Biotechnology and Molecular Biology Reviews Vol. 3 (1), pp. 001-007, February 2008
`Available online at http://www.academicjournals.org/BMBR
`ISSN 1538-2273 © 2008 Academic Journals
`
`Standard Review
`Plant terpenoids: applications and future potentials
`Sam Zwenger and Chhandak Basu*
`
`University of Northern Colorado, School of Biological Sciences, Greeley, Colorado, 80639, USA.
`
`Accepted 7 February, 2008
`
`The importance of terpenes in both nature and human application is difficult to overstate. Basic
`knowledge of terpene and isoprene biosynthesis and chemistry has accelerated the pace at which
`scientists have come to understand many plant biochemical and metabolic processes. The abundance
`and diversity of terpene compounds in nature can have ecosystem-wide influences. Although terpenes
`have permeated human civilization since the Egyptians, terpene synthesis pathways are only now being
`understood in great detail. The use of bioinformatics and molecular databases has largely contributed
`to analyzing exactly how and when terpenes are synthesized. Additionally, terpene synthesis is
`beginning to be understood in respect to the various stages of plant development. Much of this
`knowledge has been contributed by the plant model, Arabidopsis thaliana. Considering the advances in
`plant terpene knowledge and potential uses, it is conceivable that they may soon be used in
`agrobiotechnology.
`
`Key words: Terpenes, terpene synthase, secondary metabolites, transgenic plants
`
`TABLE OF CONTENT
`
`1. Introduction
`2. Terpene chemistry and biosynthesis
`3. Terpenes in nature
`4. Society and terpenes
`5. Transgenic plants and future research
`6. Conclusions
`
`INTRODUCTION
`
`Plants produce primary and secondary metabolites which
`encompass a wide array of functions (Croteau et al.,
`2000). Primary metabolites, which include amino acids,
`simple sugars, nucleic acids, and lipids, are compounds
`that are necessary for cellular processes. Secondary
`metabolites include compounds produced in response to
`stress, such as the case when acting as a deterrent
`against herbivores (Keeling, 2006). Plants can manu-
`facture many different types of secondary metabolites,
`which have been subsequently exploited by humans for
`their beneficial role in a diverse array of applications
`(Balandrin et al., 1985). Often, plant secondary metabo-
`lites may be referred to as plant natural products, in
`which case they illicit effects on other organisms. Although
`this review focuses on plant terpenes, it should be realized
`
`* Corresponding author. E-mail: chhandak.basu@unco.edu.
`
`that other organisms are able to synthesize terpenes. For
`example, the endophytic fungus isolated from St. John's
`Wort (Hypericum perforatum) was recently shown to
`produce hypericin and emodin, two types of terpene lac-
`tones (Kusari et al., 2008). There are three broad cate-
`gories of plant secondary metabolites as natural pro-
`ducts; terpenes and terpenoids (~25,000 types), alkaloids
`(~12,000 types), and phenolic compounds (~8,000 types)
`(Croteau et al., 2000).
`
`Terpene chemistry and biosynthesis
`
`Ever since techniques such as low-temperature chroma-
`tography, were used to separate plant terpenes nearly a
`half of a century ago (Clements, 1958), great strides have
`been made to explore molecular details of terpenes. For
`instance, subjecting plant vegetation to pyrolysis tech-
`niques and gas chromatography has allowed for identifi-
`
`LCY Biotechnology Holding, Inc.
`Ex. 1010
`Page 1 of 7
`
`

`

`002 Biotechnol. Mol. Biol. Rev.
`
`
`
`
`
`
`
`
`
`Terpenes are the most numerous and structurally diver-
`se plant natural products (Figure 1). For this reason, a
`system of nomenclature has been established. The
`nomenclature of terpene compounds is ostensibly com-
`plex, yet can be quickly elucidated upon closer exami-
`nation. The isoprene unit, which can build upon itself in
`various ways, is a five-carbon molecule. The single iso-
`prene unit, therefore, represents the most basic class of
`terpenes, the hemiterpenes. An isoprene unit bonded
`with a second isoprene is the defining characteristic of
`terpene, which is also a monoterpene (C10). While ses-
`quiterpenes contain three isoprene units (C15), diterpenes
`(C20) and triterpenes (C30) contain two and three terpene
`units, respectively. Tetraterpenes consist of four terpene
`units and polyterpenes are those terpenes containing
`more than four terpene units (i.e., more than eight iso-
`prene units).
