!"';.

;'>'!' "1 '1 '' '^ ; i:' i i") ':(.;n.s j'niii u! tvjir j/'iv'-i 95

A)

''X Ti Multiple Steps T19H ' " ^ ,OMe

H OCH3 0CH3 0CH3 Secologanin Tabersonine Lochnericine Horhammericine Root Specific Alkaloids

B) C)

_1500 ^1 200 if"' fiit 100 l| 50 •'^!tf .)-!n«dr^T

vO

) I

V?-'*?^

'.1 A 1.1 '.STl IT 96

Geraniol-10-hydroxlase (GJOH) converts geraniol to 10-hydroxygeraniol and is

the first committed step in iridoid biosynthesis. In situ hybridization studies showed that glOh transcripts as well as the MEP pathway were preferentially expressed in IPAP cells

[Burlat, et al., (2004)]. Other studies using laser capture micro-dissection to harvest the different cell types within Catharanthus leaves and RT-PCR suggested that glOh transcripts could also be found at some levels within epidermal, laticifer and vascular cells [Murata and De Luca, (2005)]. To document the distribution of GIOH at the biochemical level, the enzyme activity of whole leaf extracts was compared with that of epidermis enriched leaf extracts and that of carborundum associated proteins extracts

(Fig. 4). In addition the distribution of GIOH activity was compared with those of J6- hydroxytabersonine 0-methyltransferase (I6-0MT) and loganic acid methy[transferase

(LAMT) that were used as markers for the leaf epidermis MIA pathway, and to those of deacetylvindoline-4-O-acetyltransferase (DAT) and of 16-Methoxy-2,3- dihydrotabersonine-A^-methyltransferase (NMT) that were used as markers for the MIA pathway for cells within the leaf (Fig. 4) [Murata and De Luca, (2(X)5); Murata et al.,

(2008)]. While low levels of GIOH activity were found within epidermis enriched leaf extracts, whole leaf extracts were 90 times more active (Fig. 4B). This data is consistent with the IPAP localization of GJOH transcripts [Burlat, et al., (2004)] and with the production of 10-hydroxygeraniol in this specialized cell type. ;.*.;<-) •*>\ • 'W t.i , s" -t'lm^ni

)''-'. <.!; I i!''' i H ':.(!

.<»,,' ::'' ..':', /'.,.,.;" f i _»i:i 'ft- ;.^-;; c4. ?qr>3^i;

H I.-J

. ; \/M':.: .'()!' ;.r1'- ; -ivisv"*/ -^i/* ^i"''. !«:I'j

It- . >!! v: -.fi .r-i' ft K;: n ' ;!J';>'5

• '" ':' I . b : -r*,! ;i-»U, HMr.ul 'I

'< , 'iv- -,1- . , 'fji'. ?rv ' it >* >/ '-r'V:

. '(.I : . - . > J 'Cji '.J . -I!!!!

l>>:^'i'if' ';• ^ }'•'. ' *'.- W.J if ».^ ''i" ."O- (.

./.. ^ ,/;.

'I ' '• !fi. • . :» '7:ii;.ir. >i, r>i

:r, >' V . \\i. ' .'V'li.*

i;;f- ;.. ,;A\-3iHj;i>.'lii.

> ' J i ;tv- .Ij (1 v

! . v/=i *! '>,jri^ .1

''•;< ^ > 1 ! '

t^ '> i; . H' v= ^"•'t'Ni'^.t'v

.<;:' -.'s'J -U \i 97

A) B) 20 E S«colog«nln 25-1 s Vlndolln*

a>- Hi.,

= 4 rk G1CH LAMT 160MT NMT DAT 1 5 110 Carborundum Abrasion (M In)

C)

Epidermis

Figure 4: Localization of Iridoid Biosynthesis. Panel A describes the cellular localization of iridoids. Panel B: Enzyme assays of carborundum abraded leaves compared to whole leaf extracts, indicating the fold enrichement which the epidermis. Whole leaf specific activites: G10H- 184 ± 14 nmol/ng protein; LAMT: 0.69 ± 0.02 pmol/ \ig protein; 16-OMT: 1.89 ± 0.05 pmol/ pg protein; NMT: 0.47 ± 0.01 pmol/ pg protein DAT: 0.37 ± 0.02 pmol/ ng protein. Panel C: A model representing the cellular localization and spatial separation of the Iridoid biosynthetic pathway and the monoterpene indole alkaloid (MIA) biosynthetic pathway. IPAP-lnternal Phloem Assisted Parenchyma cells, MEP-methyl erythritol 4-phosphate. (A

*f

V t m

J4 i I

«a»«n& «f«nrs w^ta "-»jr* -I- ^«\r*» - . - — 1#-

»

':. I ri

.c . ..

X

! < 98

3.4.4- Feedback Inhibition oflridoid Biosynthesis

Since LAMT is subject to strong feedback inhibition by loganin [Murata, et al.,

(2008)], its possible biological role as an inhibitor was investigated based on reports that showed that availability of secologanin was a limiting factor in the biosynthesis of MIAs in Catharanthus cultures [Peebles, et ah, (2006)]. Since the present studies show that secologanin, but not loganin accumulates (from 750 to 4500 |ig/gfw) within different

Catharanthus organs, the inhibitory effect of secologanin and sucrose which have some structural similarity to loganin (Fig. 5A) were tested for their LAMT inhibitory activity.

Neither secologanin nor sucrose inhibited LAMT activity at the concentrations tested (Fig.

5B).

3.5- Discussion

3.5.7- Biological Function oflridoids

Iridoids are a special class of monoterpenes that are characteristically differentiated by their highly oxygenated pentacylic ring and the presence of a glucose moiety [Jensen and

Schripsema (2002)]. Structurally, iridoids are very effective at cross-linking proteins as they contain two aldehyde functional groups which can readily react with lysine residues

[Konno, et al., (1999)]. Remarkably, iridoids are not only produced by plants as defensive compounds, but certain insects have also evolved similar iridoid pathways for their production or to opportunistically sequester iridoids from host plants that they consume

to protect themselves from predators [Ananthakrishnan, (2001); Penuelas, et al., (2006);

Burse, et al., (2007)]. Another proposed biological role for the iridoids is in

osmoregulation although direct evidence for this is lacking [Voitsekhovskaja, et al.,

(2006); Beninger, et al., (2007)] or in carbon transport from source leaves to sink tissues. .'u,-.^ ''A^\ -\. t^.

. -'. i"fv .' J J-Jlt.

\<-' '': ! ) i" '•''rjl.," %i\i i . ' rj!

'':• ^'.A*'' r-'. ,: •.i; :,.". '(.

>' .''i'l-w. ^\iU

.' .: 1 (. Uif • i- .'Cd;- '«-..IflO

' f 1 o;

>5?t'si;/-

V;, Vi.,..r -w

';-.; *'i2 K Jit yhU)

:ii.r\'K. ' !•

' ' i

'Ami

./•' ' n a'. ) .!S f* .Oiifv

] 5.. ?i' ;:, nnitt!'*

".-..'r t 'J .><'

?f :h ;.

v,i:!,a 99

A)

O^ OMe

OH

Secologanin Sucrose

B)

O 24 I H ifl il " t H I Seocologanin Sucrose O E Q. 6

12 25 65 128 190 260

Inhibitor |i IVI)

Figure 5: Putative Inhibition of Secologanin with LAMT. Panel A- Chemical representation of the inhibitors emphasing the structural similarities between loganin, secologanin and sucrose. Panel B- Inhibition assays of MMTwith secologanin and sucrose as the putative inhibitors. !

0e

mf

H

? *

L ..J,

! - '

I

i I _,l \ .'I:

/.i\ 8\' :*%. ss i;?

iiB'y'TiiiiAO 100

For example the iridoid, antirrhinoside constitutes 39 % of the carbon transported in the

phloem compared to sucrose (48%) in Asarina barclaiana [Voitsekhovskaja, et al.,

2006]. Furthermore, when Antirrhinum majus source leaves were fed with ["^C]-C02,

15% of the fixed carbon was transported through the phloem as labelled antirrhinoside

[Beninger, et al., (2007)]. Inspection of the structure of iridoids and sucrose reveals that they are structurally similar (Fig. 5A) since they both contain sugar moieties and highly oxygenated rings. These biological functions may provide insights into the original biological roles played by iridoids such as secologanin that with the evolution of the mixed MIA pathways in plants like Catharanthus roseus also became substrates for their biosynthesis [Thomas, (1961)].

While it has been well established that iridoids such as secologanin are metabolic

intermediates in MIA biosynthesis in C. roseus, it is not known if they have additional biological roles as end-products that accumulate within the plant. To establish the incidence of secologanin within Catharanthus extracts, different organs were analyzed for the presence of iridoids and their levels were compared to those of the MIAs, vindoline and catharanthine (Fig. IC). The high levels of secologanin and the significantly different ratios of iridoids compared to these 2 MIAs in leaves and in flowers, suggest that the iridoids may have additional roles than to simply supply a precursor for MIA biosynthesis. However, futher data needs to be collected to substantiate this hypothesis. ,i:i<

?ri' i!' ,:>;obr,i

i. I'iu .\:'^0r>^, ..1;, i-i ,:-

';),,;»> ' A-'' '>-]. I'A ! r.' y:- Af^l' bi'

I; "'iq bf!'. Ki.

• ,y . ; .-/

'i;!i: afeiOM, ' i\> 00(11.

