Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5ß-reductases (P5ßR and P5ßR2) did not correlate with the cardenolide increase. The expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

RNAi-mediated gene knockdown of progesterone 5ß-reductases in Digitalis lanata reduces 5ß-cardenolide content.

KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Enhanced growth and cardenolides production in Digitalis purpurea under the influence of different LED exposures in the plant factory.

In this report, we have investigated the influence of different light qualities on Digitalis purpurea under a controlled environment. For this purpose, red (R), blue (B), fluorescent lamp (FL, control), along with combined red and blue (R:B) LEDs were used. Interestingly, the plant growth parameters such as number of leaf, longest root, width of leaf, width of stomata, width of trichome, leaf area, leaf or root fresh weight (FW), weight (DW) as well as length of trichome were maximum under R:B (8:2), and significantly larger than control plants. The stomatal conductance or anthocyanin was maximum under B LED than those under FL, however the photosynthesis rate was greater under FL. RuBisCO activity was maximum under R:B (1:1) LEDs while the quantity of the UV absorbing substances was highest under R LED than under FL. The maximum amount of cardenolides were obtained from leaf tissue under R:B (2:8) LED than those under FL. The R:B LEDs light was suitable for Digitalis plant growth, development, micro- and macro-elements, as well as cardenolides accumulation in the plant factory system. The adaptation of the growth strategy developed in this study would be useful for the production of optimized secondary metabolites in Digitalis spp.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism.

PRISEs (progesterone 5ß-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5ßR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5ßR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5ßR). The three enzymes show a high degree of sequence and structure conservation in the active site. Amino acids previously considered to allow discrimination between progesterone 5ß-reductase and iridoid synthase were interchanged among rAtSt5ßR, rCrISY and rDlP5ßR applying site-directed mutagenesis. Structural homologous substitutions had different effects, and changes in progesterone 5ß-reductase and iridoid synthase activity were not correlated in all cases. Our results help to explain fortuitous emergence of metabolic pathways and product accumulation. The fact that PRISEs are found ubiquitously in spermatophytes insinuates that PRISEs might have a more general function in plant metabolism such as, for example, the detoxification of reactive carbonyl species.

The Foxgloves (Digitalis) Revisited.

This review provides a renewed look at the genus Digitalis. Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used ("Digitalis" and "Foxglove" were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here. Intriguing new results emerged from studies related to the phylogeny and taxonomy of the genus as well as to the biosynthesis and potential medicinal uses of the key active compounds, the cardiac glycosides. Several Eastern and Western Foxgloves were studied with respect to their propagation in vitro. In this context, molecular biology tools were applied and phytochemical analyses were conducted. Structure elucidation and analytical methods, which have experienced less exciting progress, will not be considered here in great detail.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Somatic embryogenesis, pigment accumulation, and synthetic seed production in Digitalis davisiana Heywood.

Digitalis davisiana, commonly called Alanya foxglove, from Turkey, is an important medicinal herb as the main source of cardiac glycosides, cardenolides, anthraquinones, etc. It is also known in the Indian Medicine for treatment of wounds and burns. It has ornamental value as well. Overexploitation of D. davisiana has led this species to be declared protected, and thereby encouraged various methods for its propagation. In this study, an optimized and efficient plant tissue culture protocol was established using cotyledonary leaf, hypocotyl and root explants of D. davisiana. Callus tissues were obtained from the cotyledonary leaf, hypocotyl and root segments cultured on Murashige and Skoog's (MS) medium containing different plant growth regulators. The maximum number of somatic embryos were achieved by the MS medium containing 6-benzyladenine (1.0 mg/L BAP) or 2,4-dichlorophenoxy acetic acids (0.1 mg/L 2,4-D), which produced an average of 8.3 ± 1.5 or 5.3 ± 1.5 embryos per cotyledonary leaf, respectively. After 3 wk of culture in MS medium supplemented with 1.0 mg/L 2,4-D, callus showed a clear accumulation of orange pigmentation. Shoot regeneration was remarkably higher (14.3 indirect shoots) in a combination of α-naphthalene acetic acid (0.25 mg/L NAA) plus 3.0 mg/L BAP than 2.0 mg/L zeatin (10.3 ± 0.5 direct shoots) alone. The shoots were successfully rooted on MS medium supplemented with NAA (0.1-1.0 mg/L). In addition, synthetic seeds were produced by encapsulating shoot tips in 4% sodium alginate solution. Maximum conversion frequency of 76.6% was noted from encapsulated shoot tips cultured on 0.25 mg/L NAA with 1.0 mg/L BAP. The encapsulated shoot tips could be stored up to 60 days at 4 °C. Regenerated plantlets of D. davisiana were successfully acclimatized and transferred to soil. This study has demonstrated successful preservation of elite genotypes of D. davisiana.