`terpene
`McGarvey and Croteau (1995) reviewed
`biosynthesis and suggested that a more detailed study of
`the terpene synthases were needed and that this in turn
`would increase the role of terpenes in, perhaps, com-
`mercial uses such as flavor enhancers. Since their public-
`cation more than a decade ago, terpene biosyn-thesis
`enzymes have been studied in detail. For instance,
`Greenhagen et al. (2006) used mapping strategies to
`determine the variance and composition of amino acids
`within terpene synthase active sites. This is, arguably,
`very useful in determining the evolutionary divergence of
`the
`terpene synthases and elucidating relationships
`among plants. Trapp and Croteau (2001) reviewed the
`genomic organization of terpene synthase genes across
`different species. They suggest that terpene synthase
`genes may impact phylogenetic organization of some
`plants. For example, some terpene genes are more clo-
`sely related in certain plant species, in which the species
`themselves were previously thought to be distantly
`related.
`In a more recent review of terpene synthase genes,
`Zwenger and Basu (2007) performed in silico analysis of
`publicly available microarray data using Genevesitgator
`software (Zimmerman et al., 2004). Such software allows
`for assaying an organism of choice, in this case, Arabi-
`dopsis thaliana. In their study, more than 2,500 micro-
`arrays were simultaneously compared for expression of
`terpene synthase genes. Multiple biotic and abiotic fac-
`tors, which may or may not induce expression, were also
`considered in respect to terpene synthase gene expres-
`sion. Possibly even more important is the fact that exp-
`ression of terpene synthases were examined across the
`life cycle of Arabidopsis, which countered some wet lab
`experimental data previously published. In addition to
`expanding the comprehension of terpene synthase genes
`across the life cycle of Arabidopsis, the authors deter-
`mined that five terpene synthase genes, which appeared
`in many microarray analyses, were lacking in experi-
`mental studies. As discussed by the authors, further exp-
`eriments may lead to better understanding the roles of
`
`
`
`
`Figure 1. Pie chart representing the major groups of
`plant secon-dary metabolites according to Croteau et al.
`(2000). Based on their numbers and diversity, terpenes
`offer much potential in an array of industrial and
`medicinal applications.
`
`
`
`
`
`
`cation of different volatile organic compounds (VOCs)
`(Greenberg et al., 2006). Some extraction techniques
`have relied on using ultra pure water, also dubbed sub-
`critical water. Although this method works relatively well,
`it has been pointed out that increasing the temperature of
`the water decreases the stability of the terpenes (Yang et
`al., 2007). Lai et al. (2005) performed a crude extraction
`terpene trilactones from the leaves of Ginko biloba by
`refluxing the leaves in ethanol and then dissolving the
`extracts in water. They subsequently isolated trilactones
`with either column chromatography or a liquid-liquid extr-
`action using ethyl acetate as a solvent.
`Ma et al. (2007) used two-dimensional gas chromato-
`graphy time-of-flight mass spectrometry (GC x GC-TOF
`MS) to analyze volatile oils in the leaves and flowers of
`Artemisia annua. The authors concluded that the number
`of components was close to 700 and the majority was
`terpenes. In a comparative analysis they used the same
`extraction techniques but instead of GC x GC-TOF MS
`they used GC-MS, which resulted in a much lower sen-
`sitivity for molecular diversity. This illustrates the fact that
`newer isolation and identification methods are helping
`with terpene analysis.
`It has long been known that the basic unit of most
`secondary plant metabolites, including terpenes, consists
`of isoprene, a simple hydrocarbon molecule. The term
`terpene usually refers to a hydrocarbon molecule while
`terpenoid refers to a terpene that has been modified,
`such as by the addition of oxygen. Isoprenoids are, there-
`fore, the building blocks of other metabolites such as
`plant hormones, sterols, carotenoids, rubber, the phytol
`tail of chlorophyll, and turpentine.
`
`LCY Biotechnology Holding, Inc.
`Ex. 1010
`Page 2 of 7
`
`

`

`
`
`
`
`previously uncharacterized genes.