I : >- n

',:•' mm

''' ,.j.'^/t .1 101

3.5.2- The Iridoid-alkaloid ratio remains constant as the leaf matures but varies as the roots mature

Previous studies with TDC, STRJ, NMT, D4H and DAT indicated that their enzyme activities were most highly expressed within young leaves and tissues [De Luca,

et al., (1988); Vazquez-Flota and De Luca, (1998); St. Pierre, et al., (1999)]. These results suggested that MIA biosynthesis occurred within a short time period in young leaves, leading to the accumulation of MIAs. However, assays with extracts from leaves of different ages showed that the specific activity of LAMT only decreased to about 50% of the youngest first leaf in the older second, third and fourth leaf. This data suggested that regulation of the iridoid and MIA biosynthesis were under different controls as the MIA pathway was mostly expressed in the youngest leaves while LAMT was also active in older leaves. These data are corroborated by the accummulation of secologanin (Fig. 2A/-

iii) with leaf age, as well as lower levels of accumulation of vindoline and catharanthine.

3.5.3- Different levels of secologanin are detected in Catharanthus organs,

Iridoids are found in most organs and can be highly accumulated. The secoiridoids of

Oleaceae are found in the leaf and fruits (seed, pulp and peel) constituting 6-9% of the

dry weight [Soler-Rivas, et al., (2000)]. The iridoid loganin of Loganaceae (Strychnos nux vomica) can accumulate up to 5% of the fresh weight [Dewick, (2001); Zhang, et al.,

(2003)], while the metabolic precursor loganic acid only accumulates to 0.8% of the dry

weight of seeds [Guarnaccia, et al., (1974)]. Similarly, C. roseus accumulates loganic acid (0.8%) but not loganin in dormant seeds while its level drops to to 0.2% in 8 day old

seedlings and loganin levels increases to 0.1% of the fresh weight of 8 day old seedlings

[Guarnaccia, et al., (1974)]. The analyses of iridoid accumulation in different C. roseus '.'•'ii'.J

J.l'' .'. --i^','. ) ^>C',- 102

organs (Fig. 1) showed that stems, leaves, flowers and siliques accumulate secologanin, but not loganin or 7-deoxyloganin. The accumulation of secologanin varied from 0.07% of the stem fresh weight to 0.45% of the silique fresh weight. While these data suggests that iridoid pools within C. roseus are low as compared to those of other iridoid accumulating plants, they are large compared to the levels of MIA alkaloids found (Fig.

1-3).

3.5.4- Cellular Localization of Iridoid Biosynthesis

The biosynthesis of iridoids has been investigated for more that 25 years with early

literature suggesting they were derived from the mevalonic acid (MVA) pathway [Coscia,

et al. (1969)]. However feeding studies with [l-'''C]glucose combined with NMR labeling

patterns using Catharanthus roseus cell cultures [Contin, et al., (1998); Eichinger, et al.,

(1999)] and hairy roots of Ophiorrhiza pumila [Yamazaki, ef a/.,(2004)] clearly showed that the secologanin component of MIAs was derived from the 2-C-methyl-D-erythritol 4- phosphate pathway rather than the MVA pathway. In spite of these results there is a growing amont of evidence that under certain conditions, there is cross-talk between the

MEP and MVA pathways as both pathways share isopentyl pyrophosphate (IPP) as a common intermediate that could lead to the production of geraniol that is a the precursor

for all iridoids. The first committed step to iridoid biosynthesis is the oxidation of geraniol to form 10-hydroxygeraniol. This intermediate is then oxidized to the dialdehyde

10-oxogeranial which serves as substrate for the iridoid cylclase [Uesato, et al., (1986);

Uesato, et al., (1987)]; Sanchez-Iturbe, et al., (2005)]. The cyclized product is then

further oxidized to form iridotrial [Uesato, et al., (1986)]. Iridotrial is then glucosylated

and oxidized to form 7-deoxyloganic acid. This intermediate is further oxidized to form '

.i«. vnt! •v) .^in^.;:

.IT .( i- (11 :'iO ',>{. . V} '^>n tun

' ..»j -J , ' f i

., I ',><;-. •.» • ,. '. . J ii!/!nv/

ii; , '^/ ,!•,' '> ,'!«. O';

/'^liC' 1'- I'. (', .:. m-, .:*;t;>Uh»'>' .Jj^-S;''

' ;??/ ,(V,' ' Jl iljl, 3'-'''>,'.'0 i

' . 1 >i ' M .. ••V ; 'I .: . .t^ 7 .V'*!.'.r(

rK- '.,.,..;•' .•>

* ' .•'.' i ^

(. '. ^«»; - - /•: V -r - P'i ,-'] ; 'i! 'i- -.a)

• i;r;T- '-Cft-' *rt«?i.

"> r-t.

' ' ' -• I ; 1j jiu'i

' ) 1 ;,ir

'I i! ' ';^:' ' , -ii). ;*

':i.> .,^>«<^r;

hC}\th<.-% 103

loganic acid [Kanato, et al., (2001)]. Loganic acid is then methylated to form loganin

[Madyastha, et al., (1973)]. Loganin is then further oxidized to form secologanin

[Yamamato, et al., (1999)]. The biosynthetic order of the late iridoid biosynthesis (post- cyclization) seems to be species specific.

There are two hypothesis of where the iridoid' s are synthesized with C. roseus

leaves. The first hypothesis implies that the iridoids are produced in the internal phloem assisted parenchyma cells (IPAP) and an unidentified intermediate is transported to the epidermis where the last two steps of iridoid biosynthesis occur. The second hypothesis suggests that there are two independent sites of synthesis for iridoids one in the (IPAP) cells and other in the epidermis. Evidence for the first hypothesis is derived from in situ hybridization studies. Their data suggests that the transcripts for the MEP pathway genes

{1-deoxy-D-xylulose 5-phosphate synsthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (mecs)) as well as (gWh) the first step in iridoid biosynthesis were enriched in the (IPAP) cells

[Burlat, et al., (2004)]. The limitation of this data is that the in situ data is only

qualitative; it is at the experimentor's discretion to determine when hybridization has been saturated. Another limitation of in situ hybridization is the probe design, probes can bind non-specifically and need to be stringently designed especially when genes have high sequence similarity such as cytochrome P450's like GWH.

Evidence for the second hypothesis is derived from reverse transcriptase polymerase chain reaction (RT-PCR) from laser capture microdissected (LCM) leaf tissue [Murata and De Luca (2005)] as well as an expressed sequence tagged (EST) library from

carborundum abraded leaf tissue [Murata, et al., (2008)]. The RT-PCR data suggests that €ar

iU J .1

i.Ti • ;::•'>'- J i4 (Vm>ioJ !;y;;ii -irir: M rnd- ( /M /•»!.;,•. J .ft?"'*^'

niiysri'.

ij^OT .> iiv\-' h H^.'- •' -Ji JT.

i''>Oififi tU|ft*i(S; :tf|: Hi 'i: 't').-r.;'r;! "tl h::li

i

..:^!-> (>V

."•^ .V

t'.s? I'O !'^;'l"1 ^)V4-f'ji! i' :5..v:. /{.

fl'WVH- '.!', ..>/»-">(- '•'i '^ 'a.'.: Ki^; t,.

.^'\ '\-a\:-^ir i .Vj.1: .••;?,.M/;'j j.

' .\^;Vr•'. • .\. , / J \ '",^r'V' ,\'-:\"'''^\',f \ J

-. v f •- , u> SiV','/ '. •...iri 'Ja t;;. I.:-;i ul

i.f> ci Ui't; !'''.v/, » y^l U!d) -'; uii.b /.'. ] \o /K-'i^Minti ?>i

'i.;ii r;' ". ': ; . , I M

'<-;>' .'^f't^j I'i:'- (i.i;<' ,•'. I' "a- ' U;.^'.yHA> 'X]

'I. .j,'' •' •: 'f^sy u^q V'i;''.r.'.v i : /i : !':>,; Uj'- .(

-.'•. ^^:j :.,:',;; i. :sf r '• - i k

: ', ' ;! V tfi iC ., 1 , i.'-:-

>]'>; > /: •.%^'r ...i,-. I ;i -.-J <_ . r 104

the glOh transcript is expressed in the epidermis, laticifer and vascular tissue to a similar extent but is not found in the mesophyll or idioblast cells. Sequencing results of the EST

library showed that there was 1 EST for GIOH and 1 gene for the MEP pathway 4- diphosphocytidyl-2-C-methyl-D-erythritol synthase (MCT). The limiting factors with these techniques are similar as the first in that the RT-PCR is only semi-quantitative and the primers designed for amplification may not be specific to one gene but rather a few genes. For example within the epidermis enriched EST library there are 3 GlOH-likc genes each having an E-value below E"'^, and these three genes were sequences 23 times

in total [Murata, et al., (2008)]. Additionally, Oudin (2007) labeled hydroxymethylbutenyl diphosphate synthase (HDS) a marker enzyme for the MEP pathway with an immunogold antibody and localized the protein to the IPAP, mesophyll and epidermis cells. Their data suggested that these three cell-types are capable of producing MEP-derived terpenes; however the epidermis had 100-250 fewer HDS's (gold particles) as compared to the

IPAP cells.

The results obtained from metabolite, and enzymatic analysis to localize early and late steps in iridoid biosynthesis suggest that while the terminal steps in secologanin biosynthesis occur within the leaf epidermis, only a small percentage of the total iridoid pool not incorporated into MIAs accumulates in the leaf epidermis (2-5%). This data

suggests that while an intermediate from IPAP cell (Fig. 4) is transported to the leaf epidermis for elaboration into secologanin and into MLAs, a second pool of secologanin

is transported out of the leaf epidermis back into the leaf body for storage in an undetermined location. Unfortunately, this data can not account for transport of the iridoids from either the leaf body to the epidermis or from the epidermis to the leaf body ,

if!9Jy.:>

' \' V. I /..-> I

-• - -• ii.( J! '-ftotfjjif -ju'T '".'"^V^ Vf. V; '^^<\V

0(1'. J' ''i i;l ->u^.:- iri: -, v'ns,' 1^1 ^f^^ ',i'\^ !t.

