No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering
Journal of Integrative Plant Biology
Abstract
Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1–deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.
(Managing editor: Wei WANG)
... Subsequently, MVA kinase and MVA 5-phosphate kinase catalyze MVA to form MVA 5-diphosphate, which is decarboxylated to generate IPP afterwards. Because the generation of MVA is a non-reversible process, HMGR was regarded as the foremost enzyme in this biosynthesis process (Liu et al., 2005). The second way is the chloroplastic MEP pathway, which mediated by eight successive enzymes to synthetize IPP and DMAPP. ...
... Consequentially, the lowered leaf pigments contents and maximum quantum yield of primary photochemistry (F v /F m ) led to decreased photosynthesis and the contents of its photosynthates (Guo et al., 2017). Carbohydrates, such as glucose, starch, pyruvate and 3-phosphoglycerate (3-PGA), are the substrates for IPP biosynthesis through the MVA pathway and/or MEP pathway in plants (Liu et al., 2005). The decreased "source" pool of carbohydrates in C. grandis leaf under Al treatment inevitably lowered the output of photosynthates to roots, as indicated by the lower contents of carbohydrates (glucose, starch, fructose and sucrose) in C. grandis root and reduced IPP biosynthesis (Fig. 2) (Pulido et al., 2012). ...
... For evaluating the metabolisms of volatile isoprenoids in Citrus plants under Al treatment, we measured the enzyme activities of HMGR, DXR and DXS ( Fig. 4A-C). As the rate-limiting enzymes in MVA or MEP pathway, the decreased activities of HMGR and DXR in C. grandis under Al treatment might account for the reduced volatilization rate of monoterpenes and isoprene (Fig. 5A) (García-Alcázar et al., 2017;Liu et al., 2005). In contrast, the preserved activities of those two enzymes in C. sinensis under Al treatment guaranteed the relatively stable production of isoprenoids, which might confer the higher Al tolerance to C. sinensis (Fig. 4A-B; Fig. 5A). ...
Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, β-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO 2 assimilation, maximum quantum yield of primary PSII photochemistry (F v /F m), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism , such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.
... All terpenes originate from the universal C5 precursor IPP and its isomer DMAPP. In plants, both compounds are formed through two pathways [10,11]: the cytosolic MVA pathway, which comprises several enzymatic reactions from acetyl coenzymeA condensation through mevalonate iphosphate decarboxylation, and the plastidial MEP pathway starting from the reaction between pyruvate and glyceraldehyde-3-phosphate ( Figure 1). [10,11] The MVA pathway is primarily regulated at the level of the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase (HMGR) [1,12], and it is known that HMGR activity regulates the metabolic flux through the MVA pathway and the eventual production of the isoprenoid endproducts [13,14]. ...
... In plants, both compounds are formed through two pathways [10,11]: the cytosolic MVA pathway, which comprises several enzymatic reactions from acetyl coenzymeA condensation through mevalonate iphosphate decarboxylation, and the plastidial MEP pathway starting from the reaction between pyruvate and glyceraldehyde-3-phosphate ( Figure 1). [10,11] The MVA pathway is primarily regulated at the level of the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase (HMGR) [1,12], and it is known that HMGR activity regulates the metabolic flux through the MVA pathway and the eventual production of the isoprenoid endproducts [13,14]. The scenario seems to be more complex in the case of the MEP pathway. ...
... All monoterpenes are derived from geranyl diphosphate molecule (GPP). In the case of camphor, GPP is transformed via cyclisation of [10,11] The MVA pathway is primarily regulated at the level of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR) [1,12], and it is known that HMGR activity regulates the metabolic flux through the MVA pathway and the eventual production of the isoprenoid end-products [13,14]. The scenario seems to be more complex in the case of the MEP pathway. ...
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender.
... Nelle foglie i principali costituenti bioattivi sono rappresenati dall'arctina, dall'arctigenina e dall'onopordopicrina, non sono presenti in letteratura studi capaci di evidenziare gli effetti degli estratti ottenuti a partire da foglie (Liu et al,.2005). Diversi studi riportano che le radici di bardana posseggono svariate attività farmaceutiche che includono l'attività antibatterica, antimutagena, antiossidante, epatoprotettiva ed antinfiammatoria tutte riconducibili all'azione radical scavenger dei principali costituenti bioattivi (Lin et al. 1996, Maruta et al.1995. ...
... Nell'estratto delle radici in accordo con quanto riportato in letteratura (Maruta et al., 1995) sono presenti derivati dell'acido caffeilchinico (acido clorogenico, acido caffeico e cinarina) ma anche flavonoidi semplici e glicosilati (quercetina, quercetina ramnoside e luteolina) non ancora descritti. Nell'estratto idroalcolico delle foglie sono presenti piccole quantità di aretina come già descritto in letteratura (Liu et al., 2005); sono stati identificati altri composti non riportati in letteratura come acido caffeico, acido clorogenico, cinarina, rutina, quercitrina, quercetina e luteolina. Gli estratti ottenuti caratterizzati mediante analisi LC/MS/MS sono stati analizzati anche per valutare l'attività antiossidante. ...
... Anche per queste ragioni la produzione mondiale è insufficiente rispetto alle esigenze del mercato, considerando anche che l'artemisinina e i suoi derivati rappresentano la parte preponderante del trattamento contro la malaria in Africa secondo i protocolli del WHO (Shetty, 2004). A ciò si aggiunga che la sintesi chimica risulta complicata, con bassa efficienza ed alti costi (Liu et al., 2005). Per questo motivo, la comunità scientifica internazionale sta portando avanti ricerche per aumentare la produzione in vivo ed, alternativamente, per mettere a punto una produzione biotecnologica in vitro, che finora non è stata completamente esplorata. ...
Colture artificiali di piante medicinali Produzione di metaboliti secondari nelle piante medicinali in coltura artificiale Il volume raccoglie gli atti del Workshop sulle Colture Artificiali di Piante Medicinali, che si è tenuto il 20 ottobre 2006 presso la Facoltà di Agraria di Pisa. Il Workshop ha terminato il Progetto Produzione di metaboliti seconda-ri nelle piante medicinali in coltura artificiale (PROMEDICA), cofinanziato dal Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2004) e coordinato dal professore F. Tognoni del Dipartimento di Biologia delle pian-te agrarie di Pisa. Alberto Pardossi è dal 1998 professore associato di Orticoltura e floricoltura, ruolo ricoperto inizialmente presso la Facoltà di Agraria di Milano e successivamente di Pisa. Autore di circa 200 articoli a carattere scientifico o tecnico–divulgativo, Alberto Pardossi si occupa soprat-tutto di colture di serra. Franco Tognoni è ordinario di Colture protette presso la Facoltà di Agraria di Pisa, di cui è stato anche Preside. Attualmente ricopre la carica di direttore del Dipartimento di Biologia delle piante agrarie. È autore di un libro sulla coltivazione in serra e di centinaia di pubbli-cazioni scientifiche o a carattere tecnico–divulgativo. Anna Mensuali è una ricercatrice della Scuola Superiore di Studi Universitari e Perfeziona-mento Sant'Anna di Pisa. Le sue ricerche hanno riguardato soprattutto le colture in vitro e la conservazione post–raccolta dei prodotti ortofloricoli e sono state oggetto di numerose pub-blicazioni su riviste internazionali.
... Among these, some major connecting pathways between the primary metabolites and secondary metabolites are depicted in Fig. 1 and will be briefly described before discussion of their genetic control. These pathways provide the basic carbon skeleton (central precursors) for a class of secondary metabolites, while other routes are also used by plants for biosynthesis of metabolites (see, for detail , Herrmann 1995;Walker 1999;Mahmoud and Croteau 2001;Liu et al. 2005). ...
... However, genetic networks involved in most of these pathways are yet to be fully understood, and the knowledge about regulation of these pathways is yet to be generated (Goossens et al. 2003). For example, although all the genes and corresponding enzymes of the DXP pathway have been identified, their roles in the control of metabolic flux remain unknown (Liu et al. 2005). Similarly, the source and regulation of the IPP used as a common precursor in the biosynthesis of artemisinin has not been completely characterized, although successful metabolic engineering has been performed due to the availability of several key genes of this pathway (Dhingra et al. 2000). ...
... This approach led to isolation of genes involved in early steps of secondary metabolic pathways that have been shown to be common Plant Biotechnol Rep between plants and bacteria. For example, several genes encoding enzymes of DXP pathway in plants were isolated first from bacteria, and later the sequence information was used to isolate genes in plants (see Liu et al. 2005). Similarly, use of conserved regions of eukaryotic P450dependent enzymes led to the isolation of the first cDNA encoding a methylenedioxy-bridge forming enzyme (CYP719A1), which catalyzes the formation of (S)-canadine as the penultimate step in berberine biosynthesis (Ikezawa et al. 2003). ...
Medicinal and aromatic plants (MAPs) are important sources for plant secondary metabolites, which are important for human
healthcare. Improvement of the yield and quality of these natural plant products through conventional breeding is still a
challenge. However, recent advances in plant genomics research has generated knowledge leading to a better understanding of
the complex genetics and biochemistry involved in biosynthesis of these plant secondary metabolites. This genomics research
also concerned identification and isolation of genes involved in different steps of a number of metabolic pathways. Progress
has also been made in the development of functional genomics resources (EST databases and micro-arrays) in several medicinal
plant species, which offer new opportunities for improvement of genotypes using perfect markers or genetic transformation.
This review article presents an overview of the recent developments and future possibilities in genetics and genomics of MAP
species including use of transgenic approach for their improvement.
... These regulatory genes not only have limited roles in complex biosynthetic pathways, but they also play important roles in controlling initiation and formation of storage tissues (e.g., glandular trichomes). Overexpression of two transcription factors, MIXTA and Myb in tobacco, has been shown to increase the number of supernumerary trichomes on the stems, leaves and cotyledons of the plant (Liu et al. 2005). This shows that regulation of the transcription factor genes could also bring about an increase in the secondary metabolite production by increasing the availability of storage tissues. ...