`
`
`Terpenes in nature
`
`The distribution of terpenes in nature has been studied
`extensively. Indeed, the distribution of terpenes within
`species has received attention. To better understand
`terpene and other volatile organic compound emissions,
`from loblolly pine (Pinus taeda), Thompson et al. (2006)
`analyzed tree core samples. They found the highest
`concentrations of terpenes in heartwood, lowest in outer
`sapwood, and moderate levels in the inner sapwood. In a
`less invasive study by Martin et al. (2003) methyl
`jasmonate was applied onto foliage of Norway spruce
`(Picea abies) trees which led to a two fold increase of
`terpenes within the needles. In another investigation, the
`amounts of different terpenes in Scots pine (Pinus sylves-
`tris) needles varied across Finnish and Turkish regions,
`showing the diversity of terpene distribution can vary
`within a species (Semiz et al., 2007).
`Although more commonly associated with coniferous
`species, terpenes have been detected in other plant
`phyla, including angiosperms. Aside from terpenes manu-
`factured by plants in response to herbivory or stress
`factors, it has also been shown that flowers can emit
`terpenoids to attract pollinating insects (Maimone and
`Baran 2007). Interestingly, terpenoids have also been
`shown to attract beneficial mites, which feed on the herbi-
`vorous insects (Kappers et al., 2005). Terpene emissions
`and subsequent attracting mechanisms have been shown
`to play an indirect role in plant defense mechanisms in
`other studies as well. Kessler and Baldwin (2001) have
`shown that herbivorous insects can induce terpene relea-
`se from a plant, and also cause the plant to release sig-
`nals which attracts predatory species. These experi-
`ments provide not only powerful evidence for the role of
`terpenes for plant defense, but also give an exemplary
`model
`for co-evolution between plants, mites, and
`insects. Chen et al. (2003) have shown that many differ-
`ent volatiles, including terpenes, may be emitted from
`flowers of Arabidopsis. They propose that the role as
`insect attractants of at least some emitted terpenes
`seems inconclusive, but still strongly suggest they might
`play a role in reproduction.
`Of course, other studies have extended the under-
`standing of plant terpenes and insects. Johnson et al.
`(2007) examined fragrance mixtures including terpenes
`and found scent chemistry of the emitted fragrance play-
`ed a role in beetles and wasps pollinating an orchid spe-
`cies (Satyrium microrrhynchum). After performing GC-MS
`to
`identify
`fragrance compounds,
`they manipulated
`antennae to determine electrophysiological responses.
`Molecules which elicited effects included monoterpenes
`and sesquiterpenes. While the beetles were generalists in
`pollination, the wasps were more specific. However,
`Urzúa et al. (2007) studied terpenoids from an Astera-
`ceae (Haplopappus berterii) and suggested little or no
`
`
`
`Zwenger and Basu 003
`
`
`
`
`correlation between fragrance molecules and insect
`preference.
`Ecological roles of terpenes extend beyond plant-insect
`coevolution. Cheng et al. (2007) discuss ecological imp-
`acts of terpenes. These include their roles above and
`below ground in attracting predatory species upon herbi-
`vory attack. Additionally, they point out terpenes may act
`as chemical messengers which influence the expression
`of genes involved in plant defense mechanisms or even
`influence gene expression of neighboring plants.
`Terpenes have been studied with great interest, due to
`their roles in the earth's atmosphere. It has been estima-
`ted that the annual global emission of isoprenes is 500
`teragrams (Guenther et al., 2006). Therefore, it is tempt-
`ing to speculate on their interactions with solar radiation.
`Due to the abundance of citrus plantations in the mediter-
`ranean area, Thunis and Cuvelier (2000) helped identify
`the
`influence and composition of VOCs on ozone
`formation in this region and found some of the biogenic
`VOCs included (cid:1)-pinene and d-limonene. In a study by
`VanReken et al. (2006) a biogenic emissions chamber
`was used to measure terpenoids released from Holm oak
`(Quercus ilex), loblolly pine and a dilute mixture of (cid:1)-
`pinene. They suggested a large majority of emissions are
`chemically oxidized or otherwise transformed into differ-
`ent aerosol compounds. Llusiá and Penñuelas (2000)
`have examined stomatal conductance to better under-
`stand how plants interact with abiotic atmospheric condi-
`tions such as temperature, water availability, and irra-
`diance to alter the diffusive resistance of terpenes from
`plant leaves. They also describe the seasonal fluctuation
`of terpenes.