%.ii

5ft J i; iV ,. ' yj>!.i= %-.>•' '-'t.,

v.*^'-,;!. i'.i^

^ri'-',Ii.^

I

.'. :»=. ' ».Ki'6 'O. 'i^-'r. .Ji '»«!( ' )'iV.

'1.1.',.,:; .('' uJ,!'. .^i.-

'j •' :'.- ifj'/'t'U'i'; !;,," u /: ; - ./.V

-^ ;" I . ; <*»]( ('UJCj .. jl (.] iiil.;;. .1 j:jt. - A.'!^

ii .^..',. -^. "/ >i(. I ; i' vi ^ I!..-:. U iv >i\ .•(,<-'t^.!i'

:! <'•'. I :('|, ,^. ll . -I'll . .i--M (., iV'. I- .' t 'if^' !H

I v. VI •J^ 'I' i;'-;'. >'('!. ; -.^i 3i':j j'l

:)',r •.-.. !. ! • i! t V V -:M , 'I-,-:- un ,;,v

:''' :l.,-: '•J <.:t rj ii.^i jfii i;;,-Jro mtlll 105

but it does suggest that secologanin is produced in the epidermis and transported into the

'''':, \. ;i *"•; ;.;.w leaf body. i''«..:'-n:"v il'v : sCs-'JOj^AfiU'. y .«u; M!;'

To address the localization of the biosynthesis of the iridoid precursors carborundum

abrasion technique was used to prepare epidermis enriched protein samples Enzyme

analysis of GJOH, LAMT, 16-OMT, NMT and DAT (Fig. 4 panel B) indicated that LAMT

and 16-OMT are enriched in the leaf epidermis [Murata, et ai, (2008); Levac, et al,

(2008); Murata and De Luca, (2005)]. The GIOH enzyme activity data suggests that is it

not preferentially expressed in the leaf epidermis; however there is a small amount of

enzyme activity [(90:l)(Whole Leaf: Epidermis)], unlike the lack of activity found for the

other leaf body marker enzymes, NMT and DAT [Murata and De Luca, (2005)]. This

enzymatic data suggests that while there is a low level of GIOH expression in the leaf

epidermis [Murata and De Luca, (2005)], the IPAP cells may be the primary site for

production of the 10-hydroxygeraniol precursor.

3.5.5- Iridoids as a limiting factor in monoterpene indole alkaloid biosynthesis

It has been suggested that the terpene moiety is the limiting factor in monoterpene

indole alkaloid biosynthesis [Moreno, et al, (1993)] as displayed by feeding experiments

with geraniol, 10-hydroxygeraniol and loganin using Catharanthus cell suspension

cultures to increase the levels of alkaloids (tabersonine) being produced [Morgan and

Shanks (2000)]. Kinetic analysis with loganic acid methyltransferase (LAMT) showed

that the high Km for its substrate loganic acid (Km, 12.5 mM) [Madyastha, et al., (1973);

Murata, et al., (2008)] and the strong inhibition displayed by the reaction product loganin

(Ki, 215 fiM) [Murata, et al., (2008)] may limit the supply of loganin within the cell. The

present report suggests that loganin does not accumulate in any organ of C. roseus aor

rf

'.Vi ,'i

.Mill j/^aCi ,fi;>ijj

V 106

suggesting that product inhibition of loganin may not routinely occur under normal physiological conditions. However, secologanin, the primary substrate for MIA

biosynthesis, does accumulate and it constitutes between (0.07-0.45%) of the fresh weight depending on the organ analysed (Fig. 1-3). This fact and the structural similarities between loganin and secologanin (Fig. 5 panel A) led us to investigate the putative inhibition of secologanin with LAMT. The inhibition study illustrated in Fig. 5 panel B demonstrates that secologanin is not an inhibitor LAMT. The data presented here suggests that the late iridoid biosynthetic pathway is not the limiting factor in monoterpene indole alkaloid biosynthesis as there is a substantial amount of secologanin

that accumulates within the plant however it appears as though it is the cellular localization of the secologanin within the plant. Secologanin is accumulated within the

leaf body and is spatially separated from the enzymes involved in monoterepene indole

alkaloid biosynthesis.

Conclusion

This thesis illustrates that the epidermis of C. roseus is biochemically specialized.

This cell type is active in synthesizing flavonoids, alkaloids, terpenes, lipids and waxes.

The epidermis enriched EST library is an excellent source of candidate genes for

monoterpene indole alkaloids as illustrated in chapter 2 with the cloning and functional

characterization of loganic acid methyltransferase. The epidermis is active in producing

both iridoids and pentacyclic triterpenes and there is a dichotomy between the biological

precursors for tepenes as the MVA pathway is enriched rather than the MEP pathway.

I The data presented here would suggest that the biological role of iridoids within the

leaves of C. roseus is to fuel the biosynthesis of MlAs, while their role within the flowers ' '.'i.<-' 'i ./i;i i^r, - '' '' 'kiWiii^') ;n_;v,'. "^.j^.'o'i .'P»0'libfK)5 isCJi"!'

' ^ • -,•,, ^i-"':* '' ' c'-^') ';;; •••.,>;, 'i.;.;- -1., :, „[.,

!' '' I' ' ' ' '1 . :!>. ;:;' • ' V' t!'f w,!.-; ..iiiV;-*;-.:.: t ; »i;'>!n--

'- -\::J\ t f ,'^ ali! '*? ^-W^fpi to

' ;,''! ;' ;''j: ") -'i 107

is different since the MIA-secologanin ratio is 70-fold smaller. It appears as though the

late-iridoid pathway is not the limiting factor in MIA synthesis as secologanin

accumulates and accounts for up to 0.35% of the young leaf fresh weight. There appear to be several limitations in the supply of secologanin for MIA biosynthesis. The enzyme

activity data suggests supports earlier conclusions that the early-iridoid pathway gene

GIOH is not enriched within the epidermis but rather in the leaf body and in IPAP cells.

In contrast the last 2 steps in secologanin biosynthesis appear to be preferentially expressed within the leaf epidermis where MIA biosynthesis occurs leading to the production 16-methoxytabersonine. The secologanin is then either transported out of the

epidermis or is used within the epidermis to fuel the synthesis of monoterpene indole

alkaloids.

Future work

It appears that the epidermis is a metabolically dynamic cell. Future work should

further investigate the localization of the iridoid biosynthetic pathway. The epidermis

enriched cDNA library has many putative candidate genes for iridoid biosynthesis; three

GlOH-like genes, four lOHGO-Wke genes (10-hydroxygeraniol oxidoreductase), and 24

glucosyltransferase's. The functional characterization of other iridoid pathway genes will

be useful to localize this pathway. Carborundum abrasion and biochemical assays or by

in situ hybridization can be used to identify which enzymes are enriched in the leaf

epidermis as compared to those found in IPAP cells.

Gene and metabolite profiling of other monoterpene indole alkaloid producing

plants from the Apopcynaceae family would be useful in determining if the spatial

separation and cellular localization of the MIA pathway in C. roseus is unique or more ,^i _• V v-T'i, 0<-., . :

i."r.. ^ijf;: '(..•>:.

' I >n' : I Ji

-l< '•,'!'!• '^•'• J

.^.ii-

.' :,(.:; : '- > m

^^:^

'.'L

>*'

:\i..- ;.!-

':f7 -...J .'i;

!. :ri;.

• ."ij 108

common. In addition the cellular localization of the iridoid pathway from other iridoid producing plants should be studied. yf

' : 1; !• • •" , ,i'.r > MR,!

J ifbi -". -jti 109

References

Aerts, R., Schafer, A., Hesse, M., Baumann, T., and Slusarenko, A. (1996). Signalling

molecules and the synthesis of alkaloids in Catharanthus roseus seedlings.

Phytochemistry 42, All-All.

Aerts, R. and De Luca, V. (1992). Phytochrome is involved in light-regulation of

vindoline biosynthesis in Catharanthus. Plant Physiology 100, 1029-1032.

Albach, D., Soltis, P., and Soltis, D. (2001). Patterns of embryological and biochemical

evolution in the Asterids. Systematic Botany 26, 141-161.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and

Lipman, D.J. (1997). Gapped BLAST and PSIBLAST: a new generation of

protein database search programs. Nucleic Acid Res. 25, 3389-3402.

Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL

Workspace: A web-based environment for protein structure homology modeling.

Bioinformatics 21, 195-201.

Barthlott, W., Neinhuis, C, Cutler, D., Ditsch, F., Meusel, L, Theisen, L, and

Wllhelml, H. (1998). Classification and terminology of plant epicuticular waxes.

Bot. J. Lin. Soc. 126, 237-260.

Beninger, C, Cloutier, R., Monteiro, M., and Grodzinski, B. (2007). The distribution

of two major iridoids in different organs of Antirrhinum majus L. at selected

stages of development. J. Chem. Ecol. 33, 731-747.

Bringe, K., Schumacher, C.F.A., Schmitz-Eiberger, M., Steiner, U., and Oerke, E.C.

(2005). Ontogenetic variation in chemical and physical characteristics of adaxial

apple leaf surfaces. Phytochemistry 67, 161-170.

Brooks, B. R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., and

Karplus, M. (1983). "CHARMM: A program for macromolecular energy,

minimization, and dynamics calculations", J. Comput. Chem. 4, 187-217.

Broun, P. (2005). Transcriptional control of flavonoid biosynthesis: a complex network

of conserved regulators involved in multiple aspects of differentiation in

Arabidopsis. Curr. Opin. Plant Biol. 8, 111-119.

