1. Effect of Methyl Jasmonate on β-Thujaplicin Production in
Cupressus Tissue Cultures
Devika Dutt, Jetan Schlegel, Danielle Eakle, Thomas Savage
Department of Chemistry, Sacramento State University
Abstract
The goal of this experiment is to establish
whether ß-thujaplicin production in Cupressus
tissue cultures can be enhanced using methyl
jasmonate. Improving the production of this
antifungal compound requires an understanding of
the biosynthetic pathway, which can be elucidated
in experiments with Cupressus tissue cultures
producing significant amounts of β-thujaplicin.
Cultures were transferred onto fresh media in the
presence and absence of methyl jasmonate. Gas
chromatography-mass spectrometry was used to
analyze ß-thujaplicin accumulation in the cultured
cells. Methyl jasmonate did not increase the levels
of β-thujaplicin that accumulated in the cultures.
This suggests that methyl jasmonate does not
enhance the ability of callus cultures to be used as
an experimental system for elucidating the
biosynthetic pathway to β-thujaplicin.
Results
Cupressus dupreziana callus cultures produced β-thujaplicin as detected by gas
chromatography-mass spectrometry (Figure 3). However, the levels of β-thujaplicin
produced did not increase when the cultures were treated with methyl jasmonate
(Figure 4).
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Introduction
β-thujaplicin, (Figure 1) a monoterpene (ten-
carbon molecule) made by cedar trees has very
strong antifungal properties and a very broad
antimicrobial spectrum. These antimicrobial
properties have lead to its usefulness in cosmestics,
clinical products and many other areas.
Furthermore, β-thujaplicin synthesis may be
genetically engineered in plants to improve
resistance to fungal diseases.
β-thujaplicin is primarily obtained from the
heartwood of some species of Cupressaceae trees.
Unfortunately, productivity is extremely low. Thus,
improving β-thujaplicin production is vital.
Improving the production of β-thujaplicin
requires an understanding of the biosynthetic
pathway, which is currently unknown. Experiments
to elucidate the biosynthetic pathway require easy
manipulation of cell culture systems that produce
significant amounts of β-thujaplicin. The primary
goal is to establish whether β-thujaplicin production
in Cupressus tissue cultures can be enhanced with
methyl jasmonate as reported for other Cupressus
species.1, 2
Materials and Methods
The method involves taking cultures (Figure 2) and transferring the cultures onto fresh media and observing the
time course of production of β-thujaplicin in the presence and absence of methyl jasmonate. 0.5 mL of 200 µM
methyl jasmonate in buffer was injected into each Cupressus dupreziana callus on three different plates. Three
control plates were treated with with 0.5 mL buffer without methyl jasmonate. After one week, cultures were
extracted with ethyl acetate and the β-thujaplicin was derivatized to the trimethyl silyl derivative. Gas
chromatography-mass spectrometry was used to confirm β-thujaplicn production in Cupressus tissue cultures.
Gas chromatography-flame ionization detection was used to quantify the amount of β-thujaplicin produced.
References
1. Sakai, K., et al. Improved β-thujaplicin Production in Cupressus
lusitanica Suspension Cultures by Fungal Elicitor and Methyl Jasmonate.
Applications of Microbiology Biotechnology. (2001) 55: 301-305.
2. Aizu, Takayuki., Arima, Yaeno., Hanada, Katsumi., Ikenaga, Satsuki.,
Nakano, Hajime., Kaneko, Takahide., Yasushi, Tsuchida., Human
Metallothionein. Gene Expression Is Upregulated by β-thujaplicin:
Possible Involvement of Protein Kinase C and Reactive Oxygen Species.
Biological & Pharmaceutical Bulletin. (2006) Vol. 29 No. 155
Acknowledgements
We would like to give special thanks to Dr. McCarthy, Dr. Barrena,
Anna Pasqua, Pamela King, the Science Educational Equity Program for
making this research opportunity possible.
We would also like to thank the National Institute of Health for
providing the funds necessary to fund the research project under grant
number GM056645.
Conclusion
Methyl jasmonate did not increase levels of β-thujaplicin
production.The experiment does not suggest that methyl jasmonate
plays a role in β-thujaplicin production. However, a higher concentration
of the methyl jasmonate may enhance β-thujaplicin production.
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Figure 2: Callus Culture Growth of Cupressus dupreziana
Figure 1: Biosynthetic Pathway to β-thujaplicins.
Figure 4: Callus cultures treated with methyl jasmonate did not increase β-thujaplicin
production as expected.
Figure 3: Cupressus dupreziana callus cultures treated with methyl jasmonate
made β-thujaplicin. Upper panel is the total ion chromatography of the derivatized
volatiles extracted from Cupressus dupreziana callus cultures. The arrow
indicates the β-thujaplicin peak. The lower panel is the mass spectrum of the β-
thujaplicin trimethyl silyl derivative.
OPP
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