Promoter analysis of the Catharanthus roseus geraniol 10/​hydroxylase gene
involved in terpenoid indole alkaloid biosynthesis.

Suttipanta N, Pattanaik S, Gunjan S, Xie CH, Littleton J, Yuan L.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
40546, USA.

Geraniol 10/​hydroxylase (G10H) is an important enzyme in the biosynthetic
pathway of monoterpenoid alkaloids found in diverse plant species. The
Catharanthus roseus G10H controls the first committed step in biosynthesis of
terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was
isolated by a PCR/​based genome walking method. Sequence analysis revealed that
the G10H promoter contains several potential eukaryotic regulatory elements
involved in regulation of gene expression. The major transcription start site of
the promoter was mapped to an adenine 31 bp downstream of the TATA/​box. For
functional characterization, transcriptional fusions between the G10H promoter
fragments with 5' or 3' deletions and the GUS reporter gene were generated and
their expressions were analyzed in a tobacco protoplast transient expression
assay. Deletion of the promoter down to /​318 bp had little effect on GUS
activity. However, further deletion of the promoter to position /​103 resulted in
approximately 5/​fold reduction of GUS activity. Gain/​of/​function experiments
revealed the presence of three potential transcriptional enhancers located in
regions between /​191 and /​147, /​266 and /​188, and /​318 and /​266, respectively.
The G10H promoter was capable of conferring stable GUS expression in transgenic
tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS
expression was tissue/​specific, restricted to leaf and actively growing cells
around the root tip, and not detected in the hypocotyls, root cap and older
developing areas of the root. The GUS expression in both transgenic C. roseus
hairy roots and tobacco seedlings were responsive to fungal elicitor and
methyljasmonate. Compared to other known promoters of TIA pathway genes, the
G10H promoter contains unique binding sites for several transcription factors,
suggesting that the G10H promoter may be regulated by a different
transcriptional cascade.

PMID: 17321612 [PubMed /​ as supplied by publisher]

2: FEBS J. 2007 Mar;274(5):1290/​303.

Cloning, characterization and localization of a novel basic peroxidase gene from
Catharanthus roseus.

Kumar S, Dutta A, Sinha AK, Sen J.

National Centre for Plant Genome Research, JNU Campus, Aruna Asaf Ali Marg, New
Delhi, India.

Catharanthus roseus (L.) G. Don produces a number of biologically active
terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic
pathway. The final dimerization step of this pathway, leading to the synthesis
of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic
peroxidase. However, reports of the gene encoding this enzyme are scarce for C.
roseus. We report here for the first time the cloning, characterization and
localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp
partial peroxidase cDNA (CrInt1) was initially amplified from the internodal
stem tissue, using degenerate oligonucleotide primers, and cloned. The
full/​length coding region of CrPrx cDNA was isolated by screening a
leaf/​specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence
encodes a deduced translation product of 330 amino acids with a 21 amino acid
signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass
of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa,
and the pI value is 8.68. CrPrx was found to belong to a 'three intron' category
of gene that encodes a class III basic secretory peroxidase. CrPrx protein and
mRNA were found to be present in specific organs and were regulated by different
stress treatments. Using a beta/​glucuronidase/​green fluorescent protein fusion
of CrPrx protein, we demonstrated that the fused protein is localized in leaf
epidermal and guard cell walls of transiently transformed tobacco. We propose
that CrPrx is involved in cell wall synthesis, and also that the gene is induced
under methyl jasmonate treatment. Its potential involvement in the terpenoid
indole alkaloid biosynthetic pathway is discussed.

PMID: 17298442 [PubMed /​ in process]

3: Biotechnol Prog. 2007 Jan 27; [Epub ahead of print]

Effect of the Engineered Indole Pathway on Accumulation of Phenolic Compounds in
Catharanthus roseus Hairy Roots.

Chung IM, Hong SB, Peebles CA, Kim JA, San KY.

Department of Applied Life Science, Konkuk University, Seoul 143/​701, South
Korea, and Department of Bioengineering, Rice University, Houston, Texas, 77005.

Catharanthus roseus has been well/​known to contain indole alkaloids effective
for treatment of diverse cancers. We examined the intracellular accumulation
profiles of phenolic compounds in response to ectopic overexpression of
tryptophan feedback/​resistant anthranilate synthase holoenzyme (ASalphabeta) in
C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic
compounds were detected in late exponential phase ASalphabeta hairy roots.
Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g
DW over a 72/​h period, respectively. Upon induction, in parallel with a rapid
increase in tryptophan in the first 48 h, accumulation of phenolic compounds
tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which
phenolic levels decreased back to the uninduced level by 72 h. Naringin was a
predominant form that comprised about 72% and 36% of the total content of
phenolic compounds in the uninduced and induced lines, respectively. Upon
induction, accumulation of catechin drastically increased with the highest level
(3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic
acid showed no statistical difference. Catechin is a final product of the
flavonoid pathway, and thus metabolic flux into this pathway is transiently
increased by overexpression of AS. Like catechin, salicylic acid is very
sensitive to induction as it began to increase to 5/​fold within 4 h of
induction, but unlike catechin, no significant accumulation of salicylic acid
was noted after 4 h of induction. The results suggest differential regulation of
this particular biosynthesis branch within the phenolic pathway.

PMID: 17256967 [PubMed /​ as supplied by publisher]

4: Planta Med. 1998 May;64(4):390.

Rapid in vitro Multiplication of Plants from Mature Nodal Explants of
Catharanthus roseus.

Mitra A, Khan B, Rawal S.

Division of Plant Tissue Culture, National Chemical Laboratory, Pune, India.

PMID: 17253257 [PubMed /​ in process]

5: J Exp Bot. 2006 Dec 21; [Epub ahead of print]

Involvement of rapid nucleotide synthesis in recovery from phosphate starvation
of Catharanthus roseus cells.

Yin Y, Shimano F, Ashihara H.

Department of Advanced Bioscience, Graduate School of Humanities and Sciences,
Ochanomizu University, Tokyo, 112/​8610 Japan.

Growth of suspension/​cultured Catharanthus roseus cells ceased during phosphate
starvation, but the cells grew again upon addition of Pi even after long/​term
starvation. The metabolic fate of [(33)P]Pi was studied in 1/​week/​old stationary
phase cells in ordinary culture and in 1/​ or 2/​week/​old Pi/​starved cells.
Immediately after administration, the most heavily labelled organic compounds
are nucleotides, followed by sugar phosphates. Two weeks Pi starvation slowed
down the speed of incorporation of (33)P into nucleotides. The RNA, protein, and
free nucleotide content all decreased gradually during Pi starvation; however,
these compounds, especially nucleotides, increased markedly in the 24 h after
addition of Pi. These responses are found in all cells examined, although the
total amounts of these compounds were lower in the long/​term Pi/​deficient cells.
Of the nucleotides, a marked increase was observed in nucleoside triphosphates
and UDP/​glucose. The transcript level of phosphate transporter and the
activities of acid phosphatase, 5'/​ and 3'/​nucleotidase, and adenosine
nucleosidase were all reduced by the addition of Pi. In contrast, the activities
of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase, and
nicotinamidase, which are salvage enzymes of purine and pyridine nucleotides,
were markedly increased in the Pi/​fed cells. Little or no increase was observed
in adenosine kinase. In the light of these results, the possible involvement of
net nucleotide synthesis in the initial metabolic events of recovery from Pi
deficiency are discussed.

PMID: 17185741 [PubMed /​ as supplied by publisher]

6: BMC Complement Altern Med. 2006 Dec 21;6:41.

Catharanthus roseus flower extract has wound/​healing activity in Sprague Dawley
rats.

Nayak BS, Pinto Pereira LM.

Department of Pre Clinical Sciences, Biochemistry Unit, Faculty of Medical
Sciences, The University of the West Indies, St, Augustine, Trinidad.
snayak@fms.uwi.tt

BACKGROUND: Catharanthus roseus L (C. roseus) has been used to treat a wide
assortment of diseases including diabetes. The objective of our study was to
evaluate the antimicrobial and wound healing activity of the flower extract of
Catharanthus in rats. METHODS: Wound healing activity was determined in rats,
after administration (100 mg kg/​1 day/​1) of the ethanol extract of C. roseus
flower, using excision, incision and dead space wounds models. The animals were
divided into two groups of 6 each in all the models. In the excision model,
group 1 animals were topically treated with carboxymethyl cellulose as placebo
control and group 2 received topical application of the ethanol extract of C.
roseus at a dose of 100 mg/kg body weight/day. In an incision and dead space
model group 1 animals were given normal saline and group 2 received the extract
orally at a dose of 100 mg kg/​1 day/​1. Healing was assessed by the rate of wound
contraction, period of epithelization, tensile strength (skin breaking
strength), granulation tissue weight, and hydoxyproline content. Antimicrobial
activity of the flower extract against four microorganisms was also assessed
RESULTS: The extract of C. roseus significantly increased the wound breaking
strength in the incision wound model compared with controls (P < 0.001). The
extract/​treated wounds were found to epithelialize faster, and the rate of wound
contraction was significantly increased in comparison to control wounds (P <
0.001), Wet and dry granulation tissue weights, and hydroxyproline content in a
dead space wound model increased significantly (p < 0.05). Pseudomonas
aeruginosa and Staphylococcus aureus demonstrated sensitivity to C. roseus
CONCLUSION: Increased wound contraction and tensile strength, augmented
hydroxyproline content along with antimicrobial activity support the use of C.
roseus in the topical management of wound healing.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 17184528 [PubMed /​ indexed for MEDLINE]

7: Se Pu. 2006 Sep;24(5):534.

[Determination of three endogenous hormones in catharanthus roseus (L.) G. Don
using solid/​phase extraction and high performance liquid chromatography]

[Article in Chinese]

Zhao X, Tang Z, Guo X, Zu Y, Jiao Y, Sun Y, Yang L.

zxj_daisy@163.com

PMID: 17165557 [PubMed /​ in process]

8: Metab Eng. 2007 Mar;9(2):125/​132. Epub 2006 Oct 21.

Transcription factor Agamous/​like 12 from Arabidopsis promotes tissue/​like
organization and alkaloid biosynthesis in Catharanthus roseus suspension cells.