Burlat, v., Oudin, A., Courtois, M., Rideau, M., and St-Pierre, B. (2004). Co-

expression of three MEP pathway genes and geraniol 10-hydroxylase in internal ' ' * ' .'1: . '- ,<:/•,,: . IJ

•';. - ^,••J : hi''!

''-'" ' ' . , - .ir^; i

•' > :.•, ^ ,:. y; .J . ){

.-it -•. -•;?;!* *"v ''U .

': :. -i -,c ^0 A r.^

a

r ... .)(J!:\ .»;? 110

phloem parenchyma of Catharanthus roseus impHcates multicellular translocation

of intermediates during the biosynthesis of monoterpene indole alkaloids and

isoprenoid-derived primary metabolites. Plant J. 38, 131-141.

Burse, A., Schmidt, A., Frick, S., Kuhn, J., Gershenzon, J., and Boland, W. (2007).

Iridoid biosynthesis in Chrysomelina larvae: Fat body produces early terpenoid

precursors. Insect Biochem. & Mol. Biol. 37, 255-265.

Cacace, S., Schroder, G., Wehinger, E., Strack, D., Schmidt, J., and Schroder J.

(2003). A flavonol 0-methyltransferase from Catharanthus roseus performing

two sequential methylations. Phytochemistry 62, 127-137.

Chahed, K., Oudin, A., Guivarc'h, N., Hamdi, S., Chenieux, J., Rideau, M., and

Clastre, M. (20(X)). 1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle:

cDNA identification and induced gene expression in terpenoid indole alkaloid-

producing cells. Plant Physiology Biochemistry 38, 559-566.

Chatel, G., Montiel, G., Pre, M., Memelink, J., Thiersault, M., St. Pierre, B.,

Doireau, P., and Gantet, P. (2(X)3). CrMYCl, a Catharanthus roseus elicitor-

and jasmonate-responsive bHLH transcription factor that binds the G-box element

of the strictosidine synthase gene promoter. Journal of Experimental Botany 54,

2587-2588.

Coiner, H., Schroder, G., Wehinger, E., Liu, C.J., Noel, J.P., Schwab, W., and

Schroder, J. (2006). Methylation of sulfhydryl groups: a new function in a family

of plant 0-methyltransferases Plant J. 46, 193-205.

Collu, G., Unver, N., Peltenburg-Looman, A.M., van der Heijden, R., Verpoorte, R.,

and Memelink, J. (2001). Geraniol 10-hydroxylase, a cytochrome P450 enzyme

involved in terpenoid indole alkaloid biosynthesis. FEBS Lett. 508, 215-220.

Contin, A., van der Heijden, R., Lefeber, A.W.M., and Verpoorte, R. (1998).The

iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate

pathway in a Catharanthus roseus cell culture. FEBS Lett. 434, 413-416.

Contin, A., van der Heijden, R., and Verpoorte, R. (1999). Accumulation of loganin

and secologanin in vacuoles from suspension cultured Catharanthus roseus cells.

P/an/ 5c/. 147, 177-183. •H i

v;r ,.,^ ;.| I^! V ,, ., ;,:,-r;., •;,,-, .;r;- -,-<

(j .liV

• tif"'.'>>'i,;<, r.f..">yi a-.

I

-' ti.. fv^>:' .{,'» .- ^f ,x .-N -ilv; ,.H.; .Tvj.i „..i ,.;> ,

•w« - 1 . 111

Coscia, C, Guarnaccia, R., and Botta, L. (1969). Monoterpene Biosynthesis. I.

Occurrence and mevalonoid origin of gentiopicroside and loganic acid in Swertia

' caroliniensis. Biochemistry. 8, 5036-5043. '" ' ' "

Croteau, R., Satterwhite, M., Cane, D., and Chang, C. (1998). Biosynthesis of

Monoterpenes: Enantioselectivity in the enzymatic cyclization of (+)- and (-)-

linalyl pyrophosphate to (+)- and (-)- pinene and (+)- and (-)- camphene. Journal

of Biological Chemistry 263, 10063-10071. i ., ;

Damtoft, S., Godthjaelpsen, L., Jensen, S., and Nielsen, B. (1983). Age-dependent

variations of the efficiency of iridoid biosynthesis in Verbena officinalis.

'' Phytochemistry 22, 2614-2615. '

Damtoft, S., Franzyk, H., and Jensen, S. (1997). Iridoid glucosides from Picconia

excelsa. Phytochemistry 45, 743-750......

De Carolis, E., Chan, F., Balsevich, J., and De Luca, V. (1990). Isolation and

Characterization of a 2-oxoglutarate dependent dioxygenase involved in the

second to last step in vindoline biosynthesis. Plant Physiology 94, 1323-1329.

De Luca, V., Balsevich, J., Tyler, R.T., and Kurz, W.G.W. (1987). Characterization of

a novel A^-methyltransferase (NMT) from Catharanthus roseus plants. Plant Cell

/?ep. 6,458-461.

De Luca, V., and Cutler, A.J. (1987). Subcellular localization of enzymes involved in

indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 85, 1099-

1102.

De Luca, V., Fernandez, J., Campbell, D., and Kurz, W. (1988). Developmental

regulation of enzyme of indole alkaloid biosynthesis in Catharanthus roseus.

Plant Physiology 86, 447-450.

De Luca, V., Marineau, C, and Brisson, N. (1989). Molecular cloning and analysis of

cDNA encoding a plant tryptophan decarboxylase: Comparison with animal dopa

decarboxylases. Proc. Nat. Acad. Sci. USA. 86, 2582-2586.

Dethier, M., and De Luca, V. (1993). Partial purification of an A'-methyltransferase

involved in vindoline biosynthesis in Catharanthus roseus. Phytochemistry 32,

.>-?- 673-678. n-th:. . U (

'•' '" i' • ' "•.• ' -''. "< ' '' ^- "'ts*.. ;' ; " (

•••. -" +• " .' < •')',;. - .; V ^V,.: r-- , ; : ( ' 0, .:

* -^-'1 6';0i

I

{'; '. , ,'. ''>i . . . .: - ' . . , ,..- ,.> (V .

- '"' ' .-.''). ':'•(';• ^•^^> :^:; 1^-; ,;-,i'-l /I-- ! 'i,. ^-fr,..\n-> Pi.iMl-

> .;)f/ ^,. ; .' :''•/ 'r- •; 1. ..^'T ,. ; _ n^vi ,1.

- ,y<'' ', • •.. ^^ V .^ .^;. : ,

'" I •---' ?. -, u'. :. (;, .[ ^'V , / ,K;HiA 112

Dewick, P. (2004) Medicinal Natural Products: A Biosynthetic Approach, 2"*^ edition.

Hoboken: John Wiley and Sons pp.187, 291, 350-357.

Dudareva, N., Andersson, S., Orlova, I., Gatto, N., Reichelt, M., Rhodes D., Boland,

W., and Gershenzon, J. (2005). The nonmevalonate pathway supports both

monoterpene and sesquiterpene formation in snapdragon flowers. Proc. Natl.

Acad. Sci. USA. 102, 933-938.

Ebizuka, Y., Katsube, Y., Tsutsumi, T., Kushiro, T., and Shibuya, M. (2003).

Functional genomics approach to the study of triterpene biosynthesis Pure Appl.

Chem., 75, 369-314.

Eichel, J., Gonzalez, J.C., Hotze, M., Matthews, R.G., and Schroder, J. (1995).

Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus

roseus). Molecular characterization, regulation, heterologous expression, and

enzyme properties Eur. J. Biochem. 230, 1053-1058.

Eichinger, D., Bacher, A., Zenk, M.H., and Eisenreich, W. (1999). Analysis of

metabolic pathways via quantitative prediction of isotope labeling patterns: a

retrobiosynthetic '^C NMR study on the monoterpene loganin. Phytochemistry 51,

223-226.

Eisenreich, W., Rohdich, F., and Bacher, A. (2(X)1). Deoxyxylulose phosphate

pathway to terpenoids. Trends in Plant Science 6, 78-84.

Fincham, D., and Ralston, B.J. (1981). "Molecular dynamics simulation using the

CRAY-1 vector processing computer," Comput. Phys. Commun. 23, 127-134. van der Fits, L., and Memelink, J. (2000). 0RCA3, a jasmonate-responsive

transcriptional regulator of plant primary and secondary metabolism. Science,

289, 295-297. van der Fits, L., Zhang, H., Menke, F.L.H., Deneka, M., and Memelink, J. (2000).

Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive

region of the secondary metabolite biosynthetic gene Str and is induced by elicitor

via a JA-independent signal transduction Pathway. Plant Mol. Biol. 44, 675-685.

Fahn, W., Laussermair, E., Deus-Neumann, B., and Stockigt, J. (1985). Late enzymes

of vindoline biosynthesis. S-Adenosyl-L-meihionine: ll-O-demethyl-17-0- ;

-:f r

!.'», .' Hi

-iHhu^' iliiy •- iVty .'d5:.Ci .-jr.', '- •.,!! ^iX):-, .1 .r»t

•' '1-^^' ,;i . J»... ,'(,/;^

^/.»,>.- ;/:. .a''T;. ..ii." - -fl .',:

- '^' .ii/5.' .ill ::i s< ;.(i '*o Kc- 'vL '»o. _ , ..

'y--' ;''.!'U ; i ),;,i- : Mn'/Hiii 113

deacetylvindoline-11-O-methyltransferase and unspecific acetylesterase. Plant

• ^^;. .5. i- Cell Rep. 4, 331-340. ''•''> .

Frederlksen, L., Damtoft, S., and Jensen, S. (1999). Biosynthesis of iridoids lacking C-

10 and the chemotaxonomic implications of their distributions. Phytochemistry

52,1409-1420...... ,. .. , . .„„

Gang, D.R., Beuerle, T., Ullmann, P., Werck-Reichhart, D., and Pichersky, E.