Biotechnological Approaches for Biomass and Cardenolide Production in Digitalis purpurea L.

Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors. This chapter provides two biotechnology strategies for biomass and cardenolide production in D. purpurea. Firstly, we report biomass production using a temporary immersion system (TIS), combined with cardenolide extraction and quantification. Secondly, an efficient protocol for genetic transformation via Agrobacterium tumefaciens is provided. These strategies can be used independently or combined in order to increase the content of cardiac glycosides in D. purpurea and to unravel biosynthetic pathways associated to cardiac glycoside production.

Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species.

The effect of hydrogen peroxide (H2O2) on callus cultures of four Digitalis species (Digitalis lamarckii, Digitalis trojana, Digitalis davisiana and Digitalis cariensis) increased catalase (CAT), superoxide dismutase (SOD), total phenolic, proline activity and cardiotonic glycoside production. Callus derived from hypocotyl explants was cultured on Murashige and Skoog medium supplemented with 0.25 mg L(-1) indole-3-acetic acid (IAA) and 0.5 mg L(-1) thidiazuron (TDZ). After a month of culture, callus was transferred to MS medium containing 10 mM H2O2 and then incubated for 6 h. The amount of five cardenolides (Lanatoside C, Digitoxin, Digoxigenin, Gitoxigenin and Digoxin) as well as CAT, SOD, total phenolic, proline activity from Digitalis species were compared. No digoxin was detected in all treatments and control groups. The total cardenolides estimated were in the order of D. lamarckii (586.65  µg g(-1) dw), D. davisiana (506.79 µg g(-1) dw), D. cariensis (376.60 µg g(-1) dw) and D. trojana (282.39 µg g(-1) dw). It was clear that H2O2 pre-treatment resulted in an increase in enzymatic and nonenzymatic antioxidants. However, a significant negative relationship between cardenolides production and overall activities of CAT, SOD, total phenolic and proline was evident. The described protocol here will be useful for the development of new strategies for a large-scale production of cardenolides.

Interaction of digitalis-like compounds with liver uptake transporters NTCP, OATP1B1, and OATP1B3.

Digitalis-like compounds (DLCs) such as digoxin, digitoxin, and ouabain, also known as cardiac glycosides, are among the oldest pharmacological treatments for heart failure. The compounds have a narrow therapeutic window, while at the same time, DLC pharmacokinetics is prone to drug-drug interactions at the transport level. Hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and Na(+)-dependent taurocholate co-transporting polypeptide (NTCP) influence the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. The interaction of digoxin, digitoxin, and ouabain with hepatic uptake transporters has been studied before. However, here, we systematically investigated a much wider range of structurally related DLCs for their capability to inhibit or to be transported by these transporters in order to better understand the relation between the activity and chemical structure of this compound type. We studied the uptake and inhibitory potency of a series of 14 structurally related DLCs in Chinese hamster ovary cells expressing NTCP (CHO-NTCP) and human embryonic kidney cells expressing OATP1B1 and OATP1B3 (HEK-OATP1B1 and HEK-OATP1B3). The inhibitory effect of the DLCs was measured against taurocholic acid (TCA) uptake in CHO-NTCP cells and against uptake of ß-estradiol 17-ß-d-glucuronide (E217ßG) in HEK-OATP1B1 and HEK-OATP1B3 cells. Proscillaridin A was the most effective inhibitor of NTCP-mediated TCA transport (IC50 = 22 µM), whereas digitoxin and digitoxigenin were the most potent inhibitors of OATP1B1 and OAPTP1B3, with IC50 values of 14.2 and 36 µM, respectively. Additionally, we found that the sugar moiety and hydroxyl groups of the DLCs play different roles in their interaction with NTCP, OATP1B1, and OATP1B3. The sugar moiety decreases the inhibition of NTCP and OATP1B3 transport activity, whereas it enhances the inhibitory potency against OATP1B1. Moreover, the hydroxyl group at position 12 reinforces the inhibition of NTCP but decreases the inhibition of OATP1B1 and OATP1B3. To investigate whether DLCs can be translocated, we quantified their uptake in transporter-expressing cells by LC-MS. We demonstrated that convallatoxin, ouabain, dihydroouabain, and ouabagenin are substrates of OATP1B3. No transport was observed for the other compounds in any of the studied transporters. In summary, this work provides a step toward an improved understanding of the interaction of DLCs with three major hepatic uptake transporters. Ultimately, this can be of use in the development of DLCs that are less prone to transporter-mediated drug-drug interactions.

A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter ß-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.

Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey.

Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 µg ml(-1) thidiazuron (TDZ) and 0.25 µg ml(-1) indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 µg g(-1))>D. trojana (1385.75 µg g(-1))>D. cariensis (1038.65 µg g(-1))>D. davisiana (899.86 µg g(-1)) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides.

Uptake of diuron and concomitant loss of photosynthetic activity in leaves as visualized by imaging the red chlorophyll fluorescence.

The principles of the chlorophyll (Chl) fluorescence induction kinetics (known as Kautsky effect) and their change by the photosystem II herbicide diuron are presented together with the Chl fluorescence emission spectra of a normal and diuron-inhibited leaf. By imaging the Chl fluorescence emission of green leaves the successive uptake of diuron and the concomitant loss of photosynthetic quantum conversion from the leaf base to the leaf tip are documented.

Interaction of digitalis-like compounds with p-glycoprotein.

Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12ß seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a δ-lactone ring and a sugar moiety at 3ß of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5ß and 19, whereas inhibition is decreased by those at positions 1ß, 11α, 12ß, and 16ß. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding.

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine-BA; kinetin-Kin; and thidiazuron-TDZ) and auxins (indole-3-acetic acid-IAA; α-naphthaleneacetic acid-NAA; and 2,4-dichlorophenoxy acetic acid-2,4-D). Maximum multiple shoots (12.7 ± 0.6) were produced from nodal explants on MS + 7.5 µM BA. Shoots were rooted in vitro on MS containing 15 µM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 µM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.

[Effect of some biotic elicitors on flavonoids production in Digitalis lanata cell cultures]. / Efectul unor elicitori biotici asupra productiei de flavone in culturi celulare de Digitalis lanata.

UNLABELLED: A method to improve the secondary metabolites production in plant cell cultures is the use of elicitors (biotic or abiotic). In this paper we investigated the effect of two biotic elicitors, Botrytis and Sclerotinia, on the flavonoids production, an important category of secondary metabolites, in Digitalis lanata suspension cultures. MATERIAL AND METHOD: Were used two cell lines: line 11 and line 13C 100 (aged 10 days). Were used two concentrations of elicitors (1 ml and 2 ml/100 ml suspension, respectively). Flavones were determined by a spectrophotometric method, according to Romanian Pharmacopoeia no. X (Cynarae Folium monography). RESULTS: For both cell lines and for both elicitors, flavonoids accumulation grew with elicitation time and elicitor concentration. The highest flavonoids production took place after 96 hours elicitation time for the highest elicitor .concentration (2ml/100ml suspension). So, line 11 accumulated 1000 mg% flavonoids with Botrytis and 999.81 mg% with Sclerotinia. Line 13C 100 accumulated 1051,65 mg% flavonoids with Botrytis and 1025,43 mg% with Sclerotinia. For all experiments, Botrytis (both concentration) induced a higher flavonoids accumulation for line 11 than Sclerotinia. Line 13C 100 responses to elicitor action is stronger for Sclerotinia for higher elicitor concentration than for same concