[Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna].

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 µmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 µmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 µmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.

Plants are a potential source of a large number of valuable secondary metabolites. In vitro cultures are being considered as an alternative to agricultural processes for studying valuable secondary metabolites. In this way, nutritive factors are important parameters influencing the production of these compounds in plants. Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine, and the growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were investigated. As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis. A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834. The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content. The alkaloid concentrations obtained in the hairy roots were 3-20 times higher than that in the plants at 35 mM of KNO3. Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants.

Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets.

Hyoscyamine and scopolamine tropane alkaloids found in several solanaceous plants are anticholinergic drugs. Hyoscyamine 6ß-hydroxylase (H6H) catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6ß-hydroxyhyoscyamine and the second is epoxidation of 6ß-hydroxyhyoscyamine yielding scopolamine that is the final metabolite in the tropane alkaloid biosynthetic pathway. The effects of trivalent chromium as KCr (SO4)(2) on the production of tropane alkaloids and the expression of hyoscyamine 6ß-hydroxylase gene (h6h) were studied in micro-propagated Atropa belladonna L. plantlets. The results showed that chromium treatment decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents. Moreover, semiquantitave RT-PCR analysis showed that the transcript level of H6H increased under chromium treatment. This treatment also increased hyoscyamine and scopolamine contents as shown by HPLC analysis. Changes of scopolamine contents correlate with the expression levels of h6h gene under different concentrations of chromium.

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase.

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid.

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.