... The genetic network involved in the production of secondary metabolites is a puzzle, where the pieces need to be placed correctly to get the perfect picture (Goossens et al. 2003). The genes involved in the DXP (1-deoxy-D-xylulose 5-phosphate) pathway enzymes have been discovered, but their importance in regulating the metabolic flux is not yet known (Liu et al. 2005). The transcriptional factors that regulate biosynthesis of secondary metabolites in plants have been listed in recent years. ...
As suggested by the reports of the World Health Organization, the ancient knowledge of medicinal and aromatic plants still forms the basis of traditional and indigenous health system in most developing countries. Almost 80% of the world population depends on plant secondary metabolites (such as alkaloids, phenolics, terpenes, etc.) as a safer alternative than synthetic medicines. Traditional breeding method falls short to meet the huge demands, and technological intervention like biotechnology-based breeding methods (BBBMs) becomes a necessity. Plant tissue culture techniques like Agrobacterium-mediated gene transfer, bioreactor technology, etc. helps to improve yield to satisfactory levels, while methods like cryopreservation, micropropagation, synthetic seeds, etc. help in conservation and storage of the products. Formation of hairy roots from Agrobacterium rhizogenes transformation is one of the most exploited methods for mass production of secondary metabolites. Use of elicitors, improved media constitution, precursor feeding and engineering metabolic pathways helps to drive the flux towards our desired products. Emergence of medicinal plant genomics consortium and functional genomics technologies such as expressed sequence tag (EST) databases, microarrays and restriction site-associated DNA sequencing (RAD-seq) allows transcriptome profiling and study of yet unexcavated wild plant resources. Biotechnological intervention is also used to impart stress tolerance and reduce the toxicity of the metabolic products. Specific gene editing techniques like CRISPR/Cas9, COSTREL, TALENs and polyploidy generation are also being applied to medicinal and aromatic plants for improved industrial strain generation. The present book chapter represents an overview of the latest technological advancements in improving the quality of the medicinal plants to exploit maximum benefit out of them in a sustainable way.
... Isoprenoids also known as terpenoids, are plausibly the most diverse and largest family of natural plant products with more than 40,000 compounds (Sacchettini and Poulter 1997). Besides being essential for plant growth and survival, many isoprenoids have commercial value owing to their therapeutic properties (Ku and Lin 2013;Martin et al. 2013), antimicrobial and antioxidant activities (Heldt 2005), and industrial relevance as pigments, fragrances, and flavorings (Liu et al. 2005). The immense utility of terpenoids has compeled to develop strategies for improving their yield in plants such as mass propagation, metabolic engineering, and Agrobacterium mediated transformation (Liu et al. 2005;Oksman-Caldentey and Inzé 2004). ...
... Besides being essential for plant growth and survival, many isoprenoids have commercial value owing to their therapeutic properties (Ku and Lin 2013;Martin et al. 2013), antimicrobial and antioxidant activities (Heldt 2005), and industrial relevance as pigments, fragrances, and flavorings (Liu et al. 2005). The immense utility of terpenoids has compeled to develop strategies for improving their yield in plants such as mass propagation, metabolic engineering, and Agrobacterium mediated transformation (Liu et al. 2005;Oksman-Caldentey and Inzé 2004). While low cost effectiveness and less practability have been realized as the limitations of these approaches (El-Sayed and Verpoorte 2007;Oksman-Caldentey and Inzé 2004), inoculation of plant with beneficial soil microbes such as arbuscular mycorrhizal fungi (AMF), rhizobacteria, Bacillus coagulans, Trichoderma viride , and Azotobacter chlorococcum (Kumar et al. 2008), overcomes these limitations and offers a simpler alternative. ...
Terpenoids, in addition to being essential for plant growth and survival, are commercially valued for their medicinal properties, ecological significance, and used as flavors and fragrances. Prospective role of arbuscular mycorrhiza (AM) symbiosis in improving the accumulation of secondary metabolites especially terpenoids has gained recognition over the past two decades. Increased production of terpenoids in aboveground parts of AM-colonized medicinal plants has extensively been described. Understanding the mechanisms underpinning increase in accumulation of specific terpenoids in AM plants is important for obtaining higher yield and to improve the potential of AM in sustainable cultivation of plants harboring these compounds. Enhanced phosphorus uptake in the mycorrhizal plants has been largely credited for the increase in terpenoid production. However, recent findings have suggested AM mediated manipulation of morphology, biochemistry and gene transcription in plants. The review provides an update on recent findings in the field of AM research with a special focus on production of pharmaceutically important terpenoids. Several points are highlighted for future research to elucidate probable mechanisms underlying increase in terpenoids in mycorrhizal plants.
... Possible mechanisms and targets are reviewed by Meshnick [19]. Research by Eckstein-Ludweg et al. [48] shows compelling evidence that artemisinins act by inhibiting the "sarco/endoplasmic reticulum Ca 2+-ATPase" orthologue (PfATPase6) of [50][51][52], supplemented with the artemisinin pathway proposed by Bouwmeester and co-workers (big blue arrows) [29,53]. The oxidations catalyzed by CYP71AV1, the P450 enzyme characterized by Teoh et al.[1] is also indicated. ...
... (Plastids are organelles responsible for photosynthesis, for storage of products like starch and for the synthesis of many classes of molecules such as fatty acids and terpenes) Terpene synthases, like farnesyl diphosphate synthase (FPPS), then convert IPP and DMAPP to linear prenyl diphosphates of different chain length, who are used to form the different terpenes [50,52]. Matsushita et al. [54] were the first to clone the A. annua FPPS gene. ...
PART I: Polymorphism association with Obsessive-Compulsive Disorder In light of the putative role of the serotonergic, dopaminergic and possibly (nor)adrenergic system in OCD, following polymorphisms were analysed in a sample of >100 OCD patients and a control sample of >100 ethnically matched Caucasian subjects by means of a case-control study: Taq IA polymorphism in the non-coding region flanking the 3’ end of the dopamine D2 receptor (DRD2) gene; Catechol-O-Methyl Transferase (COMT) NlaIII high/low activity polymorphism; 5-HT2A 1438 A/G polymorphism; 5-HT1Dβ G861C polymorphism and the 5-HTTLPR 44-bp deletioninsertion. There was a higher frequency of the DRD2 A2 allele (p = 0.020) and a higher frequency of the low-activity COMT allele (p = 0.035) in male OCD patients compared to male controls. In addition, we observed an association of the DRD2 A2A2 genotype in patients with an early onset of OCD (p= 0.033). We studied whether the serotonin polymorphisms affect the efficacy of venlafaxine and paroxetine treatment in OCD. The response in paroxetine treated patients is associated with the GG genotype of the 5-HT2A polymorphism (χ2 = 8.66, df=2, p=0.013). In venlafaxine treated patients, response is associated with the SL genotype of the 5-HTTLPR polymorphism (χ2 = 9.71, df=2, p=0.008). PART II: proteomics on Artemisia annua Three strategies were followed to discover genes of the plant Artemisia annua L. that are involved in the production of the antimalarial artemisinin: a proteome analysis, a quantitative cDNA amplified fragment length polymorphism analysis (cDNA AFLP) and the construction of full length Expressed Sequence Tag (EST) cDNA libraries. We identified proteins that were differentially expressed between trichomes and whole leaf tissue. Proteins that are upregulated in the trichomes might be involved in artemisinin production. To narrow down the results to the most valid gene candidates, the proteome data was compared with a cDNA AFLP analysis that investigated samples of A. annua leafs, taken at different time points during a 72h time period after exposure to jasmonic acid. We were able to compile a list of EST candidates, which could be useful for further investigation.
... monoterpenes are produced by the DXP pathway. However, there are recent evidences that the pathways to some extent crosstalk at the isopentenyl diphosphate (IPP) level (Yan Liu, et al., 2005). ...
... Zhou et al. (2005) observed the derivate 3-(12-ȕ-artemisininoxy) phenoxyl succinic acid (SM735) to be strongly immunosuppressive in vitro and in vivo. Artemisinin derivatives have also been shown to have strong antineoplastic properties (Disbrow et al., 2005; Efferth, 2006; Efferth et al., 2002; Liu et al., 2005). ...
Artemisinin is aheterocyclic natural product and belongs to the natural product class of sesquiterpenoids
with an unusual 1,2,4 trioxane substructure. Artemisinin is one of the most potent antimalarial drugs available
and it serves as alead compound in the drug development process to identify new chemical derivatives
with antimalarial optimized activity and improved bioavailability. In this review we report about the latest
status of research on chemical and physical properties of the drug and its derivatives. We describe new
strategies to produce artemisinin on abiotechnological level in heterologous hosts and in plant cell
cultures. We also summarize recent reports on its pharmacokinetic profile and attempts to develop drug
delivery systems to overcome bioavailability problems and to target the drug to Plasmodium
infected erythrocytes as main target cells.
... Despite the growing market demand, medicinal plants naturally have a very low performance in producing SMs (Thakur et al. 2019). So, several techniques have been developed aimed at improving the performance of these valuable metabolites in medicinal plants, such as Agrobacterium-mediated transformation, metabolic engineering, and mass propagation (Oksman-Caldentey and Inzé 2004; Liu et al. 2005). However, each of these methods is associated with limitations such as low practicality, high cost, and low effectiveness (Oksman-Caldentey and Inzé 2004; El-Sayed and Verpoorte 2007; Kapoor et al. 2017). ...
Plants synthetize and accumulate a various class of organic compounds known as secondary metabolites (SMs). Although SMs do not play a crucial role as primary metabolites in plant growth and development, they are of high ecological significance. Humans use these compounds in the pharmaceutical industry. One of the results of mutual symbiotic association between plants and arbuscular mycorrhizal fungi (AMF) is the reprogramming of metabolic pathways and modulating the range and content of plant SMs such as phenolics, terpenoids, and alkaloids. Many of SMs act as signals for multiple interactions between plants and AMF, from the pre-symbiotic stage to the creation/formation of a functional symbiosis. This chapter briefly reviews the current research status in the field of SM changes under the influence of AMF. The plant association with AMF increases the production and accumulation of SMs directly through improving water and nutrient uptake and enhancing the photosynthetic capacity or indirectly by provoking the biosynthetic pathways of SMs through generation of signaling molecules and changes in the concentration of phytohormones. The extent to which AMF affect plant SMs depends on the plant and fungus species and environmental factors.
