Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures

Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA₃ enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical hairy root phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.In transformed roots of Cucumis sativus, the phenotype of the roots is influenced by the expression of auxin synthesis genes on T_R-DNA resulting in roots with two distinct morphologies. The pattern of expression of the enzyme ascorbate oxidase in populations of control roots of different morphologies is described. The significance of these phenotypic variations on the utility of transformed root cultures for the study of secondary metabolic pathways will be discussed.Abbreviations AO ascorbate oxidase - DW dry weight - FW fresh weight - GA₃ gibberellic acid     

Enhanced production of tropane alkaloids in <Emphasis Type="Italic">Scopolia parviflora</Emphasis> by introducing the <Emphasis Type="Italic">PMT</Emphasis> (putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferase) gene

Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane alkaloid levels were the highest in the root (0.9 mg g⁻¹ on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR) and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large yields of tropane alkaloids. These authors contributed equally to this paper (co-first authors).     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)- is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK- on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacterium rhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK- for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10^–7 or 10^–8 M PSK-. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK- was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK- influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Growth,graviresponsiveness and abscisic-acid content of Zea mays seedlings treated with Fluridone

Ten-d-old seedlings of Zea mays L. cv. Tx 5855 treated with 1-methyl-3-phenyl-5-(3-[trifluoromethyl]phenyl)-4-(1H)-pyridinone (Fluridone) were analyzed for abscisic acid (ABA) content using high-performance liquid chromatography with an analysis sensitivity of 2.5 ng ABA g^-1 fresh weight (FW). Seedlings were divided into three portions: leaves, detipped roots, and root tips (terminal 1.5 mm). Control plants (water treatment only; no Fluridone) were characterized by the following amounts of ABA: leaves, 0.114±0.024 (standard deviation) g ABA g^-1 FW; detipped roots, 0.260±0.039±g ABA g^-1 FW; root tips, no ABA detected. We did not detect any ABA in tissues of Fluridone-treated plants. Primary roots of treated and untreated seedlings were strongly graviresponsive, with no significant differences between the curvatures or the growth rates of primary roots of Fluridone-treated and control seedlings. These results indicate that 1) Fluridone completely inhibits ABA synthesis, and 2) ABA is not necessary for positive gravitropism by primary roots of Zea mays.Abbreviations ABA abscisic acid - Fluridone 1-methyl-3-phenyl-5-(3-[trifluoromethyl]phenyl)-4-(1H)-pyridinone - FW fresh weight - SD standard deviation     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Reduced gravitropic sensitivity in roots of a starch-deficient mutant ofNicotiana sylvestris

The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the -amino group or that decarboxylate -N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical -N-methylornithine.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - PCA perchloric acid - PMT putrescine N-methyltransferase     

Effect of cell-cycle inhibition on synthesis of steroidal alkaloids by Solanum aviculare plant cells

Summary Addition of 40 mg l^-1 hydroxyurea to Solanium aviculare cultures resulted in complete inhibition of growth and an increase in the levels of steroidal alkaloids in the biomass. These results demonstrate that solasodine synthesis and growth can be uncoupled, and suggest that steroidal-alkaloid production can be significantly enhanced by inhibiting growth at high cell density. Treatment with 1 mg l^-1 abscisic acid had a negligible effect on growth and product synthesis.     

Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop.

Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L^–1 myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 g g^–1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.Abbreviations MS Murashige and Skoog's (1962) medium     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathway

Excised root cultures of Gloriosa superba reached 7.5 g dry wt l^–1 and accumulated 240±40 g colchicine g^–1 cell dry wt after 4 weeks growth. While all precursors (except trans-cinnamic acid) enhanced colchicine content of root cultures without adversely affecting root growth, treatment with p-coumaric acid + tyramine (each at 20 mg l^–1) increased colchicine content to 1.9 mg g^–1 cell dry wt.     

The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkloids and anthraquinones

The influence of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth and the production of alkaloids and anthraquinones in cell suspension cultures of Cinchona ledgeriana Moens was studied. It was found that maximum growth and maximum alkaloid yield were obtained with a B₅-medium containing the normal level of nitrate and 4% sucrose, whereas the anthraquinone yield was maximum at 8% sucrose at the normal level of nitrate. Maximum contents of secondary metabolites, expressed as g.gDW^-1, were found using a medium containing 2% sucrose, but four times the normal nitrate concentration.     

Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2–3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l^–1 and 0.1 mg -naphthaleneacetic acid l^–1 produced more ajmaline (0.01 mg g^–1 dry wt) and ajmalicine (0.006 mg g^–1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g^–1 dry wt) and ajmalicine (0.0007 mg g^–1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.