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- Komaraiah, Palle, et al.
(author)
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Growth behavior in plant cell cultures based on emissions detected by a multisensor array
- 2004
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In: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 20:4, s. 1245-1250
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Journal article (peer-reviewed)abstract
- The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.
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- Brodelius, Maria, et al.
(author)
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Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
- 2002
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:14, s. 3570-3577
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Journal article (peer-reviewed)abstract
- A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.
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| 9. |
- Olsson, Mikael E., et al.
(author)
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Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L
- 2009
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In: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 70:9, s. 1123-1128
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Journal article (peer-reviewed)abstract
- A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta 11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1 alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.
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| 14. |
- Teixeira, M.C., et al.
(author)
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Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.)
- 2009
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In: Biotechnology letters. - : Springer Science and Business Media LLC. - 0141-5492 .- 1573-6776. ; 31:7, s. 1089-1101
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Journal article (peer-reviewed)abstract
- Two full-length cDNA clones of PoleFAD2 and one full-length cDNA clone of PoleFAD6, encoding omega-6 fatty acid desaturases, the key enzymes for the conversion of oleic into linoleic acid, were isolated from purslane (Portulaca oleracea L.) leaves and seeds. The deduced amino acid sequence of both isoforms of PoleFAD2 showed higher similarities to other microsomal omega-6 desaturases then to PoleFAD6 or other plastidial orthologues, and vice versa. Expression analysis by RT-PCR showed that all genes are expressed in all tissues of purslane tested, but higher levels of mRNA accumulation were detected in reproductive organs and cells that proliferate rapidly or store lipids. Wounding affected the levels of mRNA accumulation of both, FAD2 and FAD6 genes in purslane leaves, while chilling stress affected only FAD2 transcript level. The expression patterns observed reflect the discrete roles of these genes in membrane synthesis for cell division, thylakoid development, and lipid storage or in the biosynthetic pathway for the production of signaling molecules that influence plant development or defense.
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| 20. |
- Heimgartner, U, et al.
(author)
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Purification and Partial Characterization of Milk Clotting Proteases from Flowers of Cynara cardunculus
- 1990
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In: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 29:5, s. 1405-1410
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Journal article (peer-reviewed)abstract
- Three proteases (cynarases 1, 2 and 3) with milk-clotting activity have been purified from dried flowers of Cynara cardunculus. The proteases are each composed of one large and one small subunit. The native Mr of the dimeric proteins is 49 000. The three proteases are glycoproteins containing N-linked high mannose type glycans. Cynarase 3 shows the highest proteolytic and milk-clotting activity. All three enzymes express maximum activity at pH 5.1. Inhibitor studies indicate that the cynarases are of the aspartic acid type. Antibodies raised against the large subunit of cynarase 3 cross-reacts with the large subunits of the other two cynarases after destruction of the glycan structure by periodate oxidation.
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