Montiel G, Breton C, Thiersault M, Burlat V, Jay/​Allemand C, Gantet P.

Universite de Tours Unite sous Contrat reconnue par l'Institut National de la
Recherche Agronomique, Facteurs de Transcription et Ingenierie Metabolique
Vegetale, UFR des Sciences et Techniques et UFR des Sciences Pharmaceutiques,
Parc de Grandmont, 37200 Tours, France; Universite de Tours EA 2106,
Biomolecules et Biotechnologies Vegetales, UFR des Sciences et Techniques,
Laboratoire de Physiologie Vegetale, Parc de Grandmont, 37200 Tours, France;
Institut National de la Recherche Agronomique/​Orleans, Unite Amelioration,
Genetique et Physiologie Forestieres, BP 20619 ARDON, F/​45166 Olivet cedex,
France.

In Catharanthus roseus, monomeric terpenoid indole alkaloids (TIAs) are
biosynthesized in specific tissues, particularly in roots, but failed to be
produced by in vitro undifferentiated suspension cells. In this paper, we
describe the impact of the root/​specific MADS/​box transcription factor
Agamous/​like 12 (Agl12) from Arabidopsis thaliana on the differentiation of
suspension cells from C. roseus. The expression of Agl12 is sufficient to
promote an organization of suspension cells into globular parenchyma/​like
aggregates but is insufficient by itself to induce complete morphological root
differentiation. Agl12 expression selectively increases the expression of genes
encoding enzymes involved in the early biosynthesis steps of the terpenic
precursor of alkaloids. The transgenic cell lines expressing Agl12 produced
significant amounts of ajmalicine, an antihypertensive TIA that normally
accumulates in C. roseus roots. The present paper indicates that transcription
factors involved in tissue or organ differentiation may constitute new metabolic
engineering tools that could help to design in vitro cultured cells able to
produce specific valuable secondary metabolites.

PMID: 17157545 [PubMed /​ as supplied by publisher]

9: Pharmazie. 2006 Nov;61(11):952/​6.

Screening of herbal extracts for activation of the human peroxisome
proliferator/​activated receptor.

Rau O, Wurglics M, Dingermann T, Abdel/​Tawab M, Schubert/​Zsilavecz M.

Johann Wolfgang Goethe University Frankfurt, Institute of Pharmaceutical
Chemistry/ZAFES, Frankfurt/Main, Germany.

The peroxisome proliferator/​activated receptors play a pivotal role in metazoan
lipid and glucose homeostasis. Synthetic activators of PPARalpha (fibrates) and
PPARgamma (glitazones) are therefore widely used for treatment of dislipidemia
and diabetes, respectively. There is growing evidence for herbal compounds to
influence nuclear receptor signalling e.g. the PPARs. We recently reported
carnosic acid and carnosol, both being diterpenes found in the labiate herbs
sage and rosemary, to be activators of PPARgamma. The subsequent screening of a
variety of ethanolic extracts, obtained from traditionally used herbs, for PPAR
activation, led to an exceptionally high hit rate. Among 52 extracts nearly the
half significantly activated PPARgamma and 14 activated PPARalpha in addition,
whereas three of them were pan/​PPAR activators, which also activated PPARdelta.
The most active extracts, for which a concentration dependent effect could be
shown, were the extracts of Alisma plantago aquatica (ze xie/european
waterplantain), Catharanthus roseus (madagascar periwinkle), Acorus calamus
(sweet calamus), Euphorbia balsamifera (balsam spurge), Jatropha curcas
(barbados nut), Origanum majorana (marjoram), Zea mays (corn silk), Capsicum
frutescens (chilli) and Urtica dioica (stinging nettle). The results of the
present study provide a possible rationale for the traditional use of many herbs
as antidiabetics.

PMID: 17152989 [PubMed /​ indexed for MEDLINE]

10: Biotechnol Prog. 2006 Nov/​Dec;22(6):1659/​63.

Determination of biomass composition of Catharanthus roseus hairy roots for
metabolic flux analysis.

Sriram G, Gonzalez/​Rivera O, Shanks JV.

Department of Chemical and Biological Engineering, Iowa State University, 3031,
Sweeney Hall, Ames, Iowa 50011, USA.

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering,
and determination of biomass composition is indispensable to accurate flux
evaluation. We report the elemental and biomolecular composition of Catharanthus
roseus hairy roots, a pharmaceutically significant plant system and an important
metabolic engineering target. The molecular formula of the organic material in
the hairy roots was C12.0H22.7N0.4O7.6 during mid/​exponential growth. The
abundances of lipids, lignin, cellulose, hemicellulose, starch, protein,
proteinogenic amino acids, mineral ash, and moisture in the biomass were
quantified. Analysis of water/​soluble components of the biomass with 1/​D 13C and
2/​D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water/​soluble
components were composed almost entirely of /​glucans. Agropine, a frequently
reported hairy root biomass component, was not detected. Our measurements of the
biomass components quantified 83.6 +//​ 9.3% (w/w) of the biomass. Together with
previously reported abundances of indole alkaloids, we accounted for at least
85.9 +//​ 11.6% (w/w) of the carbon in the biomass, which enabled the precise
determination of 12 biomass synthesis fluxes.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 17137315 [PubMed /​ indexed for MEDLINE]

11: J Am Chem Soc. 2006 Nov 8;128(44):14276/​7.

Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus
roseus.

McCoy E, O'Connor SE.

Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139, USA.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 17076499 [PubMed /​ in process]

12: J Exp Bot. 2006;57(14):3921/​32. Epub 2006 Oct 18.

Transcriptome analysis in Catharanthus roseus leaves and roots for comparative
terpenoid indole alkaloid profiles.

Shukla AK, Shasany AK, Gupta MM, Khanuja SP.

Genetic Resources and Biotechnology Division, Central Institute of Medicinal and
Aromatic Plants, Lucknow 226015, India.

In Catharanthus roseus (L.) G. Don each tissue is known to produce a distinct
spectrum of terpenoid indole alkaloids. Since the invaluable antineoplastic
bisindole alkaloids are restricted to the aerial parts of the plant and do not
occur in its underground tissues, identification of the structural and
regulatory factors operating distinctly in the shoot/leaf of the plant will be a
necessity for modulation of bisindole alkaloid biosynthesis. This study aimed at
elucidating the differential gene expression in the two main tissues (leaf and
root) of the plant, well known for their distinct terpenoid indole alkaloid
profiles. The leaf and root transcriptomes of C. roseus were comparatively
analysed using two different approaches: (i) indirectly through construction and
characterization of separate cDNA libraries; and (ii) directly through a
strategically designed suppression subtractive hybridization, using the leaf and
root cDNA populations as tester and driver, respectively. A total of 155 ESTs
(55 and 45 from the separate leaf and root cDNA libraries, respectively, and 55
from the subtracted leaf/​specific cDNA library) were subjected to homology/​based
classification and submitted to dbEST. The direct approach yielded an EST for
sgd (strictosidine beta/​D/​glucosidase) and 16 novel ESTs. Dat (acetyl/​CoA:
4/​O/​deacetylvindoline 4/​O/​acetyl/​transferase) and sgd transcripts could not be
detected in the root system of the plant (cv. 'Dhawal') at any developmental
stage (6 d, 6 weeks, or 6 months). The growth/​related decrease in shoot/leaf dat
and sgd transcript levels was paralleled by a concomitant decrease in shoot/leaf
vindoline content.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 17050644 [PubMed /​ indexed for MEDLINE]

13: J Ethnobiol Ethnomedicine. 2006 Oct 13;2:45.

Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes
mellitus.

Lans CA.

BCICS, University of Victoria, British Columbia, V8W 2Y2, Canada.
cher2lans@netscape.net.

ABSTRACT: BACKGROUND: This paper is based on ethnobotanical interviews conducted
from 1996/​2000 in Trinidad and Tobago with thirty male and female respondents.
METHODS: A non/​experimental validation was conducted on the plants used for
urinary problems and diabetes mellitus: This is a preliminary step to establish
that the plants used are safe or effective, to help direct clinical trials, and
to inform Caribbean physicians of the plants' known properties to avoid
counter/​prescribing. RESULTS: The following plants are used to treat diabetes:
Antigonon leptopus, Bidens alba, Bidens pilosa, Bixa orellana, Bontia
daphnoides, Carica papaya, Catharanthus roseus, Cocos nucifera, Gomphrena
globosa, Laportea aestuans, Momordica charantia, Morus alba, Phyllanthus
urinaria and Spiranthes acaulis. Apium graviolens is used as a heart tonic and
for low blood pressure. Bixa orellana, Bontia daphnoides, Cuscuta americana and
Gomphrena globosa are used for jaundice. The following plants are used for
hypertension: Aloe vera, Annona muricata, Artocarpus altilis, Bixa orellana,
Bidens alba, Bidens pilosa, Bonta daphnoides, Carica papaya, Cecropia peltata,
Citrus paradisi, Cola nitida, Crescentia cujete, Gomphrena globosa, Hibiscus
sabdariffa, Kalanchoe pinnata, Morus alba, Nopalea cochinellifera, Ocimum
campechianum, Passiflora quadrangularis, Persea americana and Tamarindus
indicus.The plants used for kidney problems are Theobroma cacao, Chamaesyce
hirta, Flemingia strobilifera, Peperomia rotundifolia, Petiveria alliacea,
Nopalea cochinellifera, Apium graveolens, Cynodon dactylon, Eleusine indica,
Gomphrena globosa, Pityrogramma calomelanos and Vetiveria zizanioides. Plants
are also used for gall stones and for cooling. CONCLUSION: Chamaesyce hirta,
Cissus verticillata, Kalanchoe pinnata, Peperomia spp., Portulaca oleraceae,
Scoparia dulcis, and Zea mays have sufficient evidence to support their
traditional use for urinary problems, "cooling" and high cholesterol.Eggplant
extract as a hypocholesterolemic agent has some support but needs more study.
The plants used for hypertension, jaundice and diabetes that may be safe and
justify more formal evaluation are Annona squamosa, Aloe vera, Apium graveolens,
Bidens alba, Carica papaya, Catharanthus roseus, Cecropia peltata, Citrus
paradisi, Hibsicus sabdariffa, Momordica charantia, Morus alba, Persea
americana, Phyllanthus urinaria, Tamarindus indicus and Tournefortia
hirsutissima. Several of the plants are used for more than one condition and
further trials should take this into account.