... However, they are considered crucial for various mechanisms of plant chemical defense and their interaction with the environment. They often serve as pollinator attractants or herbivore repellents, antibiotics, or toxins [40,41]. ...
Plant in vitro cultures, including hairy roots, can be applied for controlled production of valuable natural products, such as triterpenoids and sterols. These compounds originate from the common precursor squalene. Sterols and triterpenoids distinctly differ in their functions, and the 2,3-oxidosqualene cyclization step is often regarded as a branch point between primary and secondary (more aptly: general and specialized) metabolism. Considering the crucial role of phytosterols as membrane constituents, it has been postulated that unconstrained biosynthesis of triterpenoids can occur when sterol formation is already satisfied, and these compounds are no longer needed for cell growth and division. This hypothesis seems to follow directly the growth-defense trade-off plant dilemma. In this review, we present some examples illustrating the specific interplay between the two divergent pathways for sterol and triterpenoid biosynthesis appearing in root cultures. These studies were significant for revealing the steps of the biosynthetic pathway, understanding the role of particular enzymes, and discovering the possibility of gene regulation. Currently, hairy roots of many plant species can be considered not only as an efficient tool for production of phytochemicals, but also as suitable experimental models for investigations on regulatory mechanisms of plant metabolism.
... Triterpenic acids (TA) including ursolic acid (3β-hydroxyurs-12-en-28-oic acid, UA), oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA), and betulinic acid (3β-hydroxylup-20(29)-en-28-oic acid, BA) related to the ursane, oleanane, and lupane backbones, respectively, have various biological activities (Mitaine-Offer et al., 2002;Liu et al., 2005;Somova et al., 2003). UA and OA exhibit anti-inflammatory, antimicrobial, hypoglycemic, and hepatoprotective activity (Sohn et al., 1995;Misra and Laatsch, 2000), while BA exhibits HIV inhibitory activity, antimalarial activity, and antitumor activity (Pisha et al., 1995;Bringmann et al., 1997). ...
Induced polyploidy is nowadays an important strategy in plant breeding and for the development of new crops. Salvia multicaulis Vahl is a valuable medicinal plant that produce precious bioactive metabolites including triterpenic acids (TAs) and phenolic compounds. Hence, at first, for selecting elite lines, both HPLC and GC-MC analyses were performed on fourteen S. multicaulis lines. Then, seeds of selected lines of S. multicaulis were exposed to different concentrations (0.00, 0.05, 0.1, and 0.2 %) of colchicine for 24 or 48 h. The flow cytometric analysis and chromosome counting were used to confirm ploidy level of tetraploid control (2n = 4x = 28, 2C DNA = 1.36 pg) and hexaploid (2n = 6x = 42, 2C DNA = 1.97 pg) plants after seven-month. For the first time, the effects of in vitro polyploidization on morphological characteristics, TAs and phenolic acid contents as well as on the expression of six TAs biosynthesis related genes were investigated. The highest efficiency of hexaploidy (12.76 %) was achieved 48 h after exposure to 0.1 % colchicine concentration. The hexaploid plants showed different growth traits compared with those of tetraploid control plant; indeed, hexaploid plants had leaves with a darker green color, a lower trichome density, and lower plant height and root length. Moreover, there was a significant increase in rosmarinic acid and caffeic acid content in hexaploid plants compared with tetraploid control plants. Also, the increase of oleanolic acid (1.33 fold) content in hexaploids was associated with a significant increased expression of squalene synthase (SQS) and β-amyrin synthase (BAS) genes in hexaploid plants. Nevertheless, a significant decreased expression of squalene epoxidase (SQE), mixed-function amyrin synthase (MFAS), and lupeol synthase (LUS) was observed in hexaploid plants, that led to a reduced content of ursolic acid and betulinic acid compared with tetraploid control plants. These results confirmed that polyploidization is a breeding method with stochastic results in secondary metabolites production and gene expression related to biosynthetic pathways.
... The phytohormone gibberellin is also an active enhancer of SM production [214]. The diterpenoid compound gibberellic acid is synthesized in the same pathway used for artemisinin biosynthesis [215,216]. Various studies have investigated the use of gibberellic acid as an elicitor. For example, it was used to enhance the production of caffeic acid derivatives and tanshinones in hairy root cultures of Echinacea pupurea and S. miltiorrhiza [217,218]. ...
Plant secondary metabolites (SMs) play important roles in plant survival and in creating ecological connections between other species. In addition to providing a variety of valuable natural products, secondary metabolites help protect plants against pathogenic attacks and environmental stresses. Given their sessile nature, plants must protect themselves from such situations through accumulation of these bioactive compounds. Indeed, secondary metabolites act as herbivore deterrents , barriers against pathogen invasion, and mitigators of oxidative stress. The accumulation of SMs are highly dependent on environmental factors such as light, temperature, soil water, soil fertility , and salinity. For most plants, a change in an individual environmental factor can alter the content of secondary metabolites even if other factors remain constant. In this review, we focus on how individual environmental factors affect the accumulation of secondary metabolites in plants during both biotic and abiotic stress conditions. Furthermore, we discuss the application of abiotic and biotic elicitors in culture systems as well as their stimulating effects on the accumulation of secondary metabolites. Specifically, we discuss the shikimate pathway and the aromatic amino acids produced in this pathway, which are the precursors of a range of secondary metabolites including terpenoids, alkaloids, and sulfur-and nitrogen-containing compounds. We also detail how the biosynthesis of important metabolites is altered by several genes related to secondary metabolite biosynthesis pathways. Genes responsible for secondary metabolite biosynthesis in various plant species during stress conditions are regulated by transcriptional factors such as WRKY, MYB, AP2/ERF, bZIP, bHLH, and NAC, which are also discussed here.
... Their extraction from native plants often leads to low yields, and chemical synthesis is usually not viable due to the structural complexity of these compounds. Attempts have been made to solve these problems by metabolic and genetic engineering in planta, cell-based and cell-free -synthetic systems‖ with the use of plant or microbial enzymes, as well as by the application of various in vitro cultures [10,14,15]. Triterpenoid concentrations in plant cells and tissue cultures are generally lower than those in intact plants. ...
... Their extraction from native plants often leads to low yields, and chemical synthesis is usually not viable due to the structural complexity of these compounds. Attempts have been made to solve these problems by metabolic and genetic engineering in plants, cell-based and cell-free "synthetic systems" with the use of plant or microbial enzymes, as well as by the application of various in vitro cultures [1,5,6]. Triterpenoid concentrations in plant cells and tissue cultures are generally lower than those in intact plants. ...
The aim of the study was the evaluation of the efficiency of selected abiotic elicitors, i.e., silver and cadmium ions, ultrasound, and UV-C irradiation, in the stimulation of triterpenoid biosynthesis, accumulation, and saponin secretion in Calendula officinalis hairy root cultures. Apart from the possible enhancement of triterpenoid production, the relationship between primary and secondary metabolism (represented respectively by sterols and pentacyclic triterpenes), modifications of the sterol compositional profile, and fluctuations in the total triterpenoid content were monitored in the performed experiments. The main phenomenon observed as a response to heavy metal treatment was the stimulation (up to 12-fold) of the secretion of saponins, accompanied by significant changes in sterol composition. Ultrasound stimulated the secretion of saponins (up to 11-fold); however, it exerted diverse influences on the triterpenoid content in hairy root tissue (stimulating or decreasing) depending on the duration of the exposure to the elicitor. UV-C radiation caused a slight increase in the content of both sterols and saponins in hairy root tissue, and stimulated saponin secretion up to 8.5-fold. The expected symptoms of the competition between the biosynthetic pathways of sterols and pentacyclic triterpenoids were less evident in reactions to abiotic stressors than those reported previously for biotic elicitors.
... Their extraction from native plants often leads to low yields, and chemical synthesis is usually not viable due to the structural complexity of these compounds. Attempts have been made to solve these problems by metabolic and genetic engineering in plants, cell-based and cell-free "synthetic systems" with the use of plant or microbial enzymes, as well as by the application of various in vitro cultures [1,5,6]. Triterpenoid concentrations in plant cells and tissue cultures are generally lower than those in intact plants. ...
The aim of the study was the evaluation of the efficiency of selected abiotic elicitors, i.e. silver and cadmium ions, ultrasound, and UV-C irradiation, in the stimulation of triterpenoid biosynthesis, accumulation, and saponin secretion in Calendula officinalis hairy root cultures. Apart from the possible enhancement of triterpenoid production, the relationship between primary and secondary metabolism (represented respectively by sterols and pentacyclic triterpenes), modifications of the sterol compositional profile, and fluctuations in the total triterpenoid content were monitored in the performed experiments. The main phenomenon observed as a response to heavy metal treatment was the stimulation (up to 12-fold) of the secretion of saponins, accompanied by significant changes in sterol composition. Ultrasound stimulated the secretion of saponins (up to 11-fold); however, it exerted diverse influences on the triterpenoid content in hairy root tissue (stimulating or decreasing) depending on the duration of the exposure to the elicitor. UV-C radiation caused a slight increase in the content of both sterols and saponins in hairy root tissue, and stimulated saponin secretion up to 8.5-fold. The expected symptoms of the competition between the biosynthetic pathways of sterols and pentacyclic triterpenoids were less evident in reactions to abiotic stressors than those reported previously for biotic elicitors
... The two isoprenoid pathways in plants are the backbone of saponin synthesis. The first of these pathways is the cytosol MVA pathway, producing the end product isopentenyl pyrophosphate (IPP); the other is the plastidial deoxyxylulose-5-phosphate pathway, with the end products IPP and dimethylallyl pyrophosphate (DMAPP) (Liu et al., 2005). The MVA pathway starts with the condensation of acetyl-CoA (Qureshi and Porter, 1981), whereas the MEP pathway needs pyruvate and glyceraldehydes 3-phosphate (Eisenreich et al., 1998). ...
Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is a rhizomatous, herbaceous, perennial plant that has been used for more than a thousand years in traditional Chinese medicine. It is facing extinction due to overharvesting. Steroids are the major therapeutic components in Paris roots, the commercial value of which increases with age. To date, no genomic data on the species have been available. In this study, transcriptome analysis of an 8-year-old root and a 4–year-old root provided insight into the metabolic pathways that generate the steroids. Using Illumina sequencing technology, we generated a high-quality sequence and demonstrated de novo assembly and annotation of genes in the absence of prior genome information. Approximately 87,577 unique sequences, with an average length of 614 bases, were obtained from the root cells. Using bioinformatics methods, we annotated approximately 65.51% of the unique sequences by conducting a similarity search with known genes in the National Center for Biotechnology Information’s non-redundant database. The unique transcripts were functionally classified using the Gene Ontology hierarchy and the Kyoto Encyclopedia of Genes and Genomes database. Of 3,082 genes that were identified as significantly differentially expressed between roots of different ages, 1,518 (49.25%) were upregulated and 1,564 (50.75%) were downregulated in the older root. Metabolic pathway analysis predicted that 25 unigenes were responsible for the biosynthesis of the saponins steroids. These data represent a valuable resource for future genomic studies on this endangered species and will be valuable for efforts to genetically engineer P. polyphylla and facilitate saponin-rich plant development.
... The latex is actually the cytoplasm of H. brasiliensis laticifers, and contains 30-50% (w/w) of NR formed from sequential condensation of IPP (isopentenyl diphosphate) units [6]. The rubber biosynthesis of rubber trees uses the basic precursor of sucrose in the laticifer's latex in a typical isoprenoid secondary metabolism [7], which is similar to the isoprenoid biosynthesis of other plant species using IPP as the precursor [8]. Many latexexpressed genes, such as HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase (GGPS), rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyltransferase (CPT) participate in the latex regeneration and rubber biosynthesis [9][10][11][12][13][14][15][16]; therefore, the gene expression profile and its kinetic change in H. brasiliensis laticifers is very important for investigating the related molecular events of latex metabolism and rubber biosynthesis [17]. ...
Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ -2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis.
... OMT-Tc08g011250 106 . T. cacao orthologous gene copy numbers for each enzyme that was determined, as described in Supplementary Table 17 Supplementary Fig. 18. ...
We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.
... OMT-Tc08g011250 106 . T. cacao orthologous gene copy numbers for each enzyme that was determined, as described in Supplementary Table 17 Supplementary Fig. 18. ...
... OMT-Tc08g011250 106 . T. cacao orthologous gene copy numbers for each enzyme that was determined, as described in Supplementary Table 17 Supplementary Fig. 18. ...
... OMT-Tc08g011250 106 . T. cacao orthologous gene copy numbers for each enzyme that was determined, as described in Supplementary Table 17 Supplementary Fig. 18. ...
... In this study, paclobutrazol induced up to a 25% elevation in caf and kah content, which is consistent with secondary metabolite augmentation as evident from earlier studies (Mahendranath et al. 2011). Since ent-kaurene is formed from geranylgeranyl pyrophosphate (MacMillan et al. 1997), PB may have increased the endogenous levels of entkaurene , which is hypothesized as a precursor for the caf and kah pathway (Liu et al. 2005). This is the likely explanation for the elevation in caf and kah levels under PB treatment. ...
Cafestol (caf) and kahweol (kah) are two diterpenes uniquely associated with the unsaponified lipid fraction of coffee brew and are reported to be responsible for an increase in serum cholesterol and triglyceride levels. The plant growth regulators (PGRs) indole-3-acetic acid (IAA), N
6-benzyladenine (BA), and thidiazuron (TDZ); the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA) were used to determine the variation of caf and kah in somatic embryos of Robusta coffee (Coffea canephora, CxR variety). Embryogenic (EG) medium was comprised of half-strength Murashige and Skoog basal components (½MS) supplemented with 2.85 μM IAA and 1.11 μM BA. After an 8-wk culture, somatic embryos were subjected to diterpene extraction and HPLC analysis of caf and kah profiles. TRIA-supplemented (5 μg L−1) EG medium devoid of IAA reduced the levels of caf and kah by 18–24 and 48–55%, respectively, in coffee somatic embryos. Similarly, the combination of 2.85 μM IAA, 2.27 μM TDZ, and 5–10% coconut water in ½MS basal medium drastically reduced the caf and kah levels in somatic embryos. There was 60–75% reduction in both caf and kah in the presence of 5 μM TIBA, followed by 56–62% reduction in the presence of 10 μM silver nitrate. In contrast, there was 25–32% elevation of caf and kah in EG medium supplemented with 5 μM paclobutrazol. In this study, for the first time, somaclonal variants of C. canephora with reduced levels of diterpenes caf and kah were established. Furthermore, these lines exhibited consistency in their metabolite profiles when cultivated under greenhouse conditions. In-depth investigations at physiological level are warranted in order to elucidate the actual mechanism of these PGR inhibitors on alterations in endogenous pools of diterpenes in coffee somatic embryos.
... In recent years, much progress have been made on the mechanism of artemisinin biosynthesis (Figure 1) (Liu et al., 2006; Bertea et al., 2005), especially after genes encoding key enzymes relevant to the biosynthesis of artemisinin were cloned. Precursors of artemisinin biosynthesis, namely isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), have been shown to be derived from the mevalonate (MVA) pathway and 2-C-methyl-D- erthritol 4-phosphate (MEP) pathway (Liu et al., 2005), respectively. Geranyldiphosphate (GPP) is the product of the reaction between IPP with DMAPP, and it can further react with IPP to produce farnesyldiphosphate (FPP). ...
Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua L., is considered to be the most effective antimalarial drug. To increase the production of artemisinin, the hmgr and fps genes which encode two key enzymes involved in artemisinin synthesis, were cloned from A. annua, respectively. The genes were inserted between the cauliflower mosaic virus 35S promoter and nopaline synthase terminator to construct the expression cassettes, and were then cloned into the plant expression vector pCAMBIA2300 to obtain the p2300-gfh vector containing both the hmgr and fps genes. An Agrobacterium-mediated method was used to stably transform A. annua, and 38 independent transgenic plants were obtained. Polymerase chain reaction and Southern blot analysis confirmed that the foreign genes were successfully introduced into the A. annua genome. The results obtained by high-performance liquid chromatography-evaporative light scattering detection showed that the artemisinin contents of most transgenic plants were higher than those in control plants. The highest artemisinin content in transgenic plants was about 1.8-fold that of the control plants. The results of real-time fluorescence quantitative analysis showed that the expression of hmgr and fps was enhanced in the transgenic plants analyzed. This study demonstrates that overexpression of the hmgr and fps genes are an effective approach to increase artemisinin content in A. annua L.
... Prenylation of these compounds with the C 20intermediate geranylgeranyl diphosphate (GGPP) is required in order to form their hydrophobic tails for integration into plastid membranes. GGPP, an isoprenoid present in plastid, is synthesized from four molecules of isopentenyl pyrophosphate, which originate in the cytosol (Liu et al., 2005). In tocopherol synthesis in chloroplasts, phytyl diphosphate is transferred to homogentisic acid to form the first tocopherol intermediate (Hirschberg, 1999). ...
Geranylgeranyl reductase (CHL P) is localized in plastids and catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate, a molecule required for both tocopherol and chlorophyll biosyntheses. To understand the regulation of Chl P expression in sesame, a Chl P cDNA (SiChlP) was isolated and the effects of light and phytohormones on SiChl P expression was investigated. The putative SiChl P cDNA was 1,633 bp and included a 1,394 bp open reading frame encoding a polypeptide of 465 amino acid residues. SiChl P mRNA was expressed most abundantly in leaves and cotyledons, moderately in developing capsules and seeds, but little in flowers, hypocotyls and roots of healthy seedlings and plants. Expression of SiChl P in a cotyledon was induced by light, but the level of induction was higher in red light than in far-red or blue light. However, SiChl P expression was diminished by dark, ethylene and abscisic acid.
... Thus, the production of antioxidant compounds (PHB and isoprenoids) by thiolase II is repressed under favorable conditions, characterized by high respiratory rates ( Fig. 1) (Ayub et al., 2009;Soto et al., 2011). On the other hand, several evidences indicate that the TCA cycle is inhibited by the oxidative stress induced under abiotic stress exposure in highly divergent organisms such as yeast, animals, plants and bacteria ( Fig. 1) (Baxter et al., 2007;Godon et al., 1998;Grant, 2008;Liu et al., 2005;Pomposiello and Demple, 2002). Therefore, the levels of acetyl-CoA would be increased and the amount of free CoA would be decreased under abiotic stress conditions (Fig. 1). ...
... These approaches show potential for future development, but improvements delivered by them so far have not met the global demand. Organic synthesis of artemisinin is possible, but the low yields make it economically nonviable as a means of commercial production (Avery et al., 1992;Liu et al., 2005). Therefore, the plant is still the only valid source of artemisinin. ...
Artemisinin isolated from the aerial parts of Artemisia annua L. is a promising and potent antimalarial drug. It posses remarkable activity against both chloroquinine resistant as well as chloroquinine sensitive strains of Plasmodium falciparum. It is also useful in the treatment of cerebral malaria. The relatively low content of artemisinin in A. annua and unavailability of cost effective and viable synthetic protocol however, are major obstacles to the commercial production of the drug. The enhanced production of artemisinin is hence, highly desirable, which can be achieved by adequate and judicious supply of plant nutrients. The present experiment was therefore, designed to study the effect of organic manure (15tonnesha−1) and chemical fertilizers (N40+40, P40, K40, S15+15kgha−1; nitrogen, phosphorus, potassium and sulphur) on the accumulation of artemisinin and biomass in various plant parts through the developmental stages of A. annua L. Artemisinin yield (kgha−1) was also determined through the developmental stages of A. annua L. Artemisinin content and artemisinin yield of dried leaves were increased significantly at pre-flowering stage in the plants treated with NPKS (27.3% and 53.6%) and NPK (18.2% and 33.5%), respectively, when compared with control. Maximum dry yield of leaf ranging from 2596 to 3141kgha−1 was observed at pre-flowering stage with various treatments.
... Prenylation of these compounds with the C 20intermediate geranylgeranyl diphosphate (GGPP) is required in order to form their hydrophobic tails for integration into plastid membranes. GGPP, an isoprenoid present in plastid, is synthesized from four molecules of isopentenyl pyrophosphate, which originate in the cytosol (Liu et al., 2005). In tocopherol synthesis in chloroplasts, phytyl diphosphate is transferred to homogentisic acid to form the first tocopherol intermediate (Hirschberg, 1999). ...
