Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH₄ ⁺/NO₃ ^? balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH₄ ⁺/NO₃ ^? and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.     

Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants

Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anti-cancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventitious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0???M 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0???M BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4???M) and IAA (5.7???M). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40???mol?m^?2?s^?1). The camptothecin content was determined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a considerable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction.     

Heterologous overexpression of Nothapodytes foetida strictosidine synthase enhances levels of anti-cancer compound camptothecin in Ophiorrhiza rugosa

Plant Cell, Tissue and Organ Culture (PCTOC) - Nothapodytes foetida, an endangered tree of Indian origin, is a major source of the anti-cancer monoterpenoid indole alkaloid, camptothecin (CPT)....     

Genetic Structure,Diversity and Long Term Viability of a Medicinal Plant,Nothapodytes nimmoniana Graham. (Icacinaceae), in Protected and Non-Protected Areas in the Western Ghats Biodiversity Hotspot

Background and Question

The harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species.

Methodology/Principal Findings

The analysis of adults and seedlings of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity (H_e = 0.816, H_o = 0.607, A = 18.857) than populations in adjoining non-protected areas (H_e = 0.781, H_o = 0.511, A = 15.571). Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (H_o = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (H_o = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years.

Conclusions/Significance

Overall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species.     

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.     

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.     

Influence of medium composition on callus induction and camptothecin(s) accumulation in Nothapodytes foetida

Camptothecin(s) production was examined in callus cultures derived from cotyledons of Nothapodytes foetida (Weigh) Sleumer. The calluses were grown on various combinations of Murashige and Skoog's basal media supplemented with auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a-napthalene acetic acid (NAA) and indole-3-acetic acid (IAA) with 6-benzyl aminopurine (BA)/kinetin in different concentrations. The presence of camptothecin (CPT) and 9-methoxycamptothecin (9-OMeCPT) were analyzed by HPLC in relation to the media composition. Hyper production of CPT(1.306% on dry wt. basis) was observed with a combination of 2,4-D with BA and 2,4,5-T and NAA in 1-month-old callus.     

Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.     

Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.)

Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3′ region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product.     

Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase.

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459).     

Molecular characterization of pheromone biosynthesis activating neuropeptide from the diamondback moth, Plutella xylostella (L.)

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, -NP, β-NP and γ-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-β-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas γ-NP (10 amino acids in length) was the longest among examined γ-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1 h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.     

Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.)

Tripterygium wilfordii Hook.f., known as Leigongteng (Thunder God Vine) in traditional Chinese medicine, has attracted much attention for its applications in relieving autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, and for treating cancer. Molecular analyses of the ITS and 5S rDNA sequences indicate that T. hypoglaucum and T. doianum are not distinct from T. wilfordii, while T. regelii should be recognized as a separate species. The results also demonstrate potential value of rDNA sequence data in forensic detection of adulterants derived from Celastrusangulatus in commercial samples of Leigongteng.     

Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Both these proteins contain ∼100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I , but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.