PMID: 17040567 [PubMed /​ in process]

14: Biotechnol Lett. 2006 Oct;28(20):1687/​94. Epub 2006 Aug 3.

Development of a quantitative method for determination of the optimal conditions
for protoplast isolation from cultured plant cells.

Aoyagi H.

Institute of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305/​8572,
Japan. aoyagi@sakura.cc.tsukuba.ac.jp

An index [kv: average isolation rate of viable protoplast (number/ml min)] was
established to evaluate the optimal conditions for protoplast isolation from
cultured plant cells. The optimal conditions for protoplasts isolation from
Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv
[31.7 x 10(3) (number/ml min)]. The colony/​forming efficiency of the protoplasts
was about 46%. The optimal conditions for protoplasts isolation from
Catharanthus roseus [kv = 38.1 x 10(3) (number/ml min)] and Wasabia japonica [kv
= 14.2 x 10(3) (number/ml min)] cultured cells could also be determined.
Furthermore, a method for rapid regenerating cell wall of protoplast in liquid
culture using alginate gel containing locust bean gum was developed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16955360 [PubMed /​ indexed for MEDLINE]

15: Planta. 2007 Feb;225(3):711/​8. Epub 2006 Sep 6.

Inorganic phosphate uptake in intact vacuoles isolated from suspension/​cultured
cells of Catharanthus roseus (L.) G. Don under varying Pi status.

Ohnishi M, Mimura T, Tsujimura T, Mitsuhashi N, Washitani/​Nemoto S, Maeshima M,
Martinoia E.

Department of Biology, Faculty of Science, Kobe University, Rokkodai 1/​1, Nada,
Kobe, 678/​8501, Japan.

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles
isolated from Catharanthus roseus suspension/​cultured cells was measured. Under
low Pi status, Pi uptake into the vacuole was strongly activated compared to
high Pi status. Since Pi uptake across the vacuolar membrane is correlated with
H(+) pumping, we examined the dependency of H(+) pumping on plant Pi status.
Both H(+) pumping and the activities of the vacuolar H(+)/​pumps, the V/​type
H(+)/​ATPase and the H(+)/​PPase were enhanced under low Pi status. Despite this
increase in H(+) pumping, Western blot analysis showed no distinct increase in
the amount of proton pump proteins. Possible mechanisms for the activation of Pi
uptake into the vacuole under low Pi status are discussed.

PMID: 16955272 [PubMed /​ in process]

16: Microbiology. 2006 Sep;152(Pt 9):2703/​16.

Plasmid pSci6 from Spiroplasma citri GII/​3 confers insect transmissibility to
the non/​transmissible strain S. citri 44.

Berho N, Duret S, Danet JL, Renaudin J.

UMR 1090 Genomique Developpement et Pouvoir Pathogene, INRA, Universite de
Bordeaux 2, Centre INRA de Bordeaux, 71 avenue Edouard Bourlaux, BP 81, 33883
Villenave d'Ornon Cedex, France.

The insect/​transmissible strain GII/​3 of Spiroplasma citri contains plasmids
pSci1/​6, five of which (pSci1/​5) encode adhesin/​like proteins and one (pSci6)
encodes protein P32, which has been associated with insect transmissibility. In
contrast, S. citri strains ASP/​1 and 44, which cannot be transmitted via
injection into the leafhopper vector Circulifer haematoceps, lack these proteins
and also do not carry plasmids pSci1/​6. To further study the apparent
relationship between the presence of plasmids and insect transmissibility,
plasmids from S. citri GII/​3 were introduced into the insect/​non/​transmissible
S. citri strain 44 by electrotransformation using the tetM gene as the selection
marker. Tetracycline/​resistant transformants were shown to carry one, two or
three distinct plasmids. Plasmids pSci1/​6 were all detected in the
transformants, pSci1 being the most frequently found, alone or together with
other plasmids. Selected S. citri 44 transformants having distinct plasmid
contents were submitted, separately or in combination, to experimental
transmission to periwinkle (Catharanthus roseus) plants via injection into the
leafhopper vector. The occurrence of symptomatic plants indicated that, in
contrast to S. citri 44, spiroplasmal transformants were transmitted to the host
plant, in which they multiplied. Spiroplasma cultures isolated from these
infected plants all contained pSci6, leading to the conclusion that, under the
experimental conditions used, transformation by pSci6 conferred insect
transmissibility to S. citri strain 44. This is believed to be the first report
of a phenotypic change associated with transformation of S. citri by natural
plasmids.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16946265 [PubMed /​ indexed for MEDLINE]

17: Biotechnol Lett. 2006 Oct;28(19):1567/​71. Epub 2006 Aug 2.

Preparation of mixed alginate elicitors with high activity for the efficient
production of 5'/​phosphodiesterase by Catharanthus roseus cells.

Aoyagi H, Akimoto/​Tomiyama C, Tanaka H.

Institute of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305/​8572, Japan.
aoyagi@sakura.cc.tsukuba.ac.jp

When various autoclaved microbial cells suspensions (exogenous elicitors) were
added to Catharanthus roseus cell cultures, its growth was inhibited but
5'/​phosphodiesterase (PDase) production was stimulated. The greatest effect was
with autoclaved Alteromonas macleodii: the dry cell concentration decreased from
13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A
combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate
oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen
species) minimized the cell growth inhibition but enhanced PDase production
(0.474 U/ml) about 20 times higher than the control (no addition). The method
for the preparation of mixed alginate elicitors with high activities containing
exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and
trans/​4,5/​dihydroxy/​2/​cyclopenten/​1/​one was developed. The mixed alginate
elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and
the productivity was increased 120/​fold compared to the control without cell
growth inhibition.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16937247 [PubMed /​ indexed for MEDLINE]

18: Plant Cell Environ. 2006 Sep;29(9):1742/​50.

Predicting leaf/​level fluxes of O3 and NO2: the relative roles of diffusion and
biochemical processes.

Eller AS, Sparks JP.

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY
14853/​2701, USA.

Pollutants like O(3) and NO(2) enter leaves through the stomata and cause damage
during reactions with components of biological cell membranes. The steady/​state
flux rates of these gases into the leaf are determined by a series of physical
and biochemical resistances including stomatal aperture, reactions occurring
within the cell wall and the ability of the leaf to remove the products of
apoplastic reactions. In the present study, multiple regression models
incorporating stomatal conductance, apoplastic and symplastic ascorbate
concentrations, and nitrate reductase (NR) activities were generated to explain
the observed variations in leaf/​level flux rates of O(3) and NO(2). These
measurements were made on the plant Catharanthus roseus (Madagascar periwinkle).
The best/​fit model explaining NO(2) flux included stomatal conductance,
apoplastic ascorbate and NR activity. This model explained 89% of the variation
in observed leaf fluxes and suggested physical resistances, reaction between
NO(2) and apoplastic ascorbate, and the removal rate of nitrate (generated by
reactions of NO(2) and water) from the apoplast all play controlling roles in
NO(2) flux to leaves. O(3) flux was best explained by stomatal conductance and
symplastic ascorbate explaining 66% of the total variation in leaf flux. Both
models demonstrate the importance of measuring processes other than stomatal
conductance to explain steady/​state leaf/​level fluxes of pollutant gases.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16913863 [PubMed /​ indexed for MEDLINE]

19: Plant Mol Biol. 2006 Jul;61(4/​5):719/​32.

Cloning, characterization and localization of three novel class III peroxidases
in lignifying xylem of Norway spruce (Picea abies).

Marjamaa K, Hilden K, Kukkola E, Lehtonen M, Holkeri H, Haapaniemi P, Koutaniemi
S, Teeri TH, Fagerstedt K, Lundell T.

Department of Biological and Environmental Sciences, University of Helsinki,
P.O. Box 65, 00014 Helsinki, Finland. kaisa.marjamaa@helsinki.fi

Plant class III peroxidases (POXs) take part in the formation of lignin and
maturation of plant cell walls. However, only a few examples of such peroxidases
from gymnosperm tree species with highly lignified xylem tracheids have been
implicated so far. We report here cDNA cloning of three xylem/​expressed class
III peroxidase encoding genes from Norway spruce (Picea abies). The translated
proteins, PX1, PX2 and PX3, contain the conserved amino acids required for
heme/​binding and peroxidase catalysis. They all begin with putative secretion
signal propeptide sequences but diverge substantially at phylogenetic level,
grouping to two subclusters when aligned with other class III plant peroxidases.
In situ hybridization analysis on expression of the three POXs in Norway spruce
seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm
of young, developing tracheids within the current growth ring where
lignification is occurring. Function of the putative N/​terminal secretion signal
peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions
with EGFP (enhanced green fluorescent protein) and expressing them in tobacco
protoplasts. Full/​length coding region of px1 was also heterologously expressed
in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1
peroxidase is processed via the endoplasmic reticulum (ER) most likely for
secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal
localization for participation in the maturation of the spruce tracheid
secondary cell wall.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16897487 [PubMed /​ indexed for MEDLINE]

20: FEBS Lett. 2006 Aug 7;580(18):4501/​7. Epub 2006 Jul 17.

Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus).

Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St.
Catharines, Ont., Canada L2S3A1.

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the
chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its
importance, no expressed sequence tag (EST) analysis of this plant has been
reported. Two cDNA libraries were generated from RNA isolated from the base part
of young leaves and from root tips to select 9,824 random clones for
unidirectional sequencing, to yield 3,327 related sequences and 1,696 singletons
by cluster analysis. Putative functions of 3,663 clones were assigned, from
5,023 non/​redundant ESTs to establish a resource for transcriptome analysis and
gene discovery in this medicinal plant.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16870181 [PubMed /​ indexed for MEDLINE]

21: Phytochemistry. 2006 Aug;67(16):1758/​64. Epub 2006 Jun 27.

Identification of a low vindoline accumulating cultivar of Catharanthus roseus
(L.) G. Don by alkaloid and enzymatic profiling.

Magnotta M, Murata J, Chen J, De Luca V.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St.
Catharines, Ont., Canada L2S 3A1.

