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Docimo T.,Max Planck Institute for Chemical Ecology | Docimo T.,CNR Institute of Agricultural Biology and Biotechnology | Davis A.J.,Max Planck Institute for Chemical Ecology | Luck K.,Max Planck Institute for Chemical Ecology | And 7 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2015

Erythroxylum coca (Erythroxylaceae) is the source of the tropane alkaloid cocaine. Several lines of evidence suggest that tropane alkaloid biosynthesis in E. coca differs from that in solanaceous species, but there are many gaps in our understanding of the pathways in both groups. The development of an E. coca cell culture that produces cocaine could provide a reproducible model system for discovering novel biosynthetic genes and study pathway regulation. Calli cultures were successfully established from young leaf explants on three different media: Anderson’s Rhododendron, Gamborg B5, and modified Murashige-Tucker, all supplemented with growth regulators: 2,4-D (0.6 mg L−1), indole butyric acid (0.06 mg L−1), and benzylaminopurine (0.5 mg L−1). All accumulated cocaine and cinnamoylcocaine at levels of 0.05–0.5 nmol per gram dry weight, as determined by LC–MS, several orders of magnitude below the concentration found in the intact plant. Anderson’s Rhododendron medium supported the highest level of tropane alkaloid production, as well as the highest level of the amino acids arginine, glutamate, proline and phenylalanine, all thought to be precursors of cocaine, but contained generally lower levels of hydroxycinnamate-quinate esters, such as chlorogenic acid. These differences may be ascribed to its relatively low content of nitrate or salts, or its high content of adenine. Addition of 100 μM salicylic acid or coronalon, an analog of the bioactive jasmonic acid-isoleucine conjugate, did not result in any increase in tropane alkaloid production. These E. coca calli could provide valuable material for studies on tropane alkaloid biosynthesis and regulation. © 2014, Springer Science+Business Media Dordrecht. Source

Campos N.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | Castanon S.,Tecnalia | Urreta I.,Tecnalia | Santos M.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | Torne J.M.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB
Plant Science | Year: 2013

Transglutaminases (TGases), that catalyze post-translational modification of proteins, are scarcely known in plants. As part of a project to characterize transglutaminase genes in new plant species, the identification and characterization of a TGase in rice is presented. Using differential primers, a cDNA (tgo) of 1767bp from genomic rice DNA amplification was obtained. The primers were designed from the rice DNA sequence relatively homologous to the gene encoding active maize chloroplast TGase. Amino acid sequence of the deduced rice TGase protein (TGO) indicated that it contains the enzyme catalytic triad (Cys-His-Asp), three repeats, myristoylation domains and a leucine zipper motif. The TGO recombinant protein was characterized, showing specific activity regulation, and indicating that tgo encoded for an authentic TGase. Substrate preference and Ca2+ dependent activity were also detected. In the rice plant TGO protein was immunolocalized in the grana chloroplasts, in protein vesicles near them, and in the bulliform cells. Immunoblot analyses, tgo mRNA expression, and TGase activity indicated that TGO expression in rice was light dependent and regulated by the illumination period. This work increases significantly our plant TGase understanding. Its functional role in rice, which is a good model system for C3 plants, is discussed. © 2013. Source

Fornale S.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | Rencoret J.,CSIC - Institute of Natural Resources and Agriculture Biology of Seville | Garcia-Calvo L.,University of Leon | Capellades M.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | And 6 more authors.
Plant Science | Year: 2015

Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves. © 2015 Elsevier Ireland Ltd. Source

Campos N.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | Torne J.M.,Center for Research in Agricultural Genomics Consorci IRTA UAB UB | Bleda M.J.,CSIC - Institute of Advanced Chemistry of Catalonia | Manich A.,CSIC - Institute of Advanced Chemistry of Catalonia | And 5 more authors.
Plant Science | Year: 2014

The recently cloned rice transglutaminase gene (. tgo) is the second plant transglutaminase identified to date (Campos et al. Plant Sci. 205-206 (2013) 97-110). Similarly to its counterpart in maize (. tgz), this rice TGase was localized in the chloroplast, although in this case not exclusively. To further characterise plastidial tgo functionality, proteomic and transcriptomic studies were carried out to identify possible TGO-related proteins. Some LHCII antenna proteins were identified as TGO related using an in vitro proteomic approach, as well as ATPase and some PSII core proteins by mass spectrometry. To study the relationship between TGO and other plastidial proteins, a transcriptomic in vivo Dynamic Array (Fluidigm™) was used to analyse the mRNA expression of 30 plastidial genes with respect to that of tgo, in rice plants subjected to different periods of continuous illumination. The results indicated a gene-dependent tendency in the expression pattern that was related to tgo expression and to the illumination cycle. For certain genes, including tgo, significant differences between treatments, principally at the initiation and/or at the end of the illumination period, connected with the day/night cycling of gene expression, were observed. The tgo expression was especially related to plastidial proteins involved in photoprotection and the thylakoid electrochemical gradient. © 2014 Elsevier Ireland Ltd. Source

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