(2002). Differential production of meta-hydroxylated phenylpropanoids in sweet

basil peltate glandular trichomes and leaves is controlled by the activities of

specific acyltransferases and hydroxylases. Plant Physiol. 130, 1536-1544.

Gang DR, Wang J, Dudareva N, Nam K.H, Simon J, Lewinsohn E, Pichersky E.

(2001). An investigation of the storage and biosynthesis of phenylpropenes in

sweet basil (Ocimum basilicum L.). Plant Physiol. 125, 539-555.

Geerlings, A., Ibanez, M., Memelink, J., van der Heijden, R., and Verpoorte, R.

(2000). Molecular cloning and analysis of strictosidine P-D-glucosidase, an

enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. The

Journal of Biological Chemistry 275, 3051-3056.

Gniwotta, F., Vogg, G., Gartmann, V., Carver, T.L.W., Riederer, M., and Jetter, R.

(2005). What do microbes encounter at the plant surface? Chemical composition

of pea leaf cuticular waxes. Plant Physiol. 139, 519-530.

Grncarevic, M., and Radler, F. (1971). A review of the surface lipids of grapes and

their importance in the drying process. Am. J. Enol. Viticult. 22, 80-86.

Guarnaccia, R., Botta, L., and Coscia, J. (1974). Biosynthesis of acidic iridoid

monoterpene glucosides in Vinca rosea. J. Amer. Chem. Soc. 96, 7079-7084. '

Guex, N., and Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-PdbViewer: An

environment for comparative protein modelling. Electrophoresis 18, 2714-2723.

Harvey, J.A., van Nouhuys, S., and Biere, A. (2005). Effects of quantitative variation

in allelochemicals in Plantago lanceolata on development of a generalist and a

specialist herbivore and their endoparasitoids. J. Chem. Ecol. 31, 287-302.

Hollenbach, B., Schreiber, L., Hartung, W., and Dietz, K.J. (1997). Cadmium leads

to stimulated expression of the lipid transfer protein genes in barley: implications

for the involvement of lipid transfer proteins in wax assembly. Planta 203, 9-19. ;-..:,!:

,.*! ^r-:'

--' :•{ :-. '.u /I Ki»n

>r<'i

;i'"'ff

"~- ' , ' <

' ..H ,rj',-i' 114

Hong, S.B., Hughes, E.H., Shanks, J.V., San, K.Y., and Gibson, S.I. (2003). Role of

the non-mevalonate pathway in indole alkaloid production by Catharanthus

roseus hairy roots. Biotechnol Prog 19, 1 105-1 108

Hotze M., Schroder G., and Schroder, J. (1995). Cinnamate-4-hydroxylase from

Catharanthus roseus, and a strategy for the functional expression of plant

cytochrome P450 proteins as translational fusions with P450 reductase in

Escherichia coli. FEES Lett. 374, 345-50.

Irmler, S., Schroder, G., St-Pierre, B., Crouch, N.P., Hotze, M., Schmidt, J., Strack,

D., Matern, U., and Schroder, J. (2000). Indole alkaloid biosynthesis in

Catharanthus roseus: new activities and identification of cytochrome P450

CYP72A1 as secologanin synthase. Plant J. 24, 797-804.

Jensen, S., and Schripsema, J. (2002). Chemotaxonomy and pharmacology of

Gentianaceae. Gentianaceae-Systematics and Natural History. (Cambridge

University Press).

Konno, K., Hirayama, C, Yasui, H., and Nakamura, M. (1999). Enzymatic activation

of oleuropein: A protein crosslinker used as a chemical defense in the privet tree.

Proc. Natl. Acad. Sci. USA 96, 9159-9164.

Kutchan, T.M. (2005). A role for intra- and intercellular translocation in natural product

biosynthesis. Curr. Opin. Plant Biol. 8, 292-300.

Kunst, L., and Samuels, A.L. (2003). Biosynthesis and secretion of plant cuticular wax.

Prog. Lipid Res. 42, 51-80.

Laflamme, P., St-Pierre, B., & De Luca, V. (2(X)1). Molecular and Biochemical

Analysis of a Madagascar Periwinkle Root-specific Minovincinine-19-Hydroxy-

0-Acetyltransferase. Plant Physiology 125, 189-198.

Levac, D., Murata, J., Kim W.S., and De Luca, V. (2008). Application of carborundum

abrasion for investigating leaf epidermis: Molecular cloning of Catharanthus

roseus 16-hydroxytabersonine-16-0-methyltransferase. Plant J. 53, 225-236.

Lange, B.M., Wildung, M.R., Stauber, E.J., Sanchez, C, Pouchnik, D., and Croteau,

R. (2000). Probing essential oil biosynthesis and secretion by functional

evaluation of expressed sequence tags from mint glandular trichomes. Proc. Nat.

Acad Sci. USA. 97, 2934-2939. i-> .J'/'i

" ; ;;•! 1.

^,,is '«f>T: '.J ...

,1(7 >. >!"r)!

i: f .)til

it; !'n

'{A

-' ' • -='* ..^-'/ : ' !: i.::.^. .v.: -•xn'l

":' ^'. .f ^ .'.-/ : v'^^^ > ! ?

'...^ -'"'.',".'...: ':.f. ;:' . ! , ,<;4'?Hff'

' ' ' ' -^ .»;' ^ ,.;•( '''>;'! ,.''1 u 'J -V . •!( v^!'.

• ' :-.4.' vn-j'-f i / .>»;?,- '^ , i 115

Lichtenthaler, H.K. (1999). The l-deoxy-D-xylulose-5-phosphate pathway of

isoprenoid biosynthesis in plants. Anna. Rev. Plant Physiol. Plant Mol. Biol. 50,

47-65.

Lopes, S., von Poser, G.L., Kerber, V.A., Farias, F.M., Konrath, E.L., Moreno, P.,

Sobral, M.E., Zuanazzi, J.A.S., and Henriques, A.T. (2004). Taxonomic

significance of alkaloids and iridoids glucosides in the tribe Psychotrieae

(Rubiaceae). Biochem. Syst. Ecol. 32, 1187-1195.

Madyastha, K.M., Guarnaccia, R., Baxter, C, and Coscia, C.J. (1973). 5-Adenosyl-

L-methionine: loganic acid methyltransferase. J. Biol. Chem. 248, 2497-2501.

Mahroug, S., Courdavault, V., Thiersault, M., St-Pierre, B., and Buriat, V. (2006).

Epidermis is a pivotal site of at least four secondary metabolic pathways in

Catharanthus roseus aerial organs. Planta 223, 1 191-12(X).

Martin, C, and Glover, B.J. (2007). Functional aspects of cell patterning in aerial

epidermis. Curr. Opin. Plant Biol. 10, 70-82

McKinght, T., Bergey, D., Burnett, R., and Nessler, C. (1991). Expression of

enzymatically active and correctly targeted strictosidine synthase in transgenic

tobacco plants. Planta 185, 148-152.

Meijer, A.H., Souer, E., Verpoorte, R., and Hoge, J.H. (1993). Isolation of cytochrome

P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR

strategy. Plant Mol. Biol. 22, 379-383.

Memelink, J., Verpoorte, R., and Kijne, J.W. (2001). Orcanization of jasmonate-

responsive gene expression in alkaloid metabolism. Trends Plant Sci. 6, 212-219.

Menke, F., Parchmann, S., Mueller, M., Kijne, J., and Memelink, J. (1999).

Involvment of the Octadecanoid Pathway and Protein Phosphorylation in Fungal

Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in

Catharanthus roseus. Plant Physiology 119, 1289-1296.

Millar, A.A., Clemens, S., Zachgo, S., Giblin, E.M., Taylor, D.C., and Kunst, L.

(1999). CUT], an Arabidopsis gene required for cuticular wax biosynthesis and

pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant

Cell 11, 825-838. in.

' A. '-^ -^'-'^ ,H ,oHTl»^^-' " ' "irt>F ,.M-' ;.<•»;»• '»i"i ,. -

5 ji; -! i ' .• ^ « i:*t , H £) .y .,;«hj*.ii mini ^M .ri s'WJ-w* ..M j:k*::w--f'Jij. ..

,^-l 116

Money, T., Wright, I., McCapra, F., and Scott, A. (1965) Biosynthesis of the indole

alkaloids. Proc. Nat. Acad. Sci. USA. 53, 901-903.

Moreno, P., van der Heijden, R., and Verpoorte, R. (1993). Effect of terpenoid

precursor feeding and elicitation on formation if indole alkaloids in cell

suspension cultures of Catharanthus roseus. Plant Cell Reports 12, 702-705.

Morgan, J., and Shanks, J. (2000). Determination of metabolic rate-limitations by

precursor feeding in Catharanthus roseus hairy root cultures. Journal of

Biotechnology 19, 137-145.

Murata, J., and De Luca, V. (2005). Localization of tabersonine 16-hydroxylase and

16-OH tabersonine- 16-0-methyltransferase to leaf epidermal cells defines them

as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus

roseus. Plant J. 44, 581-594.

Murata, J., Bienzle, D., Brandle, J.E., Sensen, C.W., and De Luca, V. (2006).

Expressed sequence tags from Madagascar periwinkle {Catharanthus roseus).

FEES Lett. 580, 4501-4507.

Murata, J., Roepke, J., Gordon, H., and De Luca, V. (2008). The leaf epidermome of

Catharanthus roseus reveals its biochemical specialization. The Plant Cell 20,

524-542.

Negre, F., Kish, C, Boatright, J., Underwood, B., Shibuya, K., Wagner, C, Clark,

D., and Dudareva, N. (2003). Regulation of methylbenzoate emission after

pollination in snapdragon and petunia flowers. The Plant Cell 15, 2992-3006.

Oudin, A., Mahroug, S., Courdavault, V., Hervouet, N., Zelwer, C, Rodriguez-

Concepcion, M., St-Pierre, B., and Burlat V. (2007) Spatial distribution and

hormonal regulation of gene products from methyl erythritol phosphate and

monoterpene-secoiridoid pathways in Catharanthus roseus. Plant Mol. Biol. 65:

13-30.

Pauw, B., Hilliou, F.A.O., Sandonis, V., Chatel, M.G., de Wolf, C.J.F., Champion, A.

Pre, M., van Duijn, B., Kijne, J.W., van der Fits, L., and Memelink, J. (2004).

Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis

genes in Catharanthus roseus. J. Biol. Chem. 279, 52940-52948. . mt

\k\ \v.Xf\Vil^

ffiatJ-i . ,.

11''.'! • '* 9(f hrr; ,

^ hWi\ .V Is? .

;"''(, ... .~._ b)'r. -iW?{r'-;odf,- I'll n i.'.i.'i:iT fi'OTl i,^, , . 117

Peebles, C.A.M., Hong, S.B., Gibson, S.I., Shanks, J.V., and San, K.Y. (2006). Effects

of terpenoid precursor feeding on Catharanthus roseus hairy roots over-

expressing the alpha or the alpha and beta subunits of anthranilate synthase.

Biotechnol. Bioeng. 93, 534-540.

Penuelas, J., Sardans, J., Stefanescu, C, Parella, T., and Filella, I. (2006). Lonicera

implexa leaves bearing naturally laid eggs of the specialist herbivore Euphydryas

aurinia have dramatically greater concentrations of iridoid glycosides than other

leaves. J. Chem. Ecol. 32, 1925-1933.

Pereyra, P.C, and Bowers, M.D. (1988). h-idoid glycosides as oviposition stimulants

for the buckeye butterfly, Junonia coenia (Nymphalidae). J. Chem. Ecol. 14, 917-

928.

Pott, M., Hippauf, F., Saschenbrecker, S., Chen, F., Ross, J., Kiefer, I., Slusarenko,

A., Noel, J., Pichersky, E., Effmert, U., and Piechulia, B. (2004). Biochemical

and structural characterization of benzenoid carboxyl methyltransferases involved

in floral scent production in Stephanotis floribunda and Nicotiana suaveolens.

Plant Physiology 135, 1946-1955.

Pyee, J., Yu, H., and Kolattukudy, P.E. (1994). Identification of a lipid transfer protein

as the major protein in the surface wax of broccoli {Brassica oleracea) leaves.

Arch. Biochem. Biophys. 311, 460-468.

Qi, X., Bakht, S., Qin, B., Leggett, M., Hemmings, A., Mellon, F., Eagles, J., Werck-

Reichhart, D., Schaller, H., Lesot, A., Melton, R., and Osbourn, A. (2006). A

different function for a member of an ancient and highly conserved cytochrome

P450 family from essential sterols to plant defense. Proc. Natl. Acad. Sci. USA.

103, 18848-18853.

Qin, GGu, H., Zhao, Y., Ma, Z., Shi, G., Yang, Y., Pichersky, E, Chen, H., Liu, M.,

Chen, Z., and Qu, L.J. (2005). An indole-3-acetic acid carboxyl

methyltransferase regulates Arabidopsis leaf development. Plant Cell 17, 2693-

2704.

Rischer, H., Oresic, M., Seppanen-Laakso, T., Katajamaa, M., Lammertyn, F.,

Ardiles-Diaz, W., Montagu, M.C.E., Inze, D., Oksman-Caldentey, K.M., and

Goossens, A. (2006). Gene-to-metabolite networks for terpenoid indole alkaloid rj-r

.&£'

,*'4«f tits' ' » „?*

.'r.vn.'ik 'av>a-«'»-k» v.-vVi'/.D'u*'.} tlf!>J',Ti 1<> X.'*w 118

biosynthesis in Catharanthus roseus cells. Proc. Natl. Acad. Sci. USA. 103, 5614-

5619.

Rodriguez-Concepcion, A., and Boronat, A. (2002). Elucidation of the

methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and

plastids. A metabolic milestone achieved through genomics. Plant Physiology

130, 1079-1089.

Roewer, I., Cloutier, N., Nessler, C, and De Luca, V. (1992). Transient induction of

tryptophan decarboxylase (TDC) and strictosidine synthase (SS) genes in cell

suspension cultures of Catharanthus roseus. Plant Cell Reports 11, 86-89.

Rohmer, M., Knani, M., Simonin, P., Sutter, B., and Sahm, H. (1993). Isoprenoid

biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl

diphosphate. Biochem J. 295, 517-524.

Ross, J., Nam, K., D'Auria, J., and Pichersky, E. (1999). 5-Adenosyl-L-methionine:

salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent

production and plant defense, represents a new class of plant methyltransferases.

Arch. Biochem. Biophy. 367, 9-16.

Ryckaert, J. P., Ciccotti, G., and Berendsen, H.J.C. (1977). "Numerical-integration of

cartesian equations of motion of a system with constraints - molecular-dynamics

of N-alkanes" J. Comput. Phys. 23, 327-341.

Sanchez-Iturbe, P., Galaz-Avalos, R., and Loyola-Vargas, V. (2005). Determination

and partial purifiaction of 10-oxogeranial:iridodial cyclase an enzyme catalyzing

the synthesis of iridodial from Catharanthus roseus hairy roots. International

Journal of Experimental Botany. 55-69.

Schmitz-Hoerner, R., and Weissenbock, G. (2003). Contribution of phenolic

compounds to the UV-B screening capacity of developing barley primary leaves

in relation to DNA damage and repair under elevated UV-B levels.

Phytochemistry 64, 243-255.

Schroder, G., Unterbusch, E., Kaltenbach, M., Schmidt, J., Strack, D., De Luca, V.,

and Schroder, J. (1999). Light-induced cytochrome P450-dependent enzyme in

indole alkaloid biosynthesis: tabersonine 16-hydroxylase. FEBS Lett. 458, 97-102. .

^'f

»' 1 * • \|->,W\ ''-.A', '.,'.'\ '.l'">; "Vjf. >\

Hi^^rirns*

;*.ia

' •! <> J ! f

'!:%* '31-;"'" . ,.:. :r ';ii .:'"'t'n .'M..H jj=>v^u*t-»jf <>n« ^..ii i5tw>='"'- ^

.'l^vii.'i-^ "'^ >^; ',', '/' J! 1.4 ii.»

.! ;i '/ ' i.') = :. ;.- / J 1 (t i-%<.vA\.'i> kVM OJ 119

Schroder, G., Wehinger, E., and Schroder, J. (2002). Predicting the substrates of

cloned plant 0-methyltransferases. Phytochemistry 59, 1-8.

Schroder, G., Wehinger, E., Lukacin, R., Wellmann, F., Seefelder, W., Schwab, W.,

and Schroder, J. (2004). Flavonoid methylation: a novel 4'-0-methyltransferase

from Catharanthus roseus, and evidence that partially methylated flavanones are

substrates of four different flavonoid dioxygenases. Phytochemistry 65, 1085-

1094.

Schwede, T., Kopp, J., Guex, N., and Peitsch, M.C. (2003). SWISS-MODEL: an

automated protein homology-modeling server. Nucleic Acids Res. 31, 3381-3385.

Seo, H.S., Song, J.T., Cheong, J.J., Lee, Y.H., Lee, Y.W., Hwang, L, Lee, J.S. and

Choi, Y.D. (2001). Jasmonic acid carboxyl methyltransferase: a key enzyme for

jasmonate-regulated plant responses. Proc. Natl. Acad. Sci. USA. 98, 4788-4793.

Shepherd, T., and Griffiths, D.W. (2006). The effects of stress on plant cuticular

waxes. A^ew. P/zjto/. 171, 469-499. /,••:''

Shukia, A.K., Shasany, A.K., Gupta, M.M., and Khanuja, S.P.S. (2006).

Transcriptome analysis in Catharanthus roseus leaves and roots for comparative

terpenoid indole alkaloid profiles, y. Exp. Bor. 57, 3921-3932.

Soler-Rivas, C, Espin, J., Wichers, H. (2000). Oleuropein and related compounds. J.

Sci. FoodAgric. 80, 1013-1023.

Sreevalli, Y., Kulkarni, R., and Baskaran, K. (2002). Inheritance of Flower Color in

Periwinkle: Orange-Red Corolla and White Eye. The Journal of Heredity 93, 55-

58.

Stevens, L.H., Blom, T.J.M., and Verpoorte, R. (1993). Subcellular localization of

tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in

suspension cultured cells of Catharanthus roseus and Tabemaemontana

divaricata. Plant Cell Rep. 12, 573-576.

Sterk, P., Booij, H., Schellekens, G.A., Van Kammen, and A., De Vries, S.C. (1991).

Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell 3,

907-921.

'!' !:- /r , Yn->m»-, ir, 7*'Rko \i. I ' ' J , .

' j.-i' Ci.' .w«r ;.;, . .;h,i!i^<"jii.'

/ .-.•fiy-' .1 ,511 I

-•f f

' -' ':'..*;i- - ; - :: ; l7r>iT-,»'|(v

" •i, ..»/ ;..') ,.;3

M.'q 120

Stockigt, J., and Zenk, M. (1977). Strictosidine (isovincoside): the key intermediate in

the biosynthesis on monoterpene indole alkaloids. J. Chem. Soc. Chem. Commun.

- 646-648. ' '

St. Pierre, B., and De Luca, V. (1995). A cytochrome P-450 monooxygenase catalyses

the first step in the conversion of tabersonine to vindoline in C. roseus. Plant

Physiology 109, I3\'l39.

St. Pierre, B., Laflamme, P., Alarco, A., and De Luca, V. (1998). The terminal O-

acetyltransferase in vindoline biosynthesis defines a new class of proteins

involved responsible for co-enzyme A dependent acyl transfer. The Plant Journal

14,703-713.

St-Pierre, B., Vazquez-FIota, F.A., and De Luca, V. (1999). Multicellular

compartmentation of Catharanthus roseus alkaloid biosynthesis predicts

intercellular translocation of a pathway intermediate. Plant Cell 11, 887-900.

Szabo, L. (2008). Molecular evolutionary lines in the formation of indole alkaloids

derived from secologanin. y4r^/voc, 167-181.

Thoma, S., Hecht, U., Kippers, A., Botella, J., de Vries, S., and Somerville, C. (1994)

Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer

protein from. Arabidopsis. Plant Physiol. \05,35-A5.

Thomas, R. (1961). A possible biosynthetic relationship between the cyclopentanoid

monoterpenes and the indole alkaloids. Tetrahedron Letters 16, 544-553.

Treimer, J., and Zenk, M. (1979). Purification and properties of strictosidine synthase,

the key enzyme in indole alkaloid formation. Eurpean Journal of Biochemistry

101, 225-233.

Uesato, S., Ueda, S., Kobayashi, K., Inouye, H. (1983). Mechanism of iridane skeleton

formation in the biosynthesis of iridoid glucosides in Gardenia jasminoides cell

cultures. Chem. Pharm. Bull. 31, 4185-4188.

Uesato, S., Ogawa, Y., Inouye, H., Saiki, K., and Zenk, M. (1986). Synthesis of

iridodial by cell free extracts from Rauwolfia serpentine cell suspension cultures.

Tetrahedron Letters 27, 2893-2896.

Uesato, S., Ikeda, H., Fujita, T., Inouye, H., Zenk, M. (1987). Elucidation of iridodial

formation mechanism- Partial purification and characterization of the novel ("^i.", I

1/

'? {(

» '-' . - ij!.i-,; f, :'i .. .; '1 ,i^bajjl . ? /> 121

monoterpene cyclase from Rauwolfia serpentine cell suspension cultures.

Tetrahedron Letters 28, 4431-4434.

Usia, T., Watabe, T., Kadota, S., and Tezuka, Y. (2005). Cytochrome P450 2D6

(CYP2D6) inhibitory constituents of Catharanthus roseus. Biol. Pharm. Bull. 28,

1021-1024.

Van der Heijden, R., Jabobs, D., Snoeijer, W., Hallard, D., and Verpoorte, R.

(2004). The Catharanthus Alkaloids: Pharmacognosy and Biotechnology.

Current Medicinal Chemistry 11, 607-628.

Vasquez-Flota, F., and De Luca, V. (1998). Developmental and light regulation of

desacetoxyvindoline 4-hydroxylase in Catharanthus roseus (L.) G. Don. Plant

Physiology 117, 1351-1361

Vazquez-Flota, F., and De Luca, V. (1998). Jasmonate Modulates Developmental and

Light regulated alkaloid Biosynthesis in Catharanthus roseus. Phytochemistry

49:395-402.

Veau, B., Contois, M., Oudin, A., Chenieux, J., Rideau, M., and Clastre, M. (2000).

Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in

Catharanthus roseus. Biochimica et Biophysica acta 1517, 159-163.

Voitsekhovskaja, O., Koroleva, O., Batashev, D., Knop, C, Tomos, D., Gamalei, Y.,

Heldt, H., and Lohaus, G. (2006). Phloem loading in two Scrophulariaceae

species. What can drive symplastic flow via plasmodesmata? Plant Physiology

140, 383-395. de Waal, A., Meijer, A., and Verpoorte, R. (1995). Strictosidine synthase from

Catharanthus roseus: purification and characterization of multiple forms.

Biochemistry Journal 306, 571-580.

Wagner, G.J., Wang, E., and Shepherd, R. W. (2004). New approaches for studying

and exploiting an old protuberance, the plant trichome. Ann. Bot. 93, 3-11.

Wagner, H., Bladt, S., Zgainski, E. (1984). Plant Drug Analysis: A thin layer

chromatography atlas Springer-Verlag New York.

Yamamoto, H., Kalano, N., Ooi, A., and Inoue, K. (1999). Transformation of loganin

and 7-deoxyloganin into secologanin by Lonicera japonica cell suspension

cultures. Phytochemistry 50, 417-422. ri;;

\]-y:: ^jt,

r ^h

'" . . '

!,'"';> V: .THm ^j,jim,. ..'IJi sifui

^'' '^.)?.- '.'![ //••' "'it ^t.i . ^'V. i*-{\.. // ii; Jr..

'!>! ,.*•, ,,.'/! b'"!s .!o(J , J? .nV 122

Yamamoto, H., Katano, N., Ooi, A., and Inoue, K. (2000). Secologanin synthase which

catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome

P450. Phytochemistry 53,1-12. --

Yamamoto, H., Sha, M., Kitamura, Y., Yamaguchi, M., Kitano, N., and Inoue, K.

(2002). Iridoid biosynthesis: 7-deoxyloganetic acid 1-0-glucosyltransferase in

cultured Lonicerajaponica cells. Plant Biotechnol. 19, 295-301. . ;

Yamazaki, M. Kitajima, M. Arita, H. Takayama, Sudo, H., Yamazaki, M., Aimi, N.,

and Saito K. (2004). Biosynthesis of Camptothecin. In Silico and in Vivo Tracer

Study from [l-'^C] Glucose. Plant Physiol. 134, 161-170.

Yang, Y., Yuan, J.S., Ross, J., Noel, J.P., Pichersky, E., and Chen, F. (2006) An

Arabidopsis thaliana methyltransferase capable of methylating famesoic acid.

Arch.Biochem.Biophys.. 448,123-132.

Zhang, X., Xu, Q., Xiao, H., and Liang, X. (2003). Iridoid glucosides from Stychnos

nux-vomica. Phytochemistry 64, 1341-1344.

Zhu, J., Guggisberg, A., Kalt-Hadamowsky, M., and Hesse, M. (1990)

Chemotaxonomic study of the genus Tabemaemontana (Apocynaceae) based on

their indole alkaloid content. Plant Systematics and Evolution 111, 1 3-34.

Zubieta, C, Ross, J.R., Koscheski, P., Yang, Y., Pichersky, E, Noel, J.P. (2003)

Structural basis for substrate recognition in the salicylic acid carboxyl

methyltransferase family. Plant Cell 15, 1704-1716.

I , i !

I i ! ODK. : \ ',sjiii r ''Vi" i-;i b«i« aiftic ill .n)-.')!t >'r}i.MV>"io Kig-idr-

.:'<:. .*?..t j>./: 123

Appendix I

Supplemental Table 1: CR0LF1NG genes related to isoprenoid biosynthesis. Gene li b^tJS'-?'. ii$-

';•:; 1 - : fV9t,,

I

I

i

I 4«-HL.-vi,t

j*;s»i'!i;

-30'- i

AVH.1 I ,; X-:

I "" 1,' r

1 "t ?-.,

i

I i

{ r>o.«.?'.«o'> ..,; fm,!,.

:/IO;TJ'.g!n

'.*6 # .

r

I . I tit -J--',

.fV-U? 124

FLS

F3H

CHS/STS

CHS/STS

DFR

PAL

DFR '

"• 'r-'J : , 1.

- , . ,t '. 1 '.'i i

« ,^ :i>t u- !VjB i vi^c'

V'»-

<):?•"•."

- -, .^-•^i Vj(r,>i t 125

Supplemental Table 4: CR0LF1NG genes related to lipid biosynthesis.

Name Description lipid transfer protein [Hellanthus annuus] putative lipid transfer protein [Solanum tuberosum] lipid transfer protein precursor [Davidia Involucrata] malate dehydrogenase [Nicotiana tabacum] 3-ketoacyl-CoA synthase [Gossypium hirsutum], CUT1 carboxyllc ester hydrolase/ hydrolase, acting on ester bonds [Arabidopsis thaliana] stearoyl-acyl carrier protein desaturse [Sesamum Indlcum] carboxyllc ester hydrolase/ hydrolase, acting on ester bonds [Arabidopsis thaliana] lipid transfer protein, putative [Arabidopsis thaliana] Enolase (2-phosphoglycerate dehydratase) [RIclnus communis] sphlngollpid delta-8 desaturase [Primula farinosa] putative phosphoglycerate mutase [Oryza satlva (japonica cuttlvar- group)] fatty acid elongase-llke protein (cer2- llke) [Arabidopsis thaliana] Cytosollc fatty-acid binding; Actln- crosslinklng proteins [Medicago tmncatula] fatty acid elongase-llke protein (cer2- llke) [Arabidopsis thaliana] chloroplast fatty acid desaturase 6 [Olea europaea subsp. europaea] catalytic/ hydrolase [Arabidopsis thaliana] Lipolytic enzyme, G-D-S-L [Medicago truncatula]

ACX4 (ACYL-COA OXIDASE 4); oxidoreductase [Arabidopsis thaliana] putative long-chain acyl-CoA synthetase [Arabidopsis thaliana] Omega-3 fatty acid desaturase, chloroplast precursor 3-ketoacyl-CoA thiolase 2, peroxisomal precursor, putative, expressed [Oryza sativa (japonica cultlvar-group)] 3-ketoacyl-ACP synthase [Cuphea pulcherrima] malate dehydrogenase [Glycine max]

lipid binding [Arabidopsis thaliana] acyl-CoA thioesterase/ catalytic/ hydrolase [Arabidopsis thaliana] acyl-CoA thioesterase/ catalytic/ hydrolase, acting on ester bonds [Arabidopsis thaliana]

lipid transfer protein [Hellanthus annuus] putative 3-isopropylmalate dehydratase, small subunit [Arabidopsis thaliana] Plant lipid transfer/seed storage/trypsln-alpha amylase inhibitor [Medicago truncatula] ;

>;*. ' «> ;, ;•>.. y^-tifi'. mrmlA

a I .ii^.v "^/r 1 ,

:.>' I.