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially
important horticultural flower species and is the only source of the
monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, key
pharmaceutical compounds used to combat a number of different cancers. The
present study uses high performance liquid chromatography for metabolic
profiling of the MIAs extracted from seedlings and young leaves of 50 different
flowering cultivars of C. roseus to show that, except for a single low vindoline
cultivar (Vinca Mediterranean DP Orchid), they accumulate similar levels of
MIAs. Further enzymatic studies with extracts from young leaves and from
developing seedlings show that the low vindoline cultivar has a 10/​fold lower
tabersonine/​16/​hydroxylase activity than those of C. roseus cv. Little Delicata.
It is concluded that rapid metabolic and more selective enzymatic profiling of
Catharanthus mutants could be useful for the identification of a range of
altered MIA biosynthesis lines.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16806326 [PubMed /​ indexed for MEDLINE]

22: Plant Biol (Stuttg). 2006 Sep;8(5):731/​6. Epub 2006 Jun 13.

Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase
receptor in tropical periwinkle Catharanthus roseus.

Papon N, Bremer J, Vansiri A, Glevarec G, Rideau M, Creche J.

Laboratoire des Sciences Vegetales, Faculte des Sciences Pharmaceutiques et
Biologiques, Paris, France.

Signalling pathways involving histidine kinase receptors (HKRs) are widely used
by prokaryotes and fungi to regulate a large palette of biological processes. In
plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing
transduction pathways. In this work, a full length cDNA named CRCIK was isolated
from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205
amino acid protein that belongs to the hybrid HKR family. The deduced amino acid
sequence shows the highest homology with AtHK1, an osmosensing HKR in
ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the
other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK
corresponding gene is either present in multiple copies or has very close
homologues in the genome of the tropical periwinkle. The gene is widely
expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly
induced after exposure to low temperature, pointing to a putative role in
cold/​shock signal transduction.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16773556 [PubMed /​ indexed for MEDLINE]

23: J Biosci Bioeng. 2006 Apr;101(4):287/​96.

Metabolic engineering of cell cultures versus whole plant complexity in
production of bioactive monoterpene indole alkaloids: recent progress related to
old dilemma.

Pasquali G, Porto DD, Fett/​Neto AG.

Programa de Pos/​graduacao em Biologia Celular e Molecular, Centro de
Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Goncalves
9500, Pr. 43.431, P.O. Box 15.005, CEP 91.501/​970, Porto Alegre, RS, Brazil.

Monoterpene indole alkaloids (MIAs) are a large class of plant alkaloids with
significant pharmacological interest. The sustained production of MIAs at high
yields is an important goal in biotechnology. Intensive effort has been expended
toward the isolation, cloning, characterization and transgenic modulation of
genes involved in MIA biosynthesis and in the control of the expression of these
biosynthesis/​related genes. At the same time, considerable progress has been
made in the detailed description of the subcellular/​, cellular/​, tissue/​ and
organ/​specific expressions of portions of the biosynthetic pathways leading to
the production of MIAs, revealing a complex picture of the transport of
biosynthetic intermediates among membrane compartments, cells and tissues. The
identification of the particular environmental and ontogenetic requirements for
maximum alkaloid yield in MIA/​producing plants has been useful in improving the
supply of bioactive molecules. The search for new bioactive MIAs, particularly
in tropical and subtropical regions, is continuously increasing the arsenal for
therapeutic, industrially and agriculturally useful molecules. In this review we
focus on recent progress in the production of MIAs in transgenic cell cultures
and organs (with emphasis on Catharanthus roseus and Rauvolfia serpentina
alkaloids), advances in the understanding of in planta spatial/​temporal
expression of MIA metabolic pathways, and on the identification of factors
capable of modulating bioactive alkaloid accumulation in nontransgenic
differentiated cultures and plants (with emphasis on new MIAs from Psychotria
species). The combined use of metabolic engineering and physiological modulation
in transgenic and wild/​type plants, although not fully exploited to date, is
likely to provide the sustainable and rational supply of bioactive MIAs needed
for human well being.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16716935 [PubMed /​ indexed for MEDLINE]

24: Can J Microbiol. 2006 May;52(5):419/​26.

Model plants for studying the interaction between Methylobacterium mesophilicum
and Xylella fastidiosa.

Andreote FD, Lacava PT, Gai CS, Araujo WL, Maccheroni W Jr, van Overbeek LS, van
Elsas JD, Azevedo JL.

Department of Genetics, Escola Superior de Agricultura Luiz de Queiroz,
University of Sao Paulo, ESALQ/USP, Piracicaba, Brazil.

Over the last few years, endophytic bacterial communities associated with citrus
have been studied as key components interacting with Xylella fastidiosa. In this
study, we investigated the possible interaction between the citrus endophyte
Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as
Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii
(Clevelands tobacco). The aim of this study was to establish the fate of M.
mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants,
using PCR/​DGGE (polymerase chain reaction/​/​denaturing gradient gel
electrophoresis) and plating techniques. Shifts in the indigenous endophytic
bacterial communities were observed in plants inoculated with strain SR1.6/6,
using specific primers targeting alpha/​ and beta/​Proteobacteria. Cells of strain
SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces
of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes
the importance of these tissues as main points of entrance for this organism.
The results showed that C. roseus and N. clevelandii could be used as model
plants to study the interaction between M. mesophilicum and X. fastidiosa.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16699566 [PubMed /​ indexed for MEDLINE]

25: Methods Mol Biol. 2006;318:349/​55.

Catharanthus roseus shoot cultures for the production of monoterpenoid indole
alkaloids.

Hernandez/​Dominguez E, Campos/​Tamayo F, Carrillo/​Pech M, Vazquez/​Flota F.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Mexico.

A protocol for the establishment of in vitro shoot cultures of Catharanthus
roseus is described. Shoots can be maintained for more than 1 yr without
evidence of tissue vitrification, disaggregation, or callus formation. Vindoline
was the main alkaloid accumulated, reaching values similar to those found in
leaves from field/​grown plants, after a long period of culture. An induction
methodology to reduce such waiting time is also presented.

PMID: 16673929 [PubMed /​ indexed for MEDLINE]

26: Plant J. 2006 Apr;46(2):193/​205.

Methylation of sulfhydryl groups: a new function for a family of small molecule
plant O/​methyltransferases.

Coiner H, Schroder G, Wehinger E, Liu CJ, Noel JP, Schwab W, Schroder J.

TU Munchen, FG Biomolekulare Lebensmitteltechnologie, Lise/​Meitner/​Str. 34,
D/​85354 Freising, Germany.

In plants, type I and II S/​adenosyl/​l/​methionine/​dependent O/​methyltransferases
(OMTs) catalyze most hydroxyl group methylations of small molecules. A
homology/​based RT/​PCR strategy using Catharanthus roseus (Madagascar periwinkle)
RNA previously identified six new type I plant OMT family members. We now
describe the molecular and biochemical characterization of a seventh protein. It
shares 56/​58% identity with caffeic acid OMTs (COMTs), but it failed to
methylate COMT substrates, and had no activity with flavonoids. However, the in
vitro incubations revealed unusually high background levels without added
substrates. A search for the responsible component revealed that the enzyme
methylated dithiothreitol (DTT), the reducing agent added for enzyme
stabilization. Unexpectedly, product analysis revealed that the methylation
occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34
compounds indicated a broad substrate range, with a preference for small
hydrophobic molecules. Benzene thiol (Km 220 microm) and furfuryl thiol (Km 60
microm) were the best substrates (6/​7/​fold better than DTT). Small isosteric
hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also
methylated, but the activities were at least 5/​fold lower than with thiols. The
enzyme was named C. roseus S/​methyltransferase 1 (CrSMT1). Models based on the
COMT crystal structure suggest that S/​methylation is mechanistically identical
to O/​methylation. CrSMT1 so far is the only recognized example of an
S/​methyltransferase in this protein family. Its properties indicate that a few
changes in key residues are sufficient to convert an OMT into a
S/​methyltransferase (SMT). Future functional investigations of plant
methyltransferases should consider the possibility that the enzymes may direct
methylation at sulfhydryl groups.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16623883 [PubMed /​ indexed for MEDLINE]

27: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5614/​9. Epub 2006 Mar 24.

Gene/​to/​metabolite networks for terpenoid indole alkaloid biosynthesis in
Catharanthus roseus cells.

Rischer H, Oresic M, Seppanen/​Laakso T, Katajamaa M, Lammertyn F, Ardiles/​Diaz
W, Van Montagu MC, Inze D, Oksman/​Caldentey KM, Goossens A.

VTT Technical Research Centre of Finland, Tietotie 2, FIN/​02044 VTT, Espoo,
Finland.

Rational engineering of complicated metabolic networks involved in the
production of biologically active plant compounds has been greatly impeded by
our poor understanding of the regulatory and metabolic pathways underlying the
biosynthesis of these compounds. Whereas comprehensive genome/​wide functional
genomics approaches can be successfully applied to analyze a select number of
model plants, these holistic approaches are not yet available for the study of
nonmodel plants that include most, if not all, medicinal plants. We report here
a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus
roseus), a source of the anticancer drugs vinblastine and vincristine.
Genome/​wide transcript profiling by cDNA/​amplified fragment/​length polymorphism
combined with metabolic profiling of elicited C. roseus cell cultures yielded a
collection of known and previously undescribed transcript tags and metabolites
associated with terpenoid indole alkaloids. Previously undescribed gene/​to/​gene
and gene/​to/​metabolite networks were drawn up by searching for correlations
between the expression profiles of 417 gene tags and the accumulation profiles
of 178 metabolite peaks. These networks revealed that the different branches of
terpenoid indole alkaloid biosynthesis and various other metabolic pathways are
subject to differing hormonal regulation. These networks also served to identify
a select number of genes and metabolites likely to be involved in the
biosynthesis of terpenoid indole alkaloids. This study provides the basis for a
better understanding of periwinkle secondary metabolism and increases the
practical potential of metabolic engineering of this important medicinal plant.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16565214 [PubMed /​ indexed for MEDLINE]

28: Curr Pharm Biotechnol. 2006 Feb;7(1):33/​49.

Micropropagation: a tool for the production of high quality plant/​based
medicines.

Debnath M, Malik CP, Bisen PS.

Tissue Culture Laboratory, Institute of Biotechnology and Allied Sciences,
Seedling Academy of Design, Technology And Management, Jaipur/​302025, India.

Medicinal plants are the most important source of life saving drugs for the
majority of the world's population. The biotechnological tools are important to
select, multiply and conserve the critical genotypes of medicinal plants. Plant
tissue culture techniques offer an integrated approach for the production of
standardized quality phytopharmaceutical through mass/​production of consistent
plant material for physiological characterization and analysis of active
ingredients. Micropropagation protocols for cloning of some medicinal plants
such as Catharanthus roseus (Apocynaceae), Chlorophytum borivilianum
(Liliaceae), Datura metel (Solanaceae), and Bacopa monnieri (Scrophulariaceae)
have been developed. Regeneration occurred via organogenesis and embryogenesis
in response to auxins and cytokinins. The integrated approaches of our culture
systems will provide the basis for the future development of novel, safe,
effective, and high/​quality products for consumers.

Publication Types:
Review

PMID: 16472132 [PubMed /​ indexed for MEDLINE]

29: Bioprocess Biosyst Eng. 2006 Apr;28(5):295/​313. Epub 2006 Feb 1.

Development of a kinetic metabolic model: application to Catharanthus roseus
hairy root.

Leduc M, Tikhomiroff C, Cloutier M, Perrier M, Jolicoeur M.

Department of Chemical Engineering, Ecole Polytechnique de Montreal, PO Box
6079, Centre/​ville Station, Montreal, Quebec, Canada, H3C 3A7.

A kinetic metabolic model describing Catharanthus roseus hairy root growth and
nutrition was developed. The metabolic network includes glycolysis,
pentose/​phosphate pathway, TCA cycle and the catabolic reactions leading to cell
building blocks such as amino acids, organic acids, organic phosphates, lipids
and structural hexoses. The central primary metabolic network was taken at
pseudo/​steady state and metabolic flux analysis technique allowed reducing from
31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate
was described as a function of intracellular concentration in cell building
blocks. Intracellular transport and accumulation kinetics for major nutrients
were included. The model uses intracellular nutrients as well as energy shuttles
to describe metabolic regulation. Model calibration was performed using
experimental data obtained from batch and medium exchange liquid cultures of C.
roseus hairy root using a minimal medium in Petri dish. The model is efficient
in estimating the growth rate.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16453114 [PubMed /​ indexed for MEDLINE]

30: Plant Cell Rep. 2006 Jun;25(6):607/​12. Epub 2006 Jan 24.

Precursor limitations in methyl jasmonate/​induced Catharanthus roseus cell
cultures.

Lee/​Parsons CW, Royce AJ.

Chemical Engineering Department, 342 Snell Engineering Center, Northeastern
University, 360 Huntington Avenue, Boston, MA 02115/​5000, USA. clee@coe.neu.edu

Jasmonates enhance the expression of various genes involved in terpenoid indole
alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding
to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the
precursor availability for TIA biosynthesis. C. roseus suspensions were induced
with MJ (100 microM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM),
loganin plus tryptamine, or geraniol (0.1/​1.0 mM) on day 7. While MJ increased
ajmalicine production by 3/​fold, induced cultures were still limited by
terpenoid precursors. However, both induced and non/​induced cultures became
tryptamine/​limited with excess loganin. Geraniol feeding also increased
ajmalicine production in non/​induced cultures. But MJ appeared to increase
geraniol availability in induced cultures, due presumably to the increased
expression of Dxs with MJ addition.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16432630 [PubMed /​ indexed for MEDLINE]

31: Phytomedicine. 2006 Jan;13(1/​2):67/​73. Epub 2005 Jun 29.

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants.

Usia T, Iwata H, Hiratsuka A, Watabe T, Kadota S, Tezuka Y.

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
2630/​Sugitani, Toyama 930/​0194, Japan.

Thirty samples of Indonesian medicinal plants were analyzed for their capacity
to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6
with a radiometric assay. The MeOH/​soluble fractions of 25 samples, prepared
from water extracts, demonstrated inhibitory activity more than 50% on the
metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by
CYP2D6. Among the MeOH/​soluble fractions, Piper nigrum leaf showed the highest
inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6
(98.1%). The water extracts of which MeOH/​soluble fraction showed inhibitory
activity more than 70% were fractionated with EtOAc. From the EtOAc/​soluble
fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit
(84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated
inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only
Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory
activity against CYP2D6. For samples that showed more than 70% inhibition, their
IC(50) values were determined. The most potent inhibitory activity against
CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum
leaf, while that of Catharanthus roseus showed the most potent inhibitory effect
against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate
once more the possibility of potential medicinal plant/​drug interactions.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16360935 [PubMed /​ indexed for MEDLINE]

32: J Plant Physiol. 2006 Jan;163(1):11/​8. Epub 2005 Aug 10.

Effect of salinity and different nitrogen sources on the activity of antioxidant
enzymes and indole alkaloid content in Catharanthus roseus seedlings.

Misra N, Gupta AK.

Department of Biochemistry, Bundelkhand University, Jhansi (UP), India.
neelam_misra@rediffmail.com

The activities of antioxidant enzymes viz. glutathione reductase, GR; superoxide
dismutase, SOD; peroxidase, POD; catalase, CAT and glutathione/​S/​transferase,
GST and alkaloid accumulation were investigated in leaf pairs (apical, middle,
basal) and in roots of Catharanthus roseus seedlings under the conditions of
different nitrogen sources (20 mM KNO(3) and 2 mM NH(4)Cl) and salinity, in the
absence (non/​saline control) and in the presence of 100 mM NaCl in the nutrient
solution. Salinity caused a reduction in plant biomass. The biomass production
of ammonium/​fed plants was lower than that of nitrate/​fed plants. The
antioxidant enzymes exhibited higher activity in saline/​treated plants. Changes
in antioxidant enzyme activity caused by different nitrogen sources differed in
all leaf pairs, as well as in roots of C. roseus. Ammonium/​fed plants showed
higher CAT, GR and GST activity in leaf pairs as well as in roots, while POD and
SOD activity were higher in nitrate/​fed plants. Higher peroxidase activity
concomitant with the increased accumulation of alkaloid was found in all leaf
pairs, as well as in roots of C. roseus of NO(3)(/​) fed plants as compared to
NH(4)(+) fed plants.

PMID: 16360799 [PubMed /​ indexed for MEDLINE]

33: Cell Mol Biol Lett. 2005;10(4):649/​57.

Isolation of a cDNA encoding the alpha/​subunit of CAAX/​prenyltransferases from
Catharanthus roseus and the expression of the active recombinant protein
farnesyltransferase.

Courdavault V, Burlat V, St/​Pierre B, Gantet P, Giglioli/​Guivarc'h N.

Universite Francois/​Rabelais de Tours, EA 2106 Biomolecules et Biotechnologies
Vegetales, UFR Sciences et Techniques, Parc de Grandmont, 37200 Tours, France.
courdavault@univ/​tours.fr

Crfta/ggt_Ia (AF525030), a cDNA encoding the ?/​subunit of the two types of
CaaX/​prenyltransferase (CaaX/​PTase), i.e. protein farnesyltransferase (PFT) and
type I protein geranylgeranyltransferase, was cloned from Catharanthus roseus
via a PCR strategy. Crfta/ggt_Ia is 1381/​bp long and bears a 999/​bp open reading
frame encoding a protein of 332 residues (FTA) that shares 66% identity with its
Lycopersicon esculentum orthologue. Southern blot analysis revealed that FTA is
encoded by a single gene copy per haploid genome. Co/​expression of Crfta/ggt_Ia
and Crftb encoding the beta/​subunit of PFT yielded purified active recombinant
PFT. This enzyme is able to prenylate proteins from C. roseus, and could be used
as a potent tool for prenylated protein identification.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16341273 [PubMed /​ indexed for MEDLINE]

34: Planta. 2006 May;223(6):1191/​200. Epub 2005 Dec 2.

Epidermis is a pivotal site of at least four secondary metabolic pathways in
Catharanthus roseus aerial organs.

Mahroug S, Courdavault V, Thiersault M, St/​Pierre B, Burlat V.

Universite Francois/​Rabelais de Tours, EA 2106 Biomolecules et Biotechnologies
Vegetales UFR Sciences et Techniques, Parc de Grandmont, 37200 Tours, France.

Catharanthus roseus produces a wide range of secondary metabolites, some of
which present high therapeutic values such as antitumoral monoterpenoid indole
alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA,
ajmalicine. We have recently shown that a complex multicellular organisation of
the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular,
the final steps of both the secoiridoid/​monoterpene and indole pathways
specifically occurred in the epidermis of leaves and petals. Chorismate is the
common precursor of indole and phenylpropanoid pathways. In an attempt to better
map the spatio/​temporal organisation of diverse secondary metabolisms in
Catharanthus roseus aerial organs, we studied the expression pattern of genes
encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia/​lyase
[PAL, E.C. 4.3.1.5], cinnamate 4/​hydroxylase [C4H, E.C. 1.14.13.11] and chalcone
synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that
CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL,
CrC4H and CrCHS were specifically expressed in the flavonoid/​rich upper
epidermis. Interestingly, these three genes were co/​expressed in the epidermis
(at least the upper, adaxial one) together with three MIA/​related genes,
indicating that single epidermis cells were capable of concomitantly producing a
wide range of diverse secondary metabolites (e.g. flavonoids, indoles,
secoiridoid/​monoterpenes and MIAs). These results, and data showing
co/​accumulation of flavonoids and alkaloids in single cells of C. roseus cell
lines, indicated the spatio/​temporal feasibility of putative common regulation
mechanisms for the expression of these genes involved in at least four distinct
secondary metabolisms.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16322983 [PubMed /​ indexed for MEDLINE]

35: Phytochemistry. 2006 Jan;67(2):132/​41.

Effect of long/​term phosphate starvation on the levels and metabolism of purine
nucleotides in suspension/​cultured Catharanthus roseus cells.

Shimano F, Ashihara H.

Department of Molecular Biology and Biochemistry, Graduate Division of Life
Sciences, Graduate School of Humanities and Sciences, Ochanomizu University,
Bunkyo/​ku, Tokyo 112/​8610, Japan.