... Based on these results, one might predict the existence of a " kahweolsynthase " enzyme specifically expressed in C. arabica seeds, and of a " cafestol-methylase " , responsible of the 16-OMC biosynthesis, specifically in C. canephora seeds. The identification of genes coding for these enzymes and for those of the isoprenoid biosynthesis pathway (Liu et al., 2005) should be facilitated by the screening of UniGene membranes of the Brazilian Coffee Genome Project (see Vieira et al., in this issue). ...
In commercial coffee species (Coffea arabica and Coffea canephora), fruit development is a lengthy process, characterized by
tissue changes and evolutions. For example, soon after fecundation and up to mid development, the fruit is mainly constituted
of the pericarp and perisperm tissue. Thereafter, the perisperm gradually disappears and is progressively replaced by the
endosperm (true seed). Initially present in a “liquid” state, the endosperm hardens as it ripens during the maturation phase, as
a result of accumulation of storage proteins, sucrose and complex polysaccharides representing the main reserves of the seed.
The last step of maturation is characterized by the dehydration of the endosperm and the color change of the pericarp. Important
quantitative and qualitative changes accompany fruit growth, highlighting the importance of its study to better understand
the final characteristics of coffee beans. Following a description of the coffee fruit tissues, this review presents some data
concerning biochemical, enzymatic and gene expression variations observed during the coffee fruit development. The latter will
also be analyzed in the light of recent data (electronic expression profiles) arising from the Brazilian Coffee Genome Project.
... Basically, terpenoids are derived from two isoprenoid pathways in plants, which are called the terpenoid backbone pathways. One is the cytosol MVA pathway with the end product of IPP; another is the plastidial DXP pathway with (Kirby and Keasling 2009;Yan et al. 2005). After the IPP and DMAPP pathways, the triterpene aglycone of ginsenoside/gypnoside, protopanaxadiol, is synthesized from 2,3-oxidosqualene. ...
Gynosaponins (Gypenosides) are major phyto- chemicals in Gynostemma pentaphyllum (Thunb.), with similarities to the ginsenosides present in Panax ginseng. Gynosaponins are classified as terpenoid compounds. In G. pentaphyllum, 25% of the total gynosaponins are similar to ginsenosides. In this study, we analyzed the transcrip- tional levels of the G. pentaphyllum genome to identify secondary metabolite genes. The complete transcriptomes for the roots and leaves were obtained using a GS-FLX pyro-sequencer. In total, we obtained 265,340 and all reads were well annotated according to biological databases. Using insilico analysis, 84% of sequence were well anno- tated and we obtained most of the secondary metabolite genes that represent mono-, di-, tri- and sesquiterpenoids. From our EST, most of the terpenoid genes were noted, among those few similar genes were studied in P. ginseng and these transcripts will help to characterize more triter- penoid genes in G. pentaphyllum. Also help to compare P. ginseng and G. pentaphyllum at transcriptome level.
... Isoprenoid (also called terpenoid) pathways in plants are the backbones of ginsenoside synthesis. In plants, two isoprenoid pathways are present, one is the cytosol MVA pathway with an end product of IPP, while the other is the plastidial DXP pathway with end products of IPP and DMAPP [36]. In this pathway, the triterpene aglycone of ginsenoside, protopanaxadiol, is synthesized from 2, 3-oxidosqualene. ...
Panay ginseng root has been used as a major source of ginsenoside throughout the history of oriental medicine. In recent years, scientists have found that all of its biomass, including embryogenic calli and flower buds can contain similar active ingredients with pharmacological functions. In this study, transcriptome analyses were used to identify different gene expressions from embryogenic calli and flower buds. In total, 6,226 expressed sequence tags (ESTs) were obtained from cDNA libraries of P ginseng. Insilico analysis was conducted to annotate the putative sequences using gene ontology functional analysis, Kyoto Encyclopedia of Genes and Genomes orthology biochemical analysis, and interproscan protein functional domain analysis. From the obtained results, genes responsible for growth, pathogenicity, pigments, ginsenoside pathway, and development were discussed. Almost 83.3% of the EST sequence was annotated using one-dimensional insilico analysis.
... Historically, it was believed that IPP and DMAPP were solemnly synthesized via MVA, in the cytosol. This process involves three key steps (Lichtenthaler 1999; Liu et al. 2005). First, three molecules of acetyl-CoA couple to yield 3-hydroxy-3-methylglutaryl CoA, which is subsequently reduced by the enzyme HMG CoA reductase to yield mevalonic acid. ...
The terpenoids, or isoprenoids, are a large family of natural products that are best known as constituents of the essential
oils in plants. Because of their pleasant flavor and aromatic properties, essential oils have an economic importance in perfumery,
cosmetic, pharmaceutical and various other industries. However, expression profiles of regulatory genes in essential oil production
have not been dissected entirely, which may be an interesting topic of future research. In this report, we review recent studies
on isoprenoids biosynthesis in plants. We also discuss the progress of our recent research activities on isoprenoid studies.
Medicinal chemists around the world have been inspired by nature and have successfully extracted chemicals from plants. Research on enzymatic modifications of naturally occurring compounds has played a critical role in the search for biologically active molecules to treat diseases.
This book set explores compounds of interest to researchers and clinicians. It presents a comprehensive analysis about the medicinal chemistry (drug design, structure-activity relationships, permeability data, cytotoxicity, appropriate statistical procedures, molecular modelling studies) of different compounds. Each chapter brings contributions from known scientists explaining experimental results which can be translated into clinical practice.
Volume 2 gives (1) a detailed overview of the sesquiterpenes polypharmacology, (2) an interesting journey around the world of cannabinoids that reveals the development of new synthetic Δ9-THC derivatives, (3) the design of specific formulations to overcome the volatility of small sized terpenes-based essential oils, (4) an update on the latest generations of endoperoxides endowed with antimalarial activity and finally (5) a summary of MedChem strategies to fix the most common issues in formulating terpene derivatives (like low potency and poor solubility).
The objective of this book set is to fulfill gaps in currently acquired knowledge with information from the recent years. It serves as a guide for academic and professional researchers and clinicians.
Secondary plant metabolites are necessary for the survival of plants and help to establish interactions between plants and environment. They constitute an important part of the plant’s defense mechanism against pathogenic attacks and environmental stresses, in addition to providing a valuable range of natural products. Plants being sessile in nature safeguard themselves from severe conditions around them by means of bioactive compounds produced as secondary metabolites. These metabolites serve as a deterrent to herbivores, provide a barrier against pathogen invasion, and reduce oxidative stress during stressful situations. The accumulation of these metabolites increases under environmental stress, such as soil water, minerals, temperature, salinity, light, humidity, air pollutants, heavy metals and frost, among others. Changes in the nutritional and hormonal status regulate the secondary metabolites production in plants. This article examines how the mineral nutrient elements and plant hormones influence the accumulation of secondary metabolites in plants under abiotic stress conditions.
Only during last century, the use of zero/very low calorie natural non saccharide super sweet (NSSS) principles cropped up in the mind of human being after visualizing serious health disorders cause by most popular calorie rich sucrose and lethal effects of zero/very low calorie synthetic sweeteners like asulfame K, sucralose, saccharin etc.
p>This study integrates three methods for detection of an induced response to aphid feeding. Firstly, it is shown that volatile organic compounds emitted from wild cabbage infested with the cabbage aphid Brevicoryne brassicae attract the aphid parasitoid Diaeretiella rapae and could constitute a reliable and detectable cue during parasitoid host foraging. It is found that there is no difference in attraction to aphid-infested plants when comparing field-collected and lab-reared parasitoids. Secondly, collection and analysis of compounds emitted from the aphid and Plutella xylostella damaged plants reveals several compounds exhibiting significant induction when compared to uninfested plants. Univariate techniques and multivariate canonical discriminant analysis reveal distinct volatile profiles contain several terpenes and 3-butenyl isothiocyanate as induced compounds that could be involved in the attraction of the parasitoid. It is proposed that this multivariate technique may closer resemble the method of signal recognition used by foraging insects.
Finally, global gene expression analysis using Arabidopsis thaliana microarrays coupled to a Brassica oleracea specific software filter is used to assay the expression of genes induced by aphid and Plutella feeding. These experiments reveal the aphid- and Plutella- related induction of genes involved in isoprenoid biosynthesis, involvement of the octadecanoid pathway following Plutella feeding and that although the genes induced are different, number of genes induced by the different herbivores is roughly equal. AFLP analysis illustrates the high degree of genotypic variation in wild Brassica populations when compared to crop cultivars and Arabidopsis thaliana . This suggests that the wild populations are segregating with a high level of gene flow between populations.</p
Artemisinin, a sesquiterpene endoperoxide lactone derived from Artemisia annua, is highly effective against malaria parasite Plasmodium falciparum. Artemisinin and its derivatives (collectively termed artemisinins) demonstrate additional anticancer, anti-inflammation, antiviral, and anti-SARS-CoV-2 activity. Because of the expensive medicine of artemisinins and the low content of artemisinin biosynthesis in native host, researchers have been endeavored to produce artemisinin via alternative approaches. Previous attempts give attention to increase artemisinin biosynthesis via common plant breeding techniques and on engineering cultivated plants for wide production. However, current trends are focusing on the bioengineering of artemisinin biosynthetic pathway in heterologous expression platforms. To date, in planta artemisinin production in two model plants, tobacco (Nicotiana benthamiana) and moss (Physcomitrella patens), has been reported successfully. Nevertheless, to meet the large-scale demands, de novo reconstituting of artemisinin biosynthetic pathway in heterologous microorganisms such as Escherichia coli and Saccharomyces cerevisiae has had documentary achievements. Since there are challenges for the expression of cytochrome P450s (CYPs) from the eukaryotic origin (such as CYP71AV1) in E. coli, S. cerevisiae has been proposed as a heterologous host for production of artemisinin precursors such as amorphadiene, artemisinic acid, and dihydro artemisinic acid. Moreover, the presence of the MVA biosynthetic pathway for production of universal sesquiterpenes precursor, farnesyl pyrophosphate (FPP), and relative compatibility of S. cerevisiae cell environment for the expression of plant-origin genes (enzymes) have made this microorganism as a favorable platform for artemisinins production.