,1..!.-; .-(.

«i'!l 1

iU:

'•-ft.'!/?,': . ,-, ,

"'.' ,.,*>

fill r .'i..-: "i'i

' '<»-:

-. .i*,j

Si

' 'Kit'.i!:^^ ,"

!W- ':'"vt

ji. V; J^'^k. 126

145-B08 CoA ligase

129-E03 t>-\U't"-?t,/j,. t

Mr

! :2'&),o.

Si

'••V . -WC'S

-¥«.£

,(-€.; ^i.i^^; (:''

:\A X-''t,

^'Xi.-J ../'..'/•

ii ,^

-'"-- ^ .it' 127

Supplemental Table 5: CR0LF1NG genes related to methyltransferases. ajjfi

"-3 t*'i- %,r-

*<^»,..^>'

, r.l.-'

1

I

i

*,';5v--ii. -..>

T >)<",,

,«..•:

':'$"

-v: i- :'*^^" .'-'T

'^^,,V'

>!-. >

^A-lS*j\^i'.^'-^J 128

Supplemental Table 6: CR0LF1NG genes related to acyltransferases. While DAT appears to be the only acyltransferase involved in vindoiine biosynthesis several putative acyltransferases including one with similarity to vinorine synthase from Rauvolfia serpentina (Bayer et al., 2004) were identified. Vinorene synthase catalyzes the formation of vinorine, which is an intermediate for the antiarrhythmic drug ajmaline in Rauvolfia, also shows some similarity (20%) to 0-47 from Catharanthus and to salutandinol acyltransferase (Grothe et al, 2001) from Papaver somniferum. The biological role of this enzyme in relation to MIA biosynthesis should be considered.

Name >?! V

Ail':] .'/! '-'':' ii~' ];') tXi -H'/;)!-'. ^'s'' '." tWi ' f)~i''

•5^» ., : 3.:}t

'0<'i t-

•' 1

i*' 129

Supplemental Table 7: CROLF1NG genes related Cytochrome P450-dependent monooxygenases (CYPs). Multiple steps in MIA biosynthesis involve reactions catalyzed by CYPs. There are three CYPs that have been cloned and functionally characterized: namely G10H (represented by 1 EST in CROLFING), SLS (represented 25 times in CROLFING) and T16H (represented 4 times in CROLFING) (Table 1). In addition, deoxyloganin 7 hydroxylase (DL7H) enzyme activity was first identified in C. roseus microsomes (Irmler et al., 2000) that converted 7-deoxyloganin into loganin only in the presence of NADPH. Although more studies are required, the CYPs represented by

CL19Contig3 (represented 5 times in CROLFING) and 110-F10 (represented 1 time in CROLFING) could be good DL7H candidates (Supplementary Table 7). For example the CYP represented by CL19Contig3 shows 100%, 98 % and 97 % amino acid sequence identity to CyP72e(AAA1 7732.1), CyP72C(AAA1 7746.1) and to SLS (AAA331 06.1), respectively.

Name ' lO^h*-' IS &i!d&T muH

; '-'i ! tfMr.yl .-.- ? /..4 .--. ^- -~-

,iJtft

/I' .j-'';'.6 «'«' , >'fV r- ' fgi

Ivsm •

rV>'

..' ; f,

. ;iv-) AK'TOy,.

• a-

ss^-:*:? £

I "''. ; ^'f" 'i 130

Supplemental Table 8: CR0LF1 NG genes related to glycosyltransferases Glycosylation of natural products will significantly increase their water solubility, their stability, and will affect their subcellular localization and/or transport between cells (Gachon et al., 2005). The biosynthesis of MIAs involves a glucosylation for the conversion of 7-deoxyloganetic acid into 7-deoxyloganic acid that has yet to be characterized in detail. This 7-deoxyloganetic acid 1-O-glycosyltransf erase (7DLGT), first detected in extracts from Lonicera japonica cell cultures (Yamamoto et al., 2002), appears to play an important role in stabilizing reactive iridoid intermediates that accumulate in certain members of the family. While this gene remains to be cloned, its suggested localization to Catharanthus leaf epidermis (Murata and De Luca, 2005) suggests that the 23 putative glycosyltransferases (GTs) from the CR0LF1NG dataset could harbor 7DLGT (Supplementary Table 8). In this context, it is interesting that only one of these putative GTs (CL972Contig1 ) found in both the CR0LF1 NG (2 ESTs) and root tip (CrUnlGene) dataset could be a good putative 7DLG 7 candidate to investigate.

Name * !

? ' ; f3-?*fe 131

Gachon, C.M.M., Langlois-Meurinne, M., and Saindrenan, P. (2005). Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends Plant Sci.lO, 542-549

Supplemental Table 9: CR0LF1 NG genes related to 2-oxoglutarate dependent dioxygenases. Dioxygenases are involved in the oxidation of diverse class of metabolites including flavonoids, carotenoids and lipids. In addition to four dioxygenases listed as flavonoid biosynthesis-related genes in Supplementary Table 3, five other genes with similarity to known dioxygenases were identified (Supplementary Table 9). In the case of the MIA biosynthesis, the hydroxyiation of deacetoxyvindoline was shown to be catalyzed by the dioxygenase D4H (Vazquez-Flota ei at., 1997), but this gene was not represented in the dataset. This is not surprising since previous studies localized both D4H and DAT to Catharanthus mesophyll idioblasts and laticifers (St. Pierre etal., 1999).

Name Description Hit ID Evalue #EST

1 ,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 3 (Aci-reductone dioxygenase 3) gb|AAW70407.1| CL221Contig1 [Arabidopsis thaliana] gi|1 841 4289] Glyoxalase/bleomycin resistance protein/dioxygenase CL836Contig1 [Medicago truncatula] 2-nitropropane dioxygenase-like protein [Arabidopsis CL885Contig1 thaliana] putative prolyl 4-hydroxylase, alpha subunit 147-F09 [Arabidopsis thaliana] Glyoxalase/bleomycin resistance protein/dioxygenase 032-E04 [Medicago truncatula] 'it. ,• „«, ^ , t

'-»!. ,b'

'• *i,v.'' I. v::' V-

.u - t -.i^»'"' r-

* .- - ' .'.J-* 132

Supplemental Table 10: CR0LF1NG genes related to transcription factors.

Transcriptional control of gene expression is a key regulatory step in triggering morphological and biochemical changes that lead to specialized cells for MIA biosynthesis. The CROLF1NG dataset contained all three previously characterized 0RCA3, ZCT2 and BPF1, respectively, classified into AP2/ERF, Zinc finger and bHLH type transcription factors (Table 1).

The transcriptional activator 0RCA3 is known to activate multiple MIA biosynthesis genes in Catharanthus cell cultures (van der Fits and Memelink, 2000) including the leaf epidermis localized TDC and STR (Table 1, St. Pierre et al., 1999). On the other hand, ZCT2 repressed the transcription of the reporter genes that were fused to TDC and STR promoter sequences (Pauw et al., 2004), suggesting that ZCT2 might act as a transcriptional repressor, while the biological function of BPF1 is still not clear (van der Fitz et al., 2000). Extensive studies with these transcription factors, however, only revealed the complexity of the transcriptional regulation of MIA pathway genes. For example, 0RCA3 did not seem to play a major role in controlling the expression of all the MIA pathway genes (van der Fitz and Memelink, 2000). While ORCA3 itself seems to be expressed throughout the leaf tissue (Murata and De Luca, 2005), the expression of SGD was not affected by overexpression of 0RCA3 (van der Fits and Memelink, 2000), indicating that ORCA3 might not be involved in the activation of transcription of SGD in leaf epidermis. These data imply that other transcription factors are probably required for SGD expression as well as later steps in MIA biosynthesis in leaf epidermal cells.

Category Name

TF CL250Contig1

TF CL214Contig1

TF CL504Contig1

TF C «»* f^i-<.^,\:.-^:,ft

' J-;.- an;.:.

0 -jj: ? 133

TF, bHLH CL1070Contlg1

TF, bHLH 112-E02

TF, bHLH 110-E08

TF, bHLH 104-F03

TF, bHLH 057-A08

TF, bHLH 010-C09

TF, bZIP CL918Contig1

TF, bZIP 125-H11

TF, bZIP 119-G01

TF, bZIP 104-F10

TF, CCAAT 010-A04

TF, CSN6a 119-D02

TF, GRAS 015-E07

TF.GRAS 126-G08

TF, GRAS 015-E07

TF, GT-1 059-D05

TF, hellcase 035-F02

TF, HMG CL723Contig1

TF, HMG 154-G09

TF, HSF 005-A10

TF, MADS CL361Contig1

TF, MYB CL87Contig1

TF, MYB 128-A08

TF, MYB 047-E02 TF, NAC CL613Contig1

TF, NAC 011-E12

TF, polll '

mi-

' :.UH

•,s"jii-'.

." • ''^^f t J^^^ •

'1

''1,'" '

'ikHi,

--.n

i f

WO:

-V Tl 134

TF, WRKY M'f.*-' 1; %',:

rri'w.

t:.'iry- y-.r:

r.,^T

i .--'If / 135