The effect of long/​term phosphate (Pi) starvation of up to 3 weeks on the levels
of purine nucleotides and related compounds was examined using
suspension/​cultured Catharanthus roseus cells. Levels of adenine and guanine
nucleotides, especially ATP and GTP, were markedly reduced during Pi/​starvation.
There was an increase in the activity of RNase, DNase, 5'/​ and 3'/​nucleotidases
and acid phosphatase, which may participate in the hydrolysis of nucleic acids
and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was
observed during the long/​term Pi starvation. Long/​term Pi starvation markedly
depressed the flux of transport of exogenously supplied [8/​(14)C]adenosine and
[8/​(14)C]adenine, but these labelled compounds which were taken up by the cells
were readily converted to adenine nucleotides even in Pi/​starved cells, in which
RNA synthesis from these precursors was significantly reduced. The activities of
adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase
were maintained at a high level in long/​term Pi starved cells.

PMID: 16321409 [PubMed /​ indexed for MEDLINE]

36: Bioelectromagnetics. 2006 Feb;27(2):98/​104.

Magnetic field exposure stiffens regenerating plant protoplast cell walls.

Haneda T, Fujimura Y, Iino M.

Department of Precision Engineering, Faculty of Engineering, Chiba Institute of
Technology, Chiba, Japan.

Single suspension/​cultured plant cells (Catharanthus roseus) and their
protoplasts were anchored to a glass plate and exposed to a magnetic field of
302 +//​ 8 mT for several hours. Compression forces required to produce constant
cell deformation were measured parallel to the magnetic field by means of a
cantilever/​type force sensor. Exposure of intact cells to the magnetic field did
not result in any changes within experimental error, while exposure of
regenerating protoplasts significantly increased the measured forces and
stiffened regenerating protoplasts. The diameters of intact cells or
regenerating protoplasts were not changed after exposure to the magnetic field.
Measured forces for regenerating protoplasts with and without exposure to the
magnetic field increased linearly with incubation time, with these forces being
divided into components based on the elasticity of synthesized cell walls and
cytoplasm. Cell wall synthesis was also measured using a cell wall/​specific
fluorescent dye, and no changes were noted after exposure to the magnetic field.
Analysis suggested that exposure to the magnetic field roughly tripled the
Young's modulus of the newly synthesized cell wall without any lag.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16304695 [PubMed /​ indexed for MEDLINE]

37: Plant J. 2005 Nov;44(4):581/​94.

Localization of tabersonine 16/​hydroxylase and 16/​OH
tabersonine/​16/​O/​methyltransferase to leaf epidermal cells defines them as a
major site of precursor biosynthesis in the vindoline pathway in Catharanthus
roseus.

Murata J, Luca VD.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St
Catharines, Ontario, L2S3A1 Canada.

The Madagascar periwinkle (Catharanthus roseus) produces the well known and
remarkably complex anticancer dimeric alkaloids vinblastine and vincristine,
which are derived by the coupling of vindoline and catharanthine monomers.
Recent data from in situ RNA hybridization and immunolocalization suggest that
combinatorial cell factories within the leaf are involved in vindoline
biosynthesis. In this study, the cell types responsible for vindoline
biosynthesis were identified by laser/​capture microdissection/RNA
isolation/RT/​PCR to show that geraniol hydroxylase, secologanin synthase,
tryptophan decarboxylase, strictosidine synthase, strictosidine ss/​glucosidase
and tabersonine 16/​hydroxylase can be detected preferentially in epidermal
cells. A new and complementary application of the carborundum abrasion (CA)
technique was developed to obtain epidermis/​enriched leaf extracts that can be
used to measure alkaloid metabolite levels, enzyme activities and gene
expression. The CA technique showed that tabersonine and 16/​methoxytabersonine,
together with 16/​hydroxytabersonine/​16/​O/​methyltransferase, are found
predominantly in Catharanthus leaf epidermis, in contrast to vindoline,
catharanthine and later enzymatic steps in vindoline biosynthesis. The results
show that leaf epidermal cells are biosynthetically competent to produce
tryptamine and secologanin precursors that are converted via many enzymatic
transformations to make 16/​methoxytabersonine. This alkaloid or its 2,3
dihydro/​derivative is then transported to cells (mesophyll/idioblast/laticifer)
within Catharanthus leaves to complete the last three or four enzymatic
transformations to make vindoline.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16262708 [PubMed /​ indexed for MEDLINE]

38: Biotechnol Bioeng. 2006 Feb 5;93(2):386/​90.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced
by Agrobacterium tumefaciens harboring rol ABC genes.

Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI.

Department of Biochemistry and Cell Biology, MS/​140, 6100 Main St., Rice
University, Houston, Texas 77005, USA.

We have established Catharanthus roseus hairy root cultures transgenic for the
rol ABC genes from T(L)/​DNA of the agropine/​type Agrobacterium rhizogenes strain
A4. The rol ABC hairy root lines exhibit a wild/​type hairy root syndrome in
terms of growth and morphology on solid medium. However, they differ from
wild/​type hairy root lines in that they more frequently have excellent
adaptability to liquid medium and do not appear to form calli during
cultivation. Moreover, they do not produce detectable levels of mannopine and
agropine which, in contrast, are often synthesized abundantly in wild/​type hairy
root lines. The absence of these opines does not appear to cause the rol ABC
lines to have higher levels of terpenoid indole alkaloids than wild/​type hairy
root lines. Unlike wild/​type lines, rol ABC lines produce very similar levels of
total alkaloids despite wide variations in individual alkaloid contents. This
work demonstrates that the three genes rol ABC are sufficient to induce
high/​quality hairy roots in Catharanthus roseus. (c) 2005 Wiley Periodicals,
Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16261632 [PubMed /​ indexed for MEDLINE]

39: Biotechnol Bioeng. 2006 Feb 20;93(3):534/​40.

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots
over/​expressing the alpha or the alpha and beta subunits of anthranilate
synthase.

Peebles CA, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

Among the pharmacologically important terpenoid indole alkaloids produced by
Catharanthus roseus are the anti/​cancer drugs vinblastine and vincristine. These
two drugs are produced in small yields within the plant, which makes them
expensive to produce commercially. Metabolic engineering has focused on
increasing flux through this pathway by various means such as elicitation,
precursor feeding, and introduction of genes encoding specific metabolic enzymes
into the plant. Recently in our lab, a feedback/​resistant anthranilate synthase
alpha subunit was over/​expressed in C. roseus hairy roots under the control of a
glucocorticoid inducible promoter system. Upon induction we observed a large
increase in the indole precursors, tryptophan, and tryptamine. The current work
explores the effects of over/​expressing the anthranilate synthase alpha or alpha
and beta subunits in combination with feeding with the terpenoid precursors
1/​deoxy/​D/​xylulose, loganin, and secologanin. In feeding 1/​deoxy/​D/​xylulose to
the hairy root line expressing the anthranilate synthase alpha subunit, we
observed an increase of 125% in horhammericine levels in the induced samples,
while loganin feeding increased catharanthine by 45% in the induced samples.
Loganin feeding to the hairy root line expressing anthranilate synthase alpha
and beta subunits increases catharanthine by 26%, ajmalicine by 84%,
lochnericine by 119%, and tabersonine by 225% in the induced samples. These
results suggest that the terpenoid precursors to the terpenoid indole alkaloids
are important factors in terpenoid indole alkaloid production. Copyright 2005
Wiley Periodicals, Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16240438 [PubMed /​ indexed for MEDLINE]

40: J Biosci Bioeng. 2005 Mar;99(3):208/​15.

Development of a novel system for producing ajmalicine and serpentine using
direct culture of leaves in Catharanthus roseus intact plant.

Iwase A, Aoyagi H, Ohme/​Takagi M, Tanaka H.

Life Science and Bioengineering, Graduate School of Life and Environmental
Sciences, University of Tsukuba, Tsukuba, Ibaraki 305/​8572, Japan.

Due to problems of production instability, the production of plant secondary
metabolites using dedifferentiated cells (callus) is not always feasible on an
industrial scale. To propose a new methodology, which does not use
dedifferentiated cells, a novel system for producing useful secondary
metabolites using the direct culture of intact plant leaves was developed.
Catharanthus roseus was used as a model medicinal plant to produce terpenoid
indole alkaloids (TIAs) by suspension culture of the leaves in the
phytohormone/​free MS liquid medium. Adjustment of the osmotic pressure (993 kPa
at 25 degrees C) in the medium, light irradiation (60 micromol m(/​2) s(/​1)) and
addition of glucose (10 g/l) were effective to promote the production of TIAs
such as ajmalicine (Aj) and serpentine (Sp). On the basis of semi/​quantitative
RT/​PCR analyses, it was revealed that the culture conditions promoted gene
expression of enzymes in the TIA pathway in the cultured leaves. By feeding
glucose (10 g/l) on day 10 of the culture period, Aj was produced at a
concentration of about 18 mg/l and Sp was produced at a concentration about
11/​fold that of the control. These results represent the first step in the
development of a novel production system for plant secondary metabolites.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16233779 [PubMed /​ indexed for MEDLINE]

41: J Biosci Bioeng. 2004;98(2):67/​70.

A novel hydroxylase from Catharanthus roseus participating in the hydroxylation
of 2/​hydroxybenzoic acid.

Shimoda K, Kubota N, Sano T, Hirakawa H, Hirata T.

Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita
University, 1/​1 Hasama/​machi, Oita 879/​5593, Japan. shimoda@med.oita/​u.ac.jp

A novel 55/​kDa hydroxylase was isolated from cultured cells of Catharanthus
roseus by a three/​step procedure: anion exchange chromatography, affinity
chromatography and hydroxylapatite adsorption chromatography. The enzyme
specifically catalyzed the hydroxylation of 2/​hydroxybenzoic acid to give
2,5/​dihydroxybenzoic acid. The enzyme activity was optimal at pH 7.8 and was
completely inhibited by divalent cations, such as Cu(2+) and Hg(2+). The enzyme
showed sequence similarity to certain plant flavonoid 3'/​hydroxylases.

PMID: 16233668 [PubMed]

42: J Biosci Bioeng. 2002;94(2):154/​9.

Production of 5'/​phosphodiesterase by Catharanthus roseus cells promoted by
heat/​degraded products generated from uronic acid.

Akimoto/​Tomiyama C, Aoyagi H, Ozawa T, Tanaka H.

Institute of Applied Biochemistry, University of Tsukuba, Tsuhtba, Ibaraki
305/​8572, Japan.