`Since many plants contribute to the earth's atmospheric
`composition by releasing volatile organic compounds,
`which include terpenes, they should arguably be studied
`more extensively. Future research may therefore help
`pave the road to understanding the global influence of
`terpenes.
`
`
`Society and terpenes
`
`There have been many applications of terpenes in human
`societies. Pharmaceutical and food industries have exp-
`loited them for their potentials and effectiveness as medi-
`cines and flavor enhancers. Perhaps the most widely
`known terpene is rubber, which has been used exten-
`sively by humans. Rubber is a polyterpene, composed of
`repeating subunits of isoprene. The addition of sulfur to
`rubber by Charles Goodyear led to vulcanized rubber,
`which yields various degrees of pliability depending on
`the mixture ratio (Stiehler and Wakelin, 1947). Other
`important terpenes include camphor, menthol, pyrethrins
`(insecticides), cleaners, antiallergenic agents, and sol-
`vents. Rosin (a diterpene), limonene, carvone, nepeta-
`lactone (in catnip), hecogenin (a detergent), and digitoxi-
`genin are also important terpenes (Croteau et al., 2000).
`Agriculture has also shown an increasing interest in
`
`LCY Biotechnology Holding, Inc.
`Ex. 1010
`Page 3 of 7
`
`

`

`004 Biotechnol. Mol. Biol. Rev.
`
`
`
`terpenes. In a study by Villalba et al. (2006) sheep were
`suggested to have increased tolerance for terpene con-
`sumption if they consumed more grains. They also sho-
`wed terpenes can influence ungulate herbivory on other
`plants. This may help agronomists balance diets of rumi-
`nants if they consume plants such as sagebrush (Arte-
`mesia sp.). Terpenes have also shown antimicrobial
`activities (Islam et al., 2003). This is important due to the
`increase in antibiotic resistant bacteria, which is occurring
`globally and at an alarming rate. Addition of terpenes into
`livestock feed may replace conventional antibiotic addi-
`tion, which in turn would slow the rate of antibiotic
`resistance in bacteria.
`The effect of some terpenes on microorganisms has
`been seriously studied since at least the 1980's (Andrews
`et al., 1980). Plant oils, which contain terpenes, have
`shown increasing promise in vivo, inhibiting multiple spe-
`cies of bacteria. For example, cinnamon oil has shown
`broad-spectrum activity against Pseudomonas aerugino-
`sa (Prabuseenivasan et al., 2006). The various composi-
`tions of terpenes can be markedly different from one
`species to another. For example, John et al. (2007) found
`plant oils from Neolitsea foliosa, which also showed some
`antibacterial properties, included sesquiterpenes such as
`(cid:2)-caryophyllene but lacked monoterpenes.
`Other microbes have also shown inhibition by terpenes.
`Murata et al. (2008) extracted numerous compounds from
`stem bark of the cape ash (Ekebergia capensis) growing
`in Kenya. Ten of these were triterpenes, whose structures
`were determined using spectroscopic analysis such as
`NMR (nuclear magnetic resonance). Determining the
`precise molecular activities of these triterpenes may be
`an important step towards finding newer and more effec-
`tive drugs against Plasmodium falciparum, the causative
`agent of malaria. Susceptibility to terpenes has been
`tested by Morales et al. (2003), in which extracts from
`Artemisia copa showed inhibitory effects against yeast
`(Candida albicans). They also showed that some plant
`extracts containing terpenes tested showed biotoxicity
`effects against brine shrimp (Artemia salina).
`Cumene (isopropylbenzene) is a terpene that has been
`used in bioremediation studies. In an experiment carried
`out by Suttinun et al. (2004), bacteria used in bioreme-
`diation of trichloroethylene (TCE) showed an increased
`capability to uptake TCE in the presence of cumene. In
`their study, 75% of the TCE present was successfully
`metabolized, allowing for a more robust degradation and
`bioremediation. Additional terpenes included in the study
`were limonene, carvone, and pinene. However, cumene
`showed the most beneficial effects. Without the know-
`ledge and application of cumene, such success in bio-
`remediation studies might not have been possible.