Chronic inflammation is involved in many pathological conditions such as rheumatoid arthritis, atherosclerosis, type 2 diabetes, asthma, obesity, inflammatory bowel diseases, neurodegenerative diseases, and cancer. Currently, there is a demand for effective anti-inflammatory drugs with low toxicity and reduced side effects. In this context, A. annua appears to be a promising source of anti-inflammatory compounds, as numerous studies on different models of inflammation certify. Further research is needed in order to understand the underlying mechanism of action for each compound. The anti-inflammatory activity of extracts is also promising, considering the fact they have been demonstrated to be more efficient than isolated compounds, although they raise the problem of reproducibility and standardization. In addition, no clinical trials were yet undertaken to assert the anti-inflammatory effect of A. annua in the human body.
In current study, the effect of gibberellic acid was tested for production of biomass, polyphenolics and Steviol glycosides in adventitious root cultures of Stevia rebaudiana. Adventitious cultures were induced from the roots of in vitro grown plantlets on Murashige and Skoog (MS) medium containing combination of gibberellic acid (GA 3 ; 0.5, 1.0, 1.5 and 2.0 mg/L) and naphthalene acetic acid (NAA; 0.5 mg/L). Initially, a known mass of inoculum roots were shifted into suspension media augmented with various GA 3 concentrations. The growth behavior of adventitious roots was recorded every 3 days for a period of 30 days. Maximum biomass biosynthesis (13.12 g/flask) was noticed in exponential phase on 27th day in the suspension containing 2.0 mg/L of GA 3. Other GA 3 concentrations also displayed optimum patterns of biomass accumulation as compared to the control. Adventitious roots were investigated for total phenolic content (TPC) and production (TPP), total flavonoid content (TFC) and production (TFP), and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-based antioxidant potential. Maximum phenolics (TPC 9.84 mg gallic acid equivalent (GAE)/g-dry weight (DW)) and TPP (147.6 mg/L), TFC (5.12 mg Quercitin equivalent (QE)/g-DW) and TFP (76.91 mg/L) were observed in 2.0 mg/L GA 3 treated cultures. The same concentration of gibberellic acid enhanced antioxidant activity (77.2%). Furthermore, maximum stevioside (7.13 mg/g-DW), rebaudioside-A (0.27 mg/g-DW) and dulcoside-A (0.001 mg/g-DW) were observed in roots exposed to 2.0 mg/L GA 3. This is the first report on the application of GA 3 on biomass accumulation and secondary metabolite production in S. rebaudiana. The current study will be helpful to scale up the adventitious root cultures in bioreactors for the production of biomass and pharmaceutically important secondary metabolites.
The mevalonate (MVA) pathway is responsible for the biosynthesis of cytosolic terpenes including gossypol and its derivatives, which play an important role in the cotton plant’s defense against pathogens and herbivores. In this study, we identified and cloned 17 potentially functional genes encoding enzymes that catalyze the six steps of the MVA pathway in Gossypium arboreum . Expression pattern analysis by qRT-PCR demonstrated that these genes had tissue-specific expression profiles and were most prevalently expressed in roots. Moreover, these genes were up-regulated in response to several elicitors, including methyl jasmonate and salicylic acid, as well as Verticillium dahliae infection and Helicoverpa armigera infestation. This indicates that the MVA pathway genes are involved in the signaling pathway regulated by exogenous hormones and the resistance of cotton plants to pathogens and herbivores. Our results improve the understanding of cytosolic terpene biosynthesis in Gossypium species and lay the foundation for further research on gossypol biosynthesis.
We presented a two-stage combined bioreactor system consisting of a stir-tank and an airlift column, and challenged with Rhizoma zedoariae cell suspensions for β-elemene production. Two-stage culture was initiated when the cell concentration in both vessels was maintained at an appropriate density. The cells were proliferated in stirred-tank with the maximal growth rate of 0.17 d⁻¹ to present enough cells for β-elemene synthesis. In the airlift column, continuous cell separation from culture medium was achieved by using a cell retention device based on centrifugal and gravity settling when the system was performed in perfusion mode. The results indicated that additives can efficiently promote the accumulation of β-elemene in R. zedoariae cells. In addition, the β-elemene content showed higher levels in cell lines of overexpressing 3-hydroxy-3-methylglutaryl coenzyme-A reductase, Farnesyldi phosphate synthase, and ST02C genes.
Malaria is probably as old as mankind and continues to affect millions of people throughout the world. Today, some 500 million people in Africa, India, Southeast Asia, and South America are exposed to endemic malaria, and it is estimated to cause one and half million deaths annually, one million of which are children. As a consequence, effective therapeutic agents against malaria are continuously being sought, especially against those strains of Plasmodium falciparum, which have become resistant to nearly all antimalarial drugs, including chloroquine and quinine. In the absence of reports of artemisinin resistance in malaria parasite, WHO now recommends the use of artemisinin-based combination therapies (ACTs) with formulations containing an artemisinin derivative. Artemisinin, a sesquiterpene endoperoxide lactone, is isolated from the shoots of Artemisia annua L. plants. Apart from a novel and potent antimalarial drug, artemisinin and its derivatives are also used in therapies against hepatitis, leishmaniasis, and schistosomiasis. Artemisinin also possess lethal activities against cancerous cells, fungi, and bacteria. It has also shown to be immune-suppressant in mammals and a potent herbicide. Despite of its immense commercial value, the production of artemisinin is not cost-effective because of its low concentration (0.01-1.1 %) in the plant. Moreover, its de novo synthesis is complex, uneconomical and gives low yields. Further, classical breeding and selection techniques have failed to develop high-yielding strains of A. annua L. plants. Efforts are therefore, needed to elucidate the complex pathway of artemisinin biosynthesis and its biochemical and molecular regulation. Non-conventional approaches have to be developed to evolve novel strains of the plant to optimize and scale up the production of artemisinin in bulk and make it available to ACT manufacturers at a price much lower than their current cost in turn making an important contribution toward attaining the goals of global malaria eradication programs. The details of past and current status of both conventional and non-conventional approaches for enhancing artemisinin content in A. annua L. plants and its yield have been discussed in this chapter. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.
Plant tissue culture is an important biotechnological tool, which involves biochemical and genetic manipulations to onset specific gene programming, which determines an optimal cell differentiation state for the production of bioactive compounds. Indeed, not only the metabolism but also the morphogenetic processes are modified in plant cells to drive the synthesis and accumulation of economically valuable compounds. In this chapter, we provide an overview of the current molecular approaches to control the expression of specific genes encoding putative heterologous and native enzymes as well as transcription factors to enhance the metabolic flow of specific pathways in order that notable bioactive compounds can be accumulated in plant cells at acceptable commercial levels. Such methods vary from single-gene plant transformations to the emerging multi-gene transformation (gene stacking) technologies embraced by private companies focusing primarily in the metabolic engineering of secondary metabolism. The virtues and potential of these molecular methods in their application to tissue culture systems are presented. Additionally, the importance of subcellular targeting of proteins, notably biosynthetic enzymes and pharmaceutical antibodies, for enhancing their activity and stability is also discussed. Finally, the progress in two emerging approaches for the production of bioactive molecules, the manipulation of cell differentiation and cell immortalization are expounded in this article. Thus, we present the molecular basis to control both cell differentiation and cell immortalization in plant tissue culture systems as novel avenues to control and perpetuate the gene programming which in turn creates and regulates cellular microenvironments for the optimal biosynthesis of valuable compounds. Consequently, our objective is to present how basic approaches, including the manipulation of gene expression, are amalgamated to other molecular strategies of higher hierarchy, particularly the manipulation of cell differentiation and immortalization for the synthesis of bioactive molecules in plant tissue culture platforms. © 2014 Springer Science+Business Media Dordrecht. All rights reserved.
Tetraploid plants were successfully induced for the first time in Mitracarpus hirtus L., by overnight immersion of shoot meristems in 0.1 % colchicine solution, followed by in vitro culture leading to plant regeneration. Examination of ploidy level by flow cytometric analysis and counting chromosome number at metaphase confirmed that original diploid plant (WT1) contained chromosome number as 2n = 2x = 28, whereas 2n = 4x = 56 was observed in the tetraploids induced with colchicine treatment (CC102 and CC110). Thicker root formation, larger stomata (1.3–2 times), and lower density of stomata (1.7–4 times) were observed in these tetraploid plants. After transplantation to the pot, tetraploid plant (CC110) showed higher fresh weights of aerial part and leaves (1.5 and 1.4 times respectively) than diploid. However, the methanolic extracts from leaves of tetraploid line CC102 showed inhibition against human pathogenic bacterium, S. aureus while WT1 and CC110 showed no activity. GC–MS revealed 40 unique compounds present in CC102, but absent in WT1 and CC110. Through hierarchical clustering analysis the 40 unique compounds in CC102 formed a cluster group found to correlate with anti-S. aurens activity. These results suggested that tetraploid M. hirtus CC102 created in this study provides a novel source of compounds useful in fighting infectious disease.
Per molte specie officinali, la propagazione in vitro rappresenta un metodo propagativo alternativo e/o esclusivo rispetto alla propagazione tradizionale per seme o talea. Negli ultimi decenni sono stati messi a punto i protocolli sperimentali di micropropagazione di numerose specie medicinali, e notevoli progressi sono stati fatti nell’approfondire, caso per caso, i punti critici di tale metodo innovativo di propagazione. In questo lavoro sono riportati i protocolli di propagazione in vitro di piante officinali quali il Lavandino (un ibrido sterile fra la Lavandula officinalis e la Lavandula latifolia, ad alta efficienza produttiva di olio essenziale) e la menta piperita. Inoltre, è riportato un metodo di pretrattamento e di sterilizzazione per espianti particolarmente inquinati di Echinacea angustifolia L. Infine, sono riportati gli esperimenti condotti su Passiflora incarnata L. per l’induzione di un certo grado di foto-autotrofia, attraverso la riduzione dei livelli di saccarosio e di sali minerali e la valutazione dell’efficienza fotosintetica delle colture in vitro così indotte.
