Diamine Oxidase from Cultured Roots of Hyoscyamus niger: Its Function in Tropane Alkaloid Biosynthesis

Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective K_m values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants V_max/K_m for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.     

Genetic and physiological analysis of biennialism in Hyoscyamus niger

A genetic and physiological study of biennialism in the diploid selfer Hyoscyamus niger (black henbane), an obligate long-day plant, is described. Three annual and two biennial accessions that were homozygous for their respective growth habits were selected. The early-flowering trait of two annual accessions was dominant over the late-flowering trait of the third annual accession. The late-flowering annual accession, but not the early-flowering ones, responded to vernalization. Two biennial accessions remained vegetative after more than 1 year in soil and thus had an obligate vernalization requirement. Crosses between annual and biennial accessions showed that biennialism was conferred through a single dominant gene. However, plants containing only one copy of this dominant gene were transformed from biennials into very late-flowering winter-annual plants that responded more rapidly to vernalization than biennials. Taken together, these results indicated that there were allelic differences in photoperiod-specific flowering time genes and that biennialism was a dose-dependent trait with incomplete dominance. Models for flowering time regulation in henbane involving photoperiod-, vernalization-, and most likely gibberellin-specific pathways are discussed.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

Production of scopolamine by normal root culture of Hyoscyamus niger

Summary Normal root of Hyoscyamus niger was cultivated in various nutrient conditions to optimize root growth and scopolamine production. Nitrate or phosphate concentration had little effect on root growth, while the low levels of nitrate or phosphate enhanced specific scopolamine content by 44% and 39%, respectively. Glucose or fructose was not adequate for the root culture as a carbon source, while the root growth was markedly enhanced at high sucrose concentration (up to 9%). The growth rate (0.59g dw./L.d.) and scopolamine productivity (1.98 mg/L.d.) was increased greater in roots with 9% initial sucrose concentration as compared to 3% sucrose (0.28 g dw./L.d., 0.98 mg/L.d.)     

Organization and Alkaloid Production in Tissue Cultures of Hyoscyamus niger

Callus and cell suspension cultures were established from Hyoscyamusniger seedlings and their growth requirements investigated.Stock suspensions were more aggregated in the earlier passagesbut became finer as the cultures were serially sub-cultured.On transfer to auxin-free medium, the cultures formed rootsand shoots were rarely noted. Callus initiated from differentparts of the seedlings showed no differences in alkaloid levels.Alkaloid contents were slightly increased on transfer to auxin-freemedium. Both alkaloid content and composition changed duringserial propagation. Different parts of the intact plant werealso analysed for alkaloids.     

茄科马尿泡和天仙子的花器官发生

利用扫描电镜研究了茄科 (Solanaceae)天仙子族 (Hyoscyameae)中国特有属马尿泡属 (PrzewalskiaMaxim .)马尿泡 (PrzewalskiatanguticaMaxim .)和天仙子属 (HyoscyamusL .)天仙子 (HyoscyamusnigerL .)的花器官发生和发育 ,研究结果表明 :马尿泡和天仙子花器官的发生和发育具有以下 3个共同特征 :1)符合Hofmeister规律 ,即新器官的发生首先出现在花顶已经存在的器官之间 ;2 )花冠的发育模式符合茄科植物所具有的“后合瓣”(“latesympetaly”)现象 ,即花瓣单独发生但后来又通过它们基部分生组织的融合而连合起来 ;3)花被五基数且花器官原基发生顺序为向心发育。但是它们的花萼原基具有不同的发生方式。天仙子花萼裂片原基的发生方式为环状发生 ;马尿泡花萼裂片原基的发生方式为螺旋状发生 ,但 5个花萼裂片原基在都出现后就连成了一个环。马尿泡是介于天仙子属和山莨菪属之间的类群 ,它比天仙子属原始但较山莨菪属进化。     

Phytochrome Regulation of Flowering in the Long-Day Plant, Hyoscyamus niger

A daylength extension with incandescent light is more effective in promoting flowering of long-day plants like Hyoscyamus niger than fluorescent light. A low phytochrome photoequilibrium (Pfr/P_tot), attained by a far-red irradiation at the close of long days under fluorescent light, also promotes flowering. Moreover, if flower initiation processes are initiated by several long days, a low phytochrome photoequilibrium at the end of short, postinduction photoperiods also enhances flowering. The initiation phase of flowering requires Pfr to be present whereas the development phase proceeds more rapidly in the absence of Pfr. Spectral dependence studies, therefore, could be misinterpreted if the initiation and development stages are combined into a single audit of flowering.     

茄科马尿泡和天仙子的花器官发生

利用扫描电镜研究了茄科(Solanaceae)天仙子族(Hyoscyameae)中国特有属马尿泡属( Przewalskia Maxim.)马尿泡( Przewalskia tangutica Maxim.)和天仙子属( Hyoscyamus L.)天仙子( Hyoscyamus niger L.)的花器官发生和发育,研究结果表明:马尿泡和天仙子花器官的发生和发育具有以下3个共同特征:1)符合Hofmeister规律, 即新器官的发生首先出现在花顶已经存在的器官之间;2)花冠的发育模式符合茄科植物所具有的"后合瓣"("late sympetaly")现象,即花瓣单独发生但后来又通过它们基部分生组织的融合而连合起来;3)花被五基数且花器官原基发生顺序为向心发育.但是它们的花萼原基具有不同的发生方式.天仙子花萼裂片原基的发生方式为环状发生;马尿泡花萼裂片原基的发生方式为螺旋状发生,但5个花萼裂片原基在都出现后就连成了一个环.马尿泡是介于天仙子属和山莨菪属之间的类群,它比天仙子属原始但较山莨菪属进化.     

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus: n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K_m values for putrescine and S-adenosyl-l-methionine are 277 and 203 μm, respectively, and the K_i value for S-adenosyl-l-homocysteine is 110 μm. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K_i values of 11.0, 9.1, and 10.0 μm. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6β-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.     

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A_615-630 indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 μmol of nitrite formed per min per mg of protein. The cytochrome-containing NR_I separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa α-subunit, a 64-kDa β-subunit, and a 23-kDa γ-subunit with relative band intensities indicative of a 1:1:1 α/β/γ subunit ratio and a M_r of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.     

Molecular cloning and expression profile of a jasmonate biosynthetic pathway gene allene oxide cyclase from Hyoscyamus niger

Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named as tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, [...] EC 5.3.99.6 [...]) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession: AY708383), which was 1044 bp long, with a 747 bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multi-copy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis ofjasmonates.     

Effects of Culture Conditions on Tropane Alkaloid Formation in Hyoscyamus niger Suspension Cultures

Tropane alkaloid formation was studied under various culture conditions in suspension cultures of Hyoscyamus niger L. High aeration increased cell growth and the contents of hyoscyamine and scopolamine. White’s medium inhibited cell growth but increased the hyoscyamine content. The low concentrations of nitrogen and phosphate in White’s medium were responsible for such effects. NAA promoted cell growth but inhibited hyoscyamine formation. A hyoscyamine content of 0.05% dry weight (DW) (0.14 mg in 25 ml of culture medium) was achieved in modified LS medium in which the phosphate concentration was decreased to 0.2 mm and the auxin omitted.

Effects of precursor feeding on alkaloid formation were also studied with the putative precursors of hyoscyamine. N-Methylputrescine, tropine, phenylalanine, and tropic acid increased alkaloid formation, but the promotive effect of these precursors varied from one experiment to another.     

Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger

Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger.     

Induced polygenic changes occurring simultaneously with major gene changes in black henbane (Hyoscyamus niger L.)

Summary Two macromutants [unbranched (Ub) and restricted branched (Rb)], one point mutation [yellow flowerd (Ada)] and two flowering variants (early and late) were isolated in the M₂ generation of gammairradiated progenies of black henbane (H. niger) — a medicinal plant belonging to family Solanaceae. These major gene changes were coupled with simultaneous changes in several polygenic systems conditioning continuous variation for flowering time, plant height, numbers of shoots and leaves, biomass and crude drug content. Such quantitative variation in qualitative mutants were measured for both per se means and cross-bred performance in terms of: (1) shifts in univariate means, (2) changes in pooled divergence, (3) alterations in gca effects, and (4) changes in heterotic capacity. The mutants were significantly divergent from the parental base for all the six metric traits, both individually as well as collectively. Divergence among mutants was wider (D² = 1491 to 11 469) than that for mutants versus source parent (D² = 117 to 7120). Conspicuous alterations in both the nature and the magnitude of the gca effects associated with all mutants, except L-2013 (late flowering), relative to the parental base were observed for all of the metric traits examined (gca - per se means). Similarly, the mutants manifested greater heterotic potential than the parental base. This is even more strongly reflected in mutant x mutant hybrids for all of the characters. The amount of economic heterosis for diverse traits followed the pattern: crude drug > biomass > flowering time - plant height > number of shoots - number of leaves.     

Studies of the Involvement of an Endogenous Rhythm in the Photoperiodic Response of Hyoscyamus niger

An attempt was made to determine the involvement of an endogenous circadian rhythm in the flowering response of the long-day plant Hyoscyamus niger L. grown in a modified White's medium. Both variable-cycle-length and light interruption experiments were employed in this attempt. In the variable-cycle experiments, plants were subjected to light periods of 6, 12, or 18 hours followed by varying lengths of darkness. The total lengths of the cycles varied from 12 to 72 hours. In experiments utilizing a 6-hour photoperiod, a high level of flowering occurred in cycle lengths of 12, 36, and 60 hours. Flowering was suppressed in the 24-, 48-, and 72-hour cycles. When a 12-hour photoperiod was used the flowering response was low between 24 and 36 hours and flowering did not indicate a rhythmic response. When an 18-hour photoperiod was used, the flowering response was suppressed in the 36- and 60-hour cycles.

Light-break experiments were conducted to study further the flowering response in Hyoscyamus. These experiments consisted of a 6-hour main photoperiod followed by varying lengths of darkness to make cycles of 24, 48, and 72 hours. At given intervals the dark period was interrupted by 2-hour light breaks. In a 24-hour cycle, flowering was promoted when a light break was given at either the twelfth or eighteenth hour of the cycle. In a 48-hour cycle, flowering was strongly promoted by light breaks given near the beginning or at the end of the dark period. In a 72-hour cycle, light breaks given at the eighteenth, forty-second, and sixty-sixth hour of the cycle stimulated flowering as compared with light breaks given at the thirtieth and fifty-fourth hour. These results are indicative of the involvement of an endogenous rhythm in the flowering response of Hyoscyamus niger.