Polyalginate was autoclaved at 121 degrees C for 20 min and its molecular weight
distribution was analyzed. The autoclaved alginate yielded alginate polymer,
oligomer and heat degraded products (HDPs). Each of the separated substances
promoted 5'/​phosphodiesterase (5'/​PDase) production in suspension culture of
Catharanthus roseus cells. HDPs could also be generated from other uronic acids
(galacturonic acid and glucuronic acid) by autoclave treatment. The most
effective substance in the HDPs was isolated and characterized as
trans/​4,5/​dihydroxy/​2/​cyclopenten/​1/​one (DHCP). The optimal conditions for DHCP
production were also established (autoclaving 1 mg/ml monogalacturonic acid [pH
2] at 121 degrees C for 2 h). A combination of oligo/​alginate (below 4 kDa) and
HDPs significantly promoted the production of 5'/​PDase in C. roseus. Based on
the above results, a novel alginate complex that gave a 44/​fold increase in
5'/​PDase production by C. roseus was developed.

PMID: 16233285 [PubMed]

43: J Biosci Bioeng. 1999;87(6):762/​8.

Development of an apparatus for monitoring protoplast isolation from plant
tissues based on both dielectric and optical methods.

Aoyagi H, Takayanagi T, Jitsufuchi T, Tanaka H.

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki
305/​8572, Japan.

In order to develop a method allowing objective determination of the optimal
conditions for the isolation of protoplasts, the process of protoplast isolation
from plant tissues was quantitatively evaluated. First, a specialized
spectrophotometer cuvette (working volume = 2.0 ml) was designed for the
continuous monitoring of protoplast isolation from plant tissues based on the
optical method. Homogeneous mixing of tissue sections and the protoplast
suspension in the cuvette was accomplished by means of a magnetic bar. The
cuvette was divided into upper and lower parts by a nylon mesh. Since tissue
sections in the upper part could not pass through the mesh, they did not affect
the optical path in the lower part, and only isolated protoplasts were able to
move freely between the two parts. At the optimal agitation speed (200 rpm),
mechanical damage to protoplasts of Catharanthus roseus did not occur. Increases
in the protoplast concentration during their isolation from tissue sections
(leaf and petal) could be continuously monitored by measuring the optical
density (O.D.), making it possible to estimate the end of protoplast isolation.
Degassing treatment of the tissues markedly enhanced protoplast isolation. In
order to monitor the viable protoplast concentration, a larger specialized
spectrophotometer cuvette (working volume = 25 ml) was developed which enabled
simultaneous measurement of the permittivity and O.D. of the suspension to be
carried out during protoplast isolation. Permittivity is a measure of the viable
protoplast concentration, while the O.D. shows protoplast characteristics such
as color. Using this large cuvette, the time courses of protoplast isolation
from leaf and petal sections were monitored and large amounts of viable
protoplasts were obtained. The protoplast isolation process after degassing
treatment was described by a simple first/​order reaction model and the viable
protoplast isolation rate was quantitatively evaluated from the rate constant
(k) on the basis of permittivity changes.

PMID: 16232551 [PubMed]

44: Phytochem Anal. 2005 Sep/​Oct;16(5):328/​33.

Rapid identification of vinca alkaloids by direct/​injection electrospray
ionisation tandem mass spectrometry and confirmation by high/​performance liquid
chromatography/​mass spectrometry.

Zhou H, Tai Y, Sun C, Pan Y.

Department of Chemistry, Zhejiang University, Hangzhou, People's Republic of
China.

A simple and rapid method for the identification of Vinca alkaloids from a crude
extract of Catharanthus roseus G. Don (Apocynaceae) by direct/​injection
electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been
developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine
were evaluated in a commercial extract of C. roseus using this method.
Catharanthine and its isomers 19S/​vindolinine and vindolinine were detected in
the commercial product by direct injection ESI/MS/MS and confirmed by
preparation and by HPLC/​ESI/MS. For the characterisation of different fragment
fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which
to identify some constituents in complex and mixed plant extracts.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16223089 [PubMed /​ indexed for MEDLINE]

45: J Chromatogr Sci. 2005 Oct;43(9):450/​3.

Simultaneous determination of vincristine, vinblastine, catharanthine, and
vindoline in leaves of catharanthus roseus by high/​performance liquid
chromatography.

Gupta MM, Singh DV, Tripathi AK, Pandey R, Verma RK, Singh S, Shasany AK,
Khanuja SP.

Analytical Testing Laboratory, Central Institute of Medicinal and Aromatic
Plants, Lucknow 226 015, India. guptammg@rediffmail.com

A simple reversed/​phase liquid chromatographic method is developed for the
simultaneous quantitation of the anticancerous drugs vincristine, vinblastine,
and their precursors catharanthine and vindoline using a Merck Chromolith
Performance reversed/​phase high/​performance liquid chromatography column. A
better resolution is obtained in comparison with available particulate/​type C18
columns. The column provides good reproducibility and peak symmetry.
Chromatography is carried isocratically with a mobile phase of acetonitrile/​0.1M
phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a
flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as
linearity, limits of quantitation (LOQ) and detection (LOD), precision,
accuracy, recovery, and robustness are studied. The method is selective and
linear for alkaloid concentration in the range 0.25 microg/​25 microg/mL. The LOQ
and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL,
respectively. The results of accuracy studies are good. Values for coefficient
of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery
of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak
purity and homogeneity of these compounds in plant extract is studied using a
photodiode/​array detector. This simple and rapid method of analysis is applied
for the determination of these alkaloids in a large number of leaf extracts of
Catharanthus roseus..

PMID: 16212789 [PubMed /​ indexed for MEDLINE]

46: Biotechnol Prog. 2005 Sep/​Oct;21(5):1572/​6.

Transient effects of overexpressing anthranilate synthase alpha and beta
subunits in Catharanthus roseus hairy roots.

Peebles CA, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

Catharanthus roseus produces two economically valuable anticancer drugs,
vinblastine and vincristine. These drugs are members of the terpenoid indole
alkaloids and accumulate in small quantities within the plant; thus these two
drugs are expensive to produce. Metabolic engineering efforts have focused on
increasing the alkaloids in this pathway through various means such as
elicitation, precursor feeding, and gene overexpression. Recently we
successfully expressed Arabidopsis genes encoding a feedback/​insensitive
anthranilate synthase alpha subunit under the control of the
glucocorticoid/​inducible promoter system and the anthranilate synthase beta
subunit under the control of a constitutive promoter in C. roseus hairy roots.
In this work we look at the transient behaviors of terpenoid indole alkaloids
over a 72 h induction period in late exponential growth phase cultures. Upon
induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72
h. In contrast, the lochnericine, horhammericine, and tabersonine pools
decreased and leveled out over the 72 h induction period. Visible changes within
the individual compounds usually took from 4 to 12 h.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16209565 [PubMed /​ indexed for MEDLINE]

47: J Biotechnol. 2006 Mar 9;122(1):28/​38. Epub 2005 Sep 26.

Expression of the Arabidopsis feedback/​insensitive anthranilate synthase
holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy
roots.

Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI.

Department of Biochemistry and Cell Biology, MS/​140, Rice University, Houston,
TX 77005, USA.

In plants, the indole pathway provides precursors for a variety of secondary
metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan,
tryptamine, is a building block for the biosynthesis of terpenoid indole
alkaloids. Previously, we manipulated the indole pathway by introducing an
Arabidopsis feedback/​insensitive anthranilate synthase (AS) alpha subunit (trp5)
cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a
glucocorticoid/​inducible promoter into C. roseus hairy roots [Hughes, E.H.,
Hong, S./​B., Gibson, S.I., Shanks, J.V., San, K./​Y. 2004a. Expression of a
feedback/​resistant anthranilate synthase in Catharanthus roseus hairy roots
provides evidence for tight regulation of terpenoid indole alkaloid levels.
Biotechnol. Bioeng. 86, 718/​727; Hughes, E.H., Hong, S./​B., Gibson, S.I.,
Shanks, J.V., San, K./​Y. 2004b. Metabolic engineering of the indole pathway in
Catharanthus roseus hairy roots and increased accumulation of tryptamine and
serpentine. Metabol. Eng. 6, 268/​276]. Inducible expression of either or both
transgenes did not lead to significant increases in overall alkaloid levels
despite the considerable accumulation of tryptophan and tryptamine. In an
attempt to more successfully engineer the indole pathway, a wild type
Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with
the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic
hairy roots expressing both trp5 and ASB1 show a significantly greater
resistance to feedback inhibition of AS activity by tryptophan than plants
expressing only trp5. In fact, a 4.5/​fold higher concentration of tryptophan is
required to achieve 50% inhibition of AS activity in plants overexpressing both
genes than in plants expressing only trp5. In addition, upon a 3 day induction
during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times
more tryptophan (specific yield ca. 3.0 mg g(/​1) dry weight) than the trp5 hairy
root line. Concurrently, tryptamine levels increase up to 9/​fold in the induced
trp5:ASB1 line (specific yield ca. 1.9 mg g(/​1) dry weight) as compared with
only a 4/​fold tryptamine increase in the induced trp5 line (specific yield ca.
0.3 mg g(/​1) dry weight). However, endogenous TDC activities of both trp5:ASB1
and trp5 lines remain unchanged irrespective of induction. When TDC is
ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC
hairy root line accumulates tryptamine up to 14/​fold higher than the uninduced
line. In parallel with the remarkable accumulation of tryptamine upon induction,
alkaloid accumulation levels were significantly changed depending on the
duration and dosage of induction.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16188339 [PubMed /​ indexed for MEDLINE]

48: Plant Cell Rep. 2005 Dec;24(11):677/​82. Epub 2005 Aug 11.

Ajmalicine production in methyl jasmonate/​induced Catharanthus roseus cell
cultures depends on Ca2+ level.

Lee/​Parsons CW, Erturk S.

Chemical Engineering Department, 342 Snell Engineering Center, 360 Huntington
Avenue, Northeastern University, Boston, MA 02115/​5000, USA. clee@coe.neu.edu

Cytosolic Ca(2+) and jasmonate mediate signals that induce defense responses in
plants. In this study, the interaction between Ca(2+) and methyl jasmonate (MJ)
in modulating defense responses was investigated by monitoring ajmalicine
production in Catharanthus roseus suspension cultures. C. roseus suspensions
were treated with nine combinations of CaCl(2) (3, 23, and 43 mM) and MJ (0, 10,
and 100 microM) on day 6 of growth. Increased Ca(2+) influx through the addition
of extracellular CaCl(2) suppressed ajmalicine production in MJ/​induced
cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells
were treated with a low level of calcium (3 mM) combined with a high level of MJ
(100 microM). In the presence of 3 mM CaCl(2) in the medium, the addition of
Ca(2+) chelator EGTA (1, 2.5, and 5 mM) or Ca(2+) channel blocker verapamil (1,
10, and 50 muM) to MJ/​induced (100 microM) cultures on day 6 also inhibited
ajmalicine production at higher levels of the Ca(2+) inhibitors. Hence,
ajmalicine production in MJ/​induced C. roseus cultures depended on the
intracellular Ca(2+) concentration and a low extracellular Ca(2+) concentration
(3 mM) enhanced MJ/​induced ajmalicine production.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16094527 [PubMed /​ indexed for MEDLINE]

49: Biotechnol Lett. 2005 Jun;27(11):793/​7.

Biotransformation of alpha/​santonin by cell suspension cultures of five plants.

Yang L, Dai J, Sakai J, Ando M.

College of Life and Environmental Sciences, The Central University for
Nationalities, 100081, Beijing, P.R. China.

Cell suspension cultures of five plants (Catharanthus roseus, Ginkgo biloba,
Platycodon grandiflorum, Taxus cuspidata, Phytolacca asinosa) were employed to
bioconvert the eudesmanolide compound, alpha/​santonin. Reactions occurring were
hydroxylation (C/​1, C/​11 and C/​15), reduction of the double bond [1(2) or 3(4)],
rearrangment of the eudesmanolide skeleton to a guaianolide skeleton and
lactone/​ring hydrolysis. Four new compounds were identified.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 16086262 [PubMed /​ indexed for MEDLINE]

50: Zhongguo Zhong Yao Za Zhi. 2005 May;30(10):741/​3, 755.

[A study on the hairy root culture and antitumor alkaloids production of
Catharanthus roseus]

[Article in Chinese]

Sun M, Zeng JJ.

School of Life Science, Southwest Normal University, Chongqing 400715, China.

OBJECTIVE: To establish transformation system and obtain alkaloids from the
hairy root of Catharanthus roseus. METHOD: Hairy roots were obtained by
infecting the different explants of C. roseus. Culture conditions of hairy root
were optimized. RESULT: The best transformation condition was leaf infected by
two/​day's pre/​culture and two/​day's co/​culture and additional A(S)
(hydroxyacetosyringone) 100 mg x L(/​1). The inducing rate of hairy root was up
to 86.25%. The best condition of hairy root culture was MS medium with sucrose
as carbon material and lactalbumin as nitron material. The analysis result
showed that the contents of total alkaloids in hairy roots were higher than
explants and calli. CONCLUSION: Hairy root of C. roseus will be useful for the
production of active components in C. roseus.

Publication Types:
English Abstract
Research Support, Non/​U.S. Gov't

PMID: 16075710 [PubMed /​ indexed for MEDLINE]

51: Phytochemistry. 2005 Aug;66(15):1797/​803.

Fosmidomycin analogues as inhibitors of monoterpenoid indole alkaloid production
in Catharanthus roseus cells.

Mincheva Z, Courtois M, Andreu F, Rideau M, Viaud/​Massuard MC.

EA 3857, Laboratoire de Synthese et Physicochimie Organique et Therapeutique
(SPOT), 31 Avenue Monge, 37200 Tours, France.

Substituted 3/​[2/​(diethoxyphosphoryl)propyl]oxazolo[4,5/​b]pyridine/​2(3H)/​ones
were obtained by functionalization at 6/​position with various substituents
(aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found
that these new fosmidomycin analogues inhibited the accumulation of ajmalicine,
a marker of monoterpenoid indole alkaloids production in plant cells. Some of
them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid
accumulation at the concentration of 100 microM.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16054176 [PubMed /​ indexed for MEDLINE]

52: BMC Bioinformatics. 2005 Jul 18;6:179.

Processing methods for differential analysis of LC/MS profile data.

Katajamaa M, Oresic M.

Turku Centre for Biotechnology, Tykistokatu 6, FIN/​20521, Turku, Finland.
mikko.katajamaa@btk.fi

BACKGROUND: Liquid chromatography coupled to mass spectrometry (LC/MS) has been
widely used in proteomics and metabolomics research. In this context, the
technology has been increasingly used for differential profiling, i.e. broad
screening of biomolecular components across multiple samples in order to
elucidate the observed phenotypes and discover biomarkers. One of the major
challenges in this domain remains development of better solutions for processing
of LC/MS data. RESULTS: We present a software package MZmine that enables
differential LC/MS analysis of metabolomics data. This software is a toolbox
containing methods for all data processing stages preceding differential
analysis: spectral filtering, peak detection, alignment and normalization.
Specifically, we developed and implemented a new recursive peak search algorithm
and a secondary peak picking method for improving already aligned results, as
well as a normalization tool that uses multiple internal standards.
Visualization tools enable comparative viewing of data across multiple samples.
Peak lists can be exported into other data analysis programs. The toolbox has
already been utilized in a wide range of applications. We demonstrate its
utility on an example of metabolic profiling of Catharanthus roseus cell
cultures. CONCLUSION: The software is freely available under the GNU General
Public License and it can be obtained from the project web page at:
http://mzmine.sourceforge.net/.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16026613 [PubMed /​ indexed for MEDLINE]

53: Prikl Biokhim Mikrobiol. 2005 May/​Jun;41(3):340/​6.

[Role of elements and physiologically active compounds in the regulation of
synthesis and accumulation of indole alkaloids in Catharanthus roseus L]

[Article in Russian]

Lovkova MIa, Buzuk GN, Sokolova SM, Buzuk LN.

Effects of various elements (Co, Ni, Zn, W, Mn, Cr, B, Mo, Fe, and V), natural
and synthetic auxins, cytokinins, and gibberellin on biosynthesis and
accumulation of indole alkaloids was studied at increasing concentrations in the
model system of Madagascar periwinkle seedlings (Catharanthus roseus L.). The
main types of concentration dependences for the effect of physiologically active
compounds under study were evaluated. A possible mechanism of the influence of
Zn and auxin on this process was partly clarified. The compounds were shown to
modulate various stages in the biosynthesis of monomeric indole alkaloids
(catharanthine and vindoline).

Publication Types:
English Abstract

PMID: 15977796 [PubMed /​ indexed for MEDLINE]

54: Planta Med. 2005 Jun;71(6):572/​4.

Cytokinin and ethylene control indole alkaloid production at the level of the
MEP/terpenoid pathway in Catharanthus roseus suspension cells.

Papon N, Bremer J, Vansiri A, Andreu F, Rideau M, Creche J.

Biomolecules et Biotechnologies Vegetales EA 2106, Tours, France.

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid
indole alkaloids, some of which have high therapeutic interest. The
biotechnological approach with cells in vitro remains an alternative to the
field culture of periwinkle for the production of such compounds. We previously
reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the
accumulation of alkaloids in periwinkle cell suspensions. In this work, we
investigated the effects of these hormones on the regulation of several genes of
the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene
greatly enhanced the expression of the geraniol 10/​hydroxylase gene. When given
together, these hormones also increased the expression of three genes belonging
to the methyl/​erythritol pathway. These results make it possible to consider
elements of cytokinin and ethylene signalling pathways as tools for improving
terpenoid indole alkaloid production through metabolic engineering.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15971133 [PubMed /​ indexed for MEDLINE]

55: Plant Physiol. 2005 Jul;138(3):1607/​14. Epub 2005 Jun 17.

Phytic acid synthesis and vacuolar accumulation in suspension/​cultured cells of
Catharanthus roseus induced by high concentration of inorganic phosphate and
cations.

Mitsuhashi N, Ohnishi M, Sekiguchi Y, Kwon YU, Chang YT, Chung SK, Inoue Y, Reid
RJ, Yagisawa H, Mimura T.

Japan Society for the Promotion of Science, Tokyo 102/​8471, Japan.

We have established a new system for studying phytic acid, myo/​inositol
hexakisphosphate (InsP(6)) synthesis in suspension/​cultured cells of
Catharanthus. InsP(6) and other intermediates of myo/​inositol (Ins) phosphate
metabolism were measured using an ion chromatography method. The detection limit
for InsP(6) was less than 50 nM, which was sufficient to analyze Ins phosphates
in living cells. Synthesis of Ins phosphates was induced by incubation in high
inorganic phosphate medium. InsP(6) was mainly accumulated in vacuoles and was
enhanced when cells were grown in high concentration of inorganic phosphates
with the cations K(+), Ca(2+), or Zn(2+). However, there was a strong tendency
for InsP(6) to accumulate in the vacuole in the presence of Ca(2+) and in
nonvacuolar compartments when supplied with Zn(2+), possibly due to
precipitation of InsP(6) with Zn(2+) in the cytosol. A vesicle transport
inhibitor, brefeldin A, stimulated InsP(6) accumulation. The amounts of both
Ins(3)P(1) myo/​inositol monophosphate synthase, a key enzyme for InsP(6)
synthesis, and Ins(1,4,5)P(3) kinase were unrelated to the level of accumulation
of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles
in plant cells are discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15965017 [PubMed /​ indexed for MEDLINE]

56: Plant Mol Biol. 2005 Apr;57(6):855/​70.

CaaX/​prenyltransferases are essential for expression of genes involvedin the
early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells.

Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St/​Pierre
B, Giglioli/​Guivarc'h N.

Biomolecules et Biotechnologies Vegetales, Labaratoire de Physiologie Vegetale,
UFR Science et Techniques, Universite Francois/​Rabelais de Tours, EA2106, 37200
, Parc de Grandmont, Tours, France.

CaaX/​prenyltransferases (CaaX/​PTases) catalyse the covalent attachment of
isoprenyl groups to conserved cysteine residues located at the C/​terminal CaaX
motif of a protein substrate. This post/​translational modification is required
for the function and/or subcellular localization of some transcription factors
and components of signal trans