Parole chiave: piante officinali, propagazione in vitro, microponica, Lavandino, Menta, Passiflora, Echinacea.
Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l−1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.
Plants contain numerous natural products (secondary metabolites) that may not participate directly in their growth and development but play an important role in ecological interactions with other organisms. Despite immense chemical diversity, which originates from simple carbohydrates produced because of photosynthesis, plant natural products are formed from only a few biosynthetic building blocks that consist of acetate, mevalonate, and shikimate. These basic building blocks undergo a variety of biosynthetic transformations and combinations that lead to numerous classes of plant natural products including, but not limited to, carbohydrates, fatty acids and their esters, aromatic polyketides (phenols and quinones), terpenoids and steroids, phenyl propanoids (lignans and lignin, coumarins, flavonoids, and isoflavonoids), and alkaloids. Summarized in this article are representative members of these important classes of plant natural products with special emphasis on their chemical diversity. The article concludes with a brief discussion on recent methods for the maximization of chemical diversity and the production of natural products from plants.
Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA3 treatment. The effect of GA3 application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA3 application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA3 application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA3 treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA3 continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA3-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA3-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA3 treatment enhanced artemisinin production in pot experiments and should be suitable for field application.
In Escherichia coli, 1-deoxy-d-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation
(Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer,
B. G. & Spenser, I. D. (1996) Chem. Commun. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms
2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase
yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct
protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.
Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species.
Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate. This is the first committed step of isoprenoid biosynthesis. A common feature of all known plant HMGR isoforms is the presence of two highly conserved hydrophobic sequences in the N-terminal quarter of the protein. Using an in vitro system, we showed that the two hydrophobic sequences of Arabidopsis HMGR1S function as internal signal sequences. Specific recognition of these sequences by the signal recognition particle mediates the targeting of the protein to microsomes derived from the endoplasmic reticulum. Arabidopsis HMGR is inserted into the microsomal membrane, and the two hydrophobic sequences become membrane-spanning segments. The N-terminal end and the C-terminal catalytic domain of Arabidopsis HMGR are positioned on the cytosolic side of the membrane, whereas only a short hydrophilic sequence is exposed to the lumen. Our results suggest that the plant HMGR isoforms known to date are primarily targeted to the endoplasmic reticulum and have the same topology in the membrane. This reinforces the hypothesis that mevalonate is synthesized only in the cytosol. The possibility that plant HMGRs might be located in different regions of the endomembrane system is discussed.
We isolated two full-length cDNA clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from potato (Solanum tuberosum) L. tubers. The clones, designated hmg2.2 and hmg3.3, are members of previously described gene subfamilies. In addition to being induced by arachidonic acid in tubers, hmg2.2 transcript accumulates developmentally in young flowers, and in mature sepals and ovaries, whereas transcript for hmg3.3 accumulates in mature petals and anthers. Our data suggest that members of specific HMGR-encoding gene subfamilies might be involved in both defense responses and flower development. Accumulation of different HMGR transcripts could provide some control of isoprenoid biosynthesis by producing isoforms specific for classes of end-products produced in particular tissues.
For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate/mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate. In E. coli, D-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli, designated dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA, the gene that encodes farnesyl-diphosphate synthase. D-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.
Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.
alpha-Tocopherol (vitamin E) is a lipid-soluble antioxidant synthesized only by photosynthetic organisms. alpha-Tocopherol is an essential component of mammalian diets, and intakes in excess of the U.S. recommended daily allowance are correlated with decreased incidence of a number of degenerative human diseases. Plant oils, the main dietary source of tocopherols, typically contain alpha-tocopherol as a minor component and high levels of its biosynthetic precursor, gamma-tocopherol. A genomics-based approach was used to clone the final enzyme in alpha-tocopherol synthesis, gamma-tocopherol methyltransferase. Overexpression of gamma-tocopherol methyltransferase in Arabidopsis seeds shifted oil compositions in favor of alpha-tocopherol. Similar increases in agricultural oil crops would increase vitamin E levels in the average U.S. diet.
Myb-class transcription factors implicated in cell shape regulation were overexpressed in Arabidopsis thaliana and Nicotiana tabacum in an attempt to assess the extent to which cellular differentiation programs might be shared between these distantly related plants. GLABROUS 1, a myb gene required for trichome development in Arabidopsis, did not alter the trichome phenotype of the tobacco plants in which it was overexpressed. MIXTA, which in Antirrhinum majus is reported to regulate certain aspects of floral papillae development, did not complement the glabrous 1 mutant of Arabidopsis. However, 35S:MIXTA transformants of N. tabacum displayed various developmental abnormalities, most strikingly production of supernumerary trichomes on cotyledons, leaves and stems. In addition, floral papillae were converted to multicellular trichomes. CotMYBA, a myb gene which is expressed in Gossypium hirsutum ovules and has some homology to MIXTA, was also overexpressed in the two species. A similar but distinct syndrome of abnormalities, including the production of cotyledonary trichomes, was observed in 35S:CotMYBA tobacco transformants. However, CotMYBA did not alter trichome production in Arabidopsis. These results suggest that the trichomes of Arabidopsis and Nicotiana are merely analogous structures, and that the myb genes regulating their differentiation are specific and separate.
Many authors have discussed the problem of a proper definition of secondary metabolites.1,2,3 Bennett and Bentley1 extensively discussed the history of the term secondary metabolites, with special reference to microbial metabolites. They gave the following definition:
“General metabolites (hence general metabolism): A metabolic intermediate or product, found in most living systems, essential to growth and life, and biosynthesized by a limited number of biochemical pathways. Secondary metabolites (hence secondary metabolism): a metabolic intermediate or product, found as a differentiation product in restricted taxonomic groups, not essential to growth and life of the producing organism, and biosynthesized from one or more general metabolites by a wider variety of pathways than is available in general metabolism.”
Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.
Living things use isoprenoids as pigments, hormones, molecular signals, and pheromones. The many variations of isoprenoid are generated by versatile enzymes that synthesize multiple products. In their Perspective, Sacchettini and Poulter discuss what the new structures of three of these cyclases, reported on pages [1811][1], [1815][2], and [1820][3] of this issue, reveal about the mechanisms of isoprenoid formation.
[1]: http://www.sciencemag.org/cgi/content/short/277/5333/1811
[2]: http://www.sciencemag.org/cgi/content/short/277/5333/1815
[3]: http://www.sciencemag.org/cgi/content/short/277/5333/1820
Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.
In Artemisia annua, [¹⁴C]arteannuic acid was incorporated into arteannuin-B as well as artemisinin, both in vivo and in a cell free system. Fe², 2-oxoglutarate, and peroxidase-H2O2 enhanced the incorporations under in vitro conditions. The results suggest that arteannuic acid might be a common precursor for arteannuin-B and artemisinin synthesis.
Incorporation of 13C-labeled glycerol or pyruvate into the ubiquinone Q8 of Escherichia coli mutants lacking enzymes of the triose phosphate metabolism and of (U-13C6)glucose into the triterpenoids of the hopane series of Zymomonas mobilis showed that glyceraldehyde 3-phosphate (or eventually glyceraldehyde) and a C2 unit derived from pyruvate decarboxylation were the only precursors of the C5 skeleton of isoprenic units in a novel non-mevalonate pathway for isoprenoid biosynthesis in these bacteria.
2-C-Methyl-d-erythritol 4-phosphate is transformed to 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol in the presence of cytidine 5′-triphosphate by a novel Escherichia coli enzyme, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, involved in the nonmevalonate pathway.
A nonmevalonate pathway intermediate, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol, is transformed to its 2-phospho-derivative in the presence of ATP by a novel Escherichia coli enzyme, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase.
This review first summarizes the diverse nature of isoprenoids found in plants, emphasizing the wide range of physiological functions these compounds serve. The biosynthetic origins of isoprenoids have occupied chemists and biochemists for decades, and the second section of this review recaps some of the conceptual models used to rationalize key biosynthetic reactions in the isoprenoid pathway. The third section describes briefly some of the recently developed experimental systems that have helped researchers uncover much of the biochemistry and molecular biology of isoprenoids. The fourth section compares the deduced amino acid sequences of enzymes with similar catalytic functions and attempts to correlate these sequences with the known enzymology. The fifth and final section focuses on our limited understanding of how isoprenoid biosynthesis is regulated in plants.
In the early times of isoprenoid research, a single pathway was found for the formation of the C5 monomer, isopentenyl diphosphate (IPP), and this acetate/mevalonate pathway was supposed to occur ubiquitously in all living organisms. Now, 40 years later, a totally different IPP biosynthesis route has been detected in eubacteria, green algae and higher plants. In this new pathway glyceraldehyde 3-phosphate (GAP) and pyruvate are precursors of isopentenyl diphosphate, but not acetyl-CoA and mevalonic acid. In green tissues of three higher plants it was shown that all chloroplastbound isoprenoids (β-carotene, phytyl chains of chlorophylls and nona-prenyl chain of plastoquinone-9) are formed via the GAP/pyruvate pathway, whereas the cytoplasmic sterols are formed via the acetate/mevalonate pathway. Also, isoprene, emitted by various plants at high light conditions by action of the plastid-bound isoprene synthase, is formed via the new GAP/pyruvate pathway. Thus, in higher plants, there exist two separate and biochemically different IPP biosynthesis pathways: (1) the novel alternative GAP/pyruvate pathway apparently bound to the plastidic compartment and (2) the classical cytoplasmic acetate/mevalonate pathway. This new GAP/pyruvate pathway for IPP formation allows a reasonable interpretation of previous odd results concerning the biosynthesis of chloroplast isoprenoids, which, so far, had mainly been interpreted assuming compartmentation differences. The novel GAP/pyruvate pathway for IPP formation in plastids appears as a heritage of their prokaryotic, endosymbiotic ancestors.