`Because terpenes have been incorporated into much
`antibacterial soaps, cosmetics and household products,
`descriptive studies have been published on absorption
`and penetration into skin. Due to their properties of lipid
`organization disruption, Cal et al., (2006) studied the ab-
`
`
`
`
`
`sorption kinetics of four cyclic terpenes; (cid:1)-pinene, (cid:2)-
`pinene, eucalyptol, and terpinen-4-ol. Each terpene var-
`ied in accumulation and elimination time with terpinen-4-
`ol showing the fastest penetration. Matura et al. (2005)
`investigated the role some terpenes play as causative
`agents of contact dermatitis and fragrance allergies. Out
`of approximately 1500 patients tests, just over 1% had
`reactions to oxidized linalool.
`To better understand the vast array of terpenes, gene-
`tically modified organisms have been used. For example,
`the biosynthesis of terpenes has been studied in trans-
`formed E. coli (Adam et al., 2002). As described by Adam
`et al. (2002), modification of organisms is important to
`help understand the various pathways of terpene synthe-
`sis for the purpose of producing antimicrobial and anti-
`parasitic drugs (Goulart et al., 2004).
`
`
`Transgenic plants and future research
`
`Plant tissue culture is an in vitro technique that allows
`clonal propagation of transformed clones. A review of
`tissue culture methods and applications by Vanisree et al.
`(2004) discusses the importance of inserting genes for
`plant secondary metabolites, including taxol (a diterpene
`alkaloid), a well-known anticancer agent. They point out
`that in vitro cell culture methods provides systematic ad-
`vantages such as the ability to manipulate plant environ-
`ment, control of cell growth, and regulation and extraction
`of metabolic products.
`In contrast to producing terpenes in the laboratory,
`others have suggested extending the methods to create
`transgenic crops for terpene sythesis and production.
`Genetic modification of Arabidopsis has been performed
`to study the production of different terpenoids by up-
`regulating terpene synthase genes (Aharoni et al., 2003),
`which has led to an increase in understanding of how
`terpenes might function. For example, it has been shown
`that genetic engineering of Arabidopsis plants has allo-
`wed for an increase in pest resistance (Kappers et al.,
`2005). Others who have shown terpenes to influence ins-
`ect behavior have suggested the use of terpene expres-
`sion as a possible control mechanism for aphid infesta-
`tions (Harmel et al., 2007). A study by Lweinsohn et al.
`(2001) determined that genetically modified tomatoes
`could be produced, which had enhanced levels of linalool
`and thus enhanced flavor and aroma. Degenhardt et al.
`(2003) discuss how monoterpenes and sesquiterpenes
`are the two most common terpenes emitted from plants
`post-herbivory. They suggest that finding the proper
`mixture and timing of terpene release from crop plants is
`key to creating an adequte transgenic plant. Additionally,
`the properties of terpene emission should be tightly
`regulated by an herbivore-responsive promoter.
`Genetic transformation of tobacco (Nicotiana tobacum)
`was carried out by Lücker et al. (2004) (Table 1). After
`inserting monoterpene synthase genes the plants showed
`
`
`
`LCY Biotechnology Holding, Inc.
`Ex. 1010
`Page 4 of 7
`
`

`

`Zwenger and Basu 005
`
`
`
`
`
`Table 1. Partial representation of organisms which have been genetically
`transformed with at least one terpene synthase gene
`
`
`Species
`Escherichia coli
`Candida albicans
`Arabidopsis thaliana
`Lycpersicon esculentum
`Nicotiana tobacum
`Lactuca sativa
`Mentha piperita
`
`Organism
`bacteria
`yeast
`thale cress
`tomato
`tobacco
`lettuce
`mint
`
`Citation
`Adam et al., 2002
`Jackson et al., 2006
`Aharoni et al., 2003
`Lweinsohn et al., 2001
`Lücker et al., 2004
`Wook et al., 2005
`Wildung et al., 2005
`
`
`
`
`an increase of terpene emission from leaves. To better
`understand terpene biosynthetic pathways Pateraki et al.
`(2007) isolated multiple cDNAs from Cistus creticus.
`They used polymerase chain reaction (PCR) techniques
`to amplify sequences from the plant and found additional
`terpene synthase genes after searching expressed seq-
`uence tag (EST) libraries. Expression of genes is at least
`partly dependent on their promoters. Davidovich-Rikanati
`et al. (2007) used a ripening-specific promoter to modify
`the aroma and flavor of tomatoes. Unfortunately, although
`levels of flavor-causing monoterpenes increased, the
`lycopene decreased.
`The medicinal value of terpenes has not been ignored.
`Canter et al. (2005) discuss some areas within biotech-
`nology to improve medicinal plant cultivation. These inclu-
`de incorporating agronomic traits into medicinal plants,
`pathway engineering and exploring additional transforma-
`tion systems. In a more recent examination, Tyo et al.
`(2007) describe methods for studying and engineering
`cells such as using 'omics' technologies, screening libra-
`ries, and synthetic and computational systems biology. As
`they have mentioned, some of these technologies are
`currently being used to extend comprehension of meta-
`bolic pathways in plants. In a study by Yao et al. (2008)
`bioinformatics helped characterize a terpene synthase
`pathway after comparative analysis of isolated cDNA from
`a cultured callus line of an endangered medicinal plant
`(Camptotheca acuminata) native to China. Similar to
`many other bioinformatic-based approaches, they bene-
`fited from NCBI's (National Center for Biotechnology
`Information) BLAST (Basic Local Alignment Search Tool),
`which can help understand phylogenetic relationships
`among nucleotide sequences (Altschul et al., 1990).
`Melvin Calvin, the Nobel laureate known for his contri-
`bution to the scientific understanding of the carbon fixa-
`tion pathways in plant chloroplasts, studied the tropical
`copiaba (Copaifera langsdorfii) for its natural biofuel
`production (Calvin, 1980). Although it has not yet been
`examined, diesel from C. langsdorfii is largely composed
`of terpenes. The current interest in biofuels is not only in
`the United States but also other countries such as Brazil
`and the European Union. This has sparked new interest
`in finding renewable sources, or plants which may
`contribute to biofuels. Considering this global interest in
`
`biofuels, research describing the up-regulation of terpene
`synthase genes in C. langsdorfii may prove very bene-
`ficial. This research would be very useful, for instance, in
`providing a more cost effective extraction and by-pass
`typical conversion of biomass (corn ethanol) to more
`contemporary biofuels (Demirbas and Balat, 2006).
`Therefore, future studies may include terpene production
`in the diesel tree or related biofuel plants.
`
`
`Conclusions
`
`Many terpenes remain to be discovered so they will
`undoubtedly intrigue scientists for years, as their applica-
`tions are only beginning to be fully realized. Arguably,
`society has benefited tremendously from terpenes. In
`addition, understanding the function of genes in terpene
`production could lead to discovering novel compounds or
`pathways, which might reveal new important aspects for
`many human applications. For instance, the ability to up-
`regulate terpene synthesis in C. langsdorfii could result in
`an increase in the diesel-like resin harvested from this
`tree, might prove beneficial to the global market of bio-
`fules. As we continue into the agrobiotechnology age, it is
`highly likely the applications and potentials of terpenes
`will be further explored.
`
`
`REFERENCES
`
`Adam P, Hecht S, Eisenreich W, Kaiser J, Grawert T, Arigoni D, Adelbert
`B, Rohdich F (2002). Biosynthesis of terpenes: Studies on 1-hydroxy-
`2methyl-2-(E)-butenyl 4-diphosphate reductase. Proc. Nat. Acad. Sci.
`99: 12108-12113.
`Aharoni A, Giri AP, Deuerlein S, Griepink F, de Kogel W, Verstappen F,
`Verhoeven HA, Jongsma MA, Schwab W, Bouwmeester HJ (2003).
`Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.
`Plant Cell. 15: 2866-2884.
`Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic local
`alignment search tool. J. Mol. Biol. 215: 403–410.
`Andrews RE, Parks LW, Spence KD (1980). Some effects of Douglas fir
`terpenes on certain microorganisms. App. Environ. Microbiol. 40:
`301-304.
`Balandrin MF, Klocke JA, Wurtele ES, W.H Bollinger (1985). Natural
`plant chemicals: Sources of industrial and medicinal materials.
`Science. 228:1154-1160.
`Cal K, Kupiec K, Sznitowska M (2005). Effect of physicochemical
`properties of cyclic terpenes on their ex vivo skin absorption and
`elimination kinetics. J. Dermatol. Sci. 41:137-142.
`
`
`
`LCY Biotechnology Holding, Inc.
`Ex. 1010
`Page 5 of 7
`
`

`

`006 Biotechnol. Mol. Biol. Rev.
`
`
`
`Calvin M (1980). Hydrocarbons from plants: Analytical methods and
`observations. Naturwissenschaften. 67: 525-533.
`Canter PH, Thomas H, Ernst E (2005). Bringing medicinal plants into
`cultivation: Opportunities and challenges for biotechnology. Trends in
`Biotechnol. 23: 180-185.
`Chen F, Dorothea T, D'Auria JC, Farooq A, Pichersky E, Gershenzon J.
`(2003). Biosynthesis and emission of terpenoid volatiles from
`Arabidopsis flowers. The Plant Cell. 15: 481-494.
`Cheng A, Lou Y, Mao Y, Lu S, Wang L, Chen X (2007). Plant terpenoids:
`Biosythesis and ecological functions. J. Integrative Plant Biol. 49:
`179-186.
`Cho DW, Park YD, Chung KH (2005). Agrobacterium-mediated
`transformation of lettuce with a terpene synthase gene. J. Korean
`Soc. Hortic. Sci. 46: 169-175.
`Clements RL (1958). Low-temperature chromatography as a means for
`separating terpene hydrocarbons. Science. 128: 899-900.
`Croteau R, Kutchan, TM, Lewis NG, (2000). Natural products
`(secondary metabolites). In Buchanan B, Gruissem W, Jones R
`(Eds.), Biochemistry and molecular biology of plants. Rockville, MD:
`American Society of Plant Physiologists. pp. 1250-1318.
`Davdidovich-Rikanati R, Sitrit Y, Tadmor Y, Iijima Y, Bilenko N, bar E,
`Carmona B, Fallik E, Dudai N, Simon J, Pichersky E, Lewinsohn E
`(2007). Enrichment of tomato flavor by diversion of the early plastidial
`terpenoid pathway. Nat. Biotechnol. 25: 899-901.
`Degenhardt J, Gershenzon J, Baldwin IT, Kessler A (2003). Attracting
`friends to feast on foes: Engineering terpene emission to make crop
`plants more attractive to herbivore enemies. Curr. Opin. Biotechnol.
`14: 169-176.
`Demirbas MF, Balat M (2006) Advances on the production and
`utilization trends of bio-fuels: A global perspective. Energy Convers.
`Manage. 47: 2371-2381.
`Goulart HR, Kimura EA, Peres VJ, Couto AS, Duarte FA, Katzin AM
`(2004). Terpenes arrest parasite development and inhibit biosynthesis
`of isoprenoids in Plasmodium falciparum. Antimicrob. Agents and
`Chemother. 48: 2502-2509.
`Greenberg JP, Friedli H, Guenther AB, Hanson D, Harley P, Karl T
`(2006). Volatile organic emissions from the distillation and pyrolysis of
`vegetation. Atmos. Chem. and Phys. 6: 81-91.
`Greenhagen BT, O'Maille PE, Noel JP, Chappell J (2006). Identifying
`and manipulating structural determinates linking catalytic specificities
`in terpene synthases. Proc. Nat. Acad. Sci. 103: 9826-9831.
`Guenther A, Karl T, Harley P, Wiedinmyer C, Palmer PI, Geron C
`(2006). Estimates of global terrestrial isoprene emissions using
`MEGAN (model of emissions of gases and aerosols from nature).
`Atmos. Chem. Phys. 5: 715–737.
`Harmel N, Almohamad R, Fauconnier M, Du Jardin P, Verheggen F,
`Marlier M, Haubruge E, Francis F (2007). Role of terpenes from
`aphid-infested potato on searching and oviposition behavior of
`Episyrphus balteatus. Insect Sci. 14: 57-63.
`Islam AK, Ali MA, Sayeed A, Salam SM, Islam A, Rahman M, Khan GR,
`Khatun S (2003). An antimircrobial terpenoid from Caesalpinia
`pulcherrima Swartz.: Its characterization, antimicrobial and cytotoxic
`activities. Asian J. Plant Sci. 2: 17-24.
`Jackson BE, Hart-Wells EA, Matsuda SPT (2003). Metabolically
`engineering yeast to produce sesquiterpenes in yeast. 5: 1629-1632.
`John AJ, Karunakran VP, George V (2007). Chemical composition an
`antibacterial activity of Neolitsea foliosa (Nees) Gamble var. caesia
`(Meisner) Gamble. J. Essent. Oil Res. 19: 498-500.
`Johnson SD, Ellis A, Dötterl B (2007). Specialization for pollination by
`beetles and wasps: The role of lollipop hairs and fragrance in
`Satyrium microrrhynchum (Orchidaceae). A. J. Bot. 94: 47-55.
`Kappers IF, Aharoni A, Van Herpen T, Luckerhoff L, Dicke M,
`Bouwmeester HJ
`(2005). Genetic engineering of
`terpenoid
`metabolism attracts bodyguards to Arabidopsis. Science. 309: 2070-
`2072.
`Keeling CI, Bohlmann J (2006). Genes, enzymes, and chemicals of
`terpenoid diversity in the constitutive and induced defence of conifers
`against insects and pathogens. New. Phytol. 170: 657-675.
`Kessler A, Baldwin T (2001). Defensive function of herbivore-induced
`plant volatile emission in nature. Science. 291: 2141-2144.
`Kusari S, Lamshöft M, Zühlke S, Spitelleractivity M (2008). An endo-
`phytic fungus from Hypericum perforatum that produces hypericin. J.
`
`
`
`
`
`
`Nat. Prod. In press.
`Lai S, Chen I, Tsai M (2005). Preparative isolation of terpene trilactones
`from Ginkgo biloba leaves. J. Chromatgr. A .1092: 125-134.
`Lewinsohn E, Schalechet F, Wilkinson J, Matsui K, Tadmor Y, Nam K,
`Amar O, Lastochkin E, Larkov O, Ravid U, Hiatt W, Gepstein S,
`Pichersky E (2001). Enhanced levels of the aroma and flavor
`compound S-linalool by metabolic engineering of the terpenoid
`pathway in tomato fruits. Plant Physiol. 127: 1256-1265.
`Llusiá J, Penñuelas J (2000). Seasonal patterns of terpene content and
`emission from seven Mediterranean woody species in field condi-
`tions. Am. J. Bot. 87:133-140.
`Lücker J, Schwab W, van Hautum B, Blaas J, van der Plas L, Bouwme-
`ester HJ, Verhoeven HA (2004). Increased and altered fragrance of
`tobacco plants after metabolic engineering using three monoterpene
`synthases from lemon. Plant Physiol. 134: 510-519.
`Ma C, Want H, Lu X, Li H, Liu B, Xu G (2007). Analysis of Artemisia
`annua L. volatile oil by comprehensive two-dimensional gas chroma-
`tography time-of-flight mass spectrometry. J. Chromatogr. A. 1150:
`50-53.
`Martin D, Tholl D, Gershenzon J, Bohlmann J (2003). Induction of
`volatile terpene biosynthesis and diurnal emission by methyl jas-
`monate in foliage of Norway spruce. Plant Phys. 132: 1586-1599.
`Matura M, Sköld M, Börje A, Andersen KE, Bruze M, Frosch P, Goos-
`sens A, Johansen JD, Svedman C, White IR, Karlberg A (2005).
`Selected oxidized fragrance terpenes are common contact allergens.
`Contact Dermat. 52: 320-328.
`McGarvey DJ, Croteau R (1995). Terpenoid metabolism. Plant Cell. 7:
`1015-1026.
`Morales G, Sierra P, Mancilla A, Paredes A, Loyola LA, Gallardo O,
`Borquez J (2002). Secondary metabolites from four medicinal plants
`from northern Chile: Antimicrobial activity and biotoxicity against
`Artemia salina. J. Chil. Chem. Soc. 48:13-18.
`Murata T, Miyase T, Muregi FW, Naoshima-Ishibashi Y,| Umehara K,
`Warashina T, Kanou S, Mkoji GM, Terada M, Ishih A (20