ABSTRACT
A new cDNA encoding hyoscyamine 6beta-hydroxylase (H6H, EC 1.14.11.11), a bifunctional enzyme catalyzing the last two steps in the biosynthesis of scopolamine, was isolated from Atropa baetica roots (GenBank accession no. EF442802). The full cDNA sequence showed an ORF of 1035bp, coding for a protein with 344 amino acid residues. Sequence analyses at the nucleotide level showed that this ORF shares high identity with other H6H from different plant species, such as Anisodus tanguticus and Hyoscyamus niger with 90% identity, and an almost total identity with A. belladonna (98%). Tissue expression analyses showed that the gene transcript was tissue dependent, appearing exclusively in roots, thus being the only biosynthetic site for the production of scopolamine. Furthermore, Southern hybridization experiments revealed that this gene was not part of a multigene family as appears in low copy number. Phylogenetic tree analysis indicated that A. baetica H6H had a very close relationship with A. belladonna and to a lesser extent with H. niger.
Subject(s)
Atropa/genetics , Mixed Function Oxygenases/genetics , Atropa/enzymology , Base Sequence , DNA, Complementary , Gene Expression Profiling , Mixed Function Oxygenases/isolation & purification , Molecular Sequence Data , Phylogeny , Plant Proteins/classification , Plant Roots/enzymology , Plant Roots/genetics , Scopolamine/biosynthesis , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Elicitation of transgenic Atropa baetica overexpressing the h6h gene with salicylic acid (SA), acetylsalicylic acid (ASA),or methyl jasmonate (MeJ) was conducted to boost tropane alkaloid yields. Scopolamine (1) amounts increased after treatment with ASA and MeJ, but not with SA. The highest enhancement of 1 was achieved with MeJ followed by ASA dissolved in EtOH. Transcriptomic analyses showed a direct relationship between content of 1 and gene expressions;the engineered h6h gene and other biosynthetic genes were stimulated. ASA dissolved in EtOH showed a high h6h gene expression, increasing 25-fold and 5-fold compared to controls; tr-I also displayed a 5-fold increase. The controls to which EtOH was added showed a 5-fold increase in h6h gene expression and 125-fold for pmt, demonstrating that EtOH also functioned as an enhancer of 1. MeJ was the best elicitor, displaying a 25-fold increase in h6h expression level, not affecting the expression of the other three genes analyzed, and it appears to possibly stimulate the phenylpropanoids branch of the tropane alkaloid pathway.
Subject(s)
Alkaloids/metabolism , Atropa/metabolism , Plant Roots/metabolism , Plants, Genetically Modified/metabolism , Salicylates/metabolism , Scopolamine/analysis , Tropanes/metabolism , Animals , Atropa/genetics , Gene Expression , Molecular Structure , Phenylpropionates/metabolism , Plants, Genetically Modified/genetics , Rhizobium/geneticsABSTRACT
Agrobacterium-mediated transformation of leaf tissues of Atropa belladonna with an adenosine 5'-diphosphate (ADP)-ribosylation factor gene of carrot, arf-001, was performed employing pBCR82 as an expression vector. This vector co-expresses rol gene cluster together with arf-001, and thus, the transformed host cells were obtained as hairy roots. Two cell lines of the transformed belladonna were established as the liquid cultures of hairy root tissues, and expression of arf-001 and accumulation of its product in the cells were confirmed by RT-PCR and Western blot analyses, respectively. A marked increase in extracellular protein concentrations was observed in the transformed belladonna root cultures as compared with the controls transformed with an empty vector. However, the secretion of the proteins of the transformants was markedly reduced in the presence of a physiological concentration of monensin. These results suggest that over-expression of arf-001 in belladonna results in the enhancement of secretory activity in the transformed cells.
Subject(s)
ADP-Ribosylation Factors/biosynthesis , ADP-Ribosylation Factors/genetics , Atropa/chemistry , Atropa/genetics , Blotting, Western , Extracellular Space/drug effects , Extracellular Space/metabolism , Gene Expression/drug effects , Genetic Vectors , Plant Roots/chemistry , RNA, Plant/biosynthesis , RNA, Plant/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rhizobium/geneticsABSTRACT
In order to investigate the production of tropane alkaloids by hairy roots of Atropa baetica, transgenic for the gene h6h encoding the enzyme hyoscyamine 6beta-hydroxylase, solvent extraction with chloroform and with dichloromethane of the metabolites present in the liquid medium and in the root tissue was compared. The extraction of scopolamine from the liquid medium was equally effective with either solvent, giving maximum values of around 850 microg/flask. For the roots, three different extraction methods were employed: A, employing chloroform:methanol: (25%) ammonia (15:5:1) for initial extraction, followed by treatment with sulfuric acid and ammonia, and using chloroform for the final extraction and washes; B, as A but using dichloromethane for extraction and washes; and C, as B but substituting chloroform for dichloromethane in the extraction cocktail. Scopolamine was the most abundant metabolite (present in amounts of 3250-3525 microg/g dry weight) and presented similar extraction efficiencies with all of the extraction methods employed. The highest amounts of hyoscyamine and the intermediate 6beta-hydxoxyhyoscyamine were present on day 31 (800 and 975 microg/g dry weight, respectively) and no statistical differences between the three extraction methods employed were detected. This study confirms that, for the extraction of tropane alkaloids, dichloromethane can replace the commonly employed chloroform, the use of which incurs major health, security and regulation problems.