Tomato plants transformed with a copy of the fruit-expressed phytoene synthase cDNA under control of the CaMV 35S promoter showed ectopic production of carotenoids. High expressers were reduced in stature. The dwarf character was inherited with an inverse relationship between expression of phytoene synthase and plant height. Severely affected plants also showed reduced chlorophyll content in young leaves. These dwarfs showed a 30-fold reduction in levels of gibberellin A1 (GA1) and growth was partially restored by treatment with exogenous GA3. Qualitative and quantitative changes in carotenoids were also found. It is proposed that the dwarf phenotype results from the over-production of phytoene synthase, which converts geranylgeranyl diphosphate to phytoene and thereby diverts this intermediate away from the gibberellin (GA) and phytol biosynthetic pathways.
The subcellular compartmentation of isopentenyl diphosphate (IPP) synthesis was examined in secretory cells isolated from glandular trichomes of peppermint (Mentha x piperita L. cv. Black Mitcham). As a consequence of their anatomy and the conditions of their isolation, the isolated secretory cells are non-specifically permeable to low-molecular-weight water-soluble metabolites. Thus, the cytoplasm is readily accessible to the exogenous buffer whereas the selective permeability of subcellular organelles is maintained. With the appropriate choice of exogenous substrates, this feature allows the assessment of cytoplasmic and organellar (e.g. plastidic) metabolism in situ. Glycolytic substrates such as [14C]glucose-6-phosphate and [14C]pyruvic acid are incorporated into both monoterpenes and sesquiterpenes with a monoterpene:sesquiterpene ratio that closely mimics that observed in vivo, indicating that the correct subcellular partitioning of these substrates is maintained in this model system. Additionally, exogenous [14C]mevalonic acid and [14C]IPP, which are both intitially metabolized in the cytoplasm, produce an abnormally high proportion of sesquiterpenes. In contrast, incubation with either [14C]citrate or [14C]acetyl-CoA results in the accumulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) with no detectable isoprenoids formed. Taken together, these results indicate that the cytoplasmic mevalonic acid pathway is blocked at HMG-CoA reductase and that the IPP utilized for both monoterpene and sesquiterpene biosynthesis is synthesized exclusively in the plastids.
A transgenic system was developed for Artemisia annua L. via Agrobacterium rhizogenes-mediated transformation. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS) placed under a CaMV 35S promoter
was transferred into Artemisia annua using Agrobacterium rhizogenes strain ATCC15834. Among the 150 hairy root lines established, 16 lines showed resistance to kanamycin (20 mg l-1). The intergration of FDS gene was confirmed by PCR and Southern blot analysis, and analysis of Northern blot revealed that
the foreign FDS gene was expressed at the transcriptional level in three hairy root lines (F-1, F-24 and F-26 root line).
F-1, F-24 and F-26 root lines grew faster than the control hairy root line. However, on the MS medium growth of F-26 root
line was abnormal in that callus frequently formed. Analysis of artemisinin demonstrated that about 2–3 mg g-1 DW of artemisinin were then detected in the three root lines, which is about 3–4 times higher than that in the control hairy
roots.
In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5′-untranslated end, and a 427-bp 3′-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC–MS analysis identified the olefins as (E)-β-farnesene (0.8%), amorpha-4,11-diene (91.2%), amorpha-4,7(11)-diene (3.7%), γ-humulene (1.0%), β-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and α-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 μM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.
An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A. annua via A. tumefaciens strain LB4404. Leaf or leaf discs were used as explants to be infected with A. tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots. Forty-five lines of resistance kanamycin shoots transformed with FDS were established. Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive. Southern blot analysis suggested the foreign FDS gene had been integrated into the A. annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines). Analysis of artemisinin demonstrated that about 8∼10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2–3 times higher than that in the control.
Chamomile sesquiterpenes were labelled with 13C by injection of an [1-13C]glucose solution into the anthodia of the plant. The sesquiterpenes bisabololoxide A and chamazulene were isolated from the hydrodistillate of the labelled flowers. Analysis of the labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that two of the isoprene building blocks were predominantly formed via the new triose\pyruvate pathway, whereas the third unit was of mixed origin, being derived from both the mevalonic acid pathway and the triose\pyruvate pathway.
Intact tubers of potato ( Solanum tuberosum L. cv. Irish Cobbler and an interspecific hybrid between S. tuberosum and S. demissum cv. Rishiri) contain a very low activity of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. The activity increased first in response to slicing, and again in response to additional treatments such as inoculation with an incompatible race of Phytophthora infestans , application of a hyphal wall component of the fungus or HgCl 2 solution, and then decreased. Both the first and the second increases in activity in response to slicing and additional treatment with a hyphal wall component to elicit phytoalexin production were inhibited by blasticidin S. Properties of HMG-CoA reductase induced by slicing and by additional treatment with HgCl 2 or fungal inoculation were investigated.
Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.
The herb Artemisia annua has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria.
In 1971, Chinese chemists isolated from the leafy portions of the plant the substance responsible for its reputed medicinal
action. This compound, called qinghaosu (QHS, artemisinin), is a sesquiterpene lactone that bears a peroxide grouping and,
unlike most other antimalarials, lacks a nitrogen-containing heterocyclic ring system. The compound has been used successfully
in several thousand malaria patients in China, including those with both chloroquine-sensitive and chloroquine-resistant strains
of Plasmodium falciparum. Derivatives of QHS, such as dihydroqinghaosu, artemether, and the water-soluble sodium artesunate,
appear to be more potent than QHS itself. Sodium artesunate acts rapidly in restoring to consciousness comatose patients with
cerebral malaria. Thus QHS and its derivatives offer promise as a totally new class of antimalarials.
Flower colour is determined primarily by the production of pigments, usually anthocyanins or carotenoids, but the shade and intensity of the colour are often changed by other factors such as vacuolar compounds, pH and metal ions. Pigmentation can also be affected by the shape of epidermal cells, especially those facing prospective pollinators. A conical shape is believed to increase the proportion of incident light that enters the epidermal cells, enhancing light absorption by the floral pigments, and thus the intensity of their colour. We have identified a gene (mixta) that affects the intensity of pigmentation of epidermal cells in Antirrhinum majus petals. The cells of the corolla lobes fail to differentiate into their normal conical form in mixta mutants. We have cloned the mixta gene by transposon tagging; its sequence reveals that it encodes a Myb-related protein that probably participates in the transcriptional control of epidermal cell shape.
Arteannuin B, which co-occurs with artemisinin, the potent antimalarial principle of the Chinese medicinal herb Artemisia annua (Asteraceae), has been converted to the latter using crude and semi-purified cell-free extracts of the leaf homogenates of the plant. Detection procedures to quantitate this bioconversion, including one that is novel which uses gcms, are detailed.
How much vitamin E is enough? An established use of supplemental vitamin E in humans is in the prevention and therapy of deficiency symptoms. The cause of vitamin E deficiency, characterized by peripheral neuropathy and ataxia, is usually malabsorption-a result of fat malabsorption or genetic abnormalities in lipoprotein metabolism. Genetic abnormalities in the hepatic alpha-tocopherol transfer protein also cause vitamin E deficiency-defects in this protein cause an impairment in plasma vitamin E transport. Impaired delivery of vitamin E to tissues, thereby, results in deficiency symptoms. Also discussed is the use of supplemental vitamin E in chronic diseases such as ischemic heart disease, atherosclerosis, diabetes, cataracts, Parkinson's disease, Alzheimer's disease, and impared immune function, as well as in subjects receiving total parenterol nutrition. In healthy individuals, a daily intake of about 15-30 mg of alpha-tocopherol is recommended to obtain "optimal plasma alpha-tocopherol concentrations" (30 microM or greater).
Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids. By labeling experiments using [1-(13)C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (beta-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.
For more than three centuries we have relied on the extracts of the bark of Cinchona species to treat malaria. Now, it seems we may be changing to the leaves of a Chinese weed, Artemisia annua, and its active compound artemisinin. Artemisinin-derived drugs have been proved particularly effective treatments for severe malaria, even for multidrug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on a global scale. Therefore, many research groups have directed their investigations toward the enhancement of artemisinin production in A. annua cell cultures or whole plants in order to overproduce artemisinin or one of its precursors. This article provides a brief review of the state of art of the different aspects in A. annua research.
For thousands of years, physicians in China have treated fever with a decoction of the plant qinghao (1). In 1972 Chinese chemists isolated the active febrifuge from this plant, determining its structure, 1, by single crystal x-ray crystallography (2, 3). The discovery was timely, for the world sorely needs a better treatment for malaria. More than 270 million people suffer from the disease, two to three million dying each year. The majority of the deaths are of children under 5 years of age, who are especially sensitive because of their lack of immunity to the disease.
Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.
Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.
Recent studies have uncovered the existence of an alternative, non-mevalonate pathway for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the two building blocks of terpene biosynthesis.
Dihydroartemisinic acid (2) was isolated as a natural product from Artemisia annua in a 66% yield, and its structure was confirmed by 1H and 13C NMR spectroscopy. Compound 2 could be chemically converted to artemisinin (4) under conditions that may also be present in the living plant. The results suggest that the conversion of 2 into 4 in the living plant might be a nonenzymatic conversion.
Two distinct pathways are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids. The classical acetate/mevalonate pathway operates in the cytosol, whereas plastidial isoprenoids originate via a novel mevalonate-independent route that involves a transketolase-catalyzed condensation of pyruvate and D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate as the first intermediate. Based on in vivo feeding experiments, rearrangement and reduction of deoxyxylulose phosphate have been proposed to give rise to 2-C-methyl-D-erythritol-4-phosphate as the second intermediate of this pyruvate/glyceraldehyde-3-phosphate pathway (1-3). The cloning of an Escherichia coli gene encoding an enzyme capable of converting 1-deoxy-D-xylulose-5-phosphate to 2-C-erythritol-4-phosphate was recently reported (4). A cloning strategy was developed for isolating the gene encoding a plant homolog of this enzyme from peppermint (Mentha x piperita), and the identity of the resulting cDNA was confirmed by heterologous expression in E. coli. Unlike the microbial reductoisomerase, the plant ortholog encodes a preprotein bearing an N-terminal plastidial transit peptide that directs the enzyme to plastids where the mevalonate-independent pathway operates in plants. The peppermint gene comprises an open reading frame of 1425 nucleotides which, when the plastidial targeting sequence is excluded, encodes a deduced enzyme of approximately 400 amino acid residues with a mature size of about 43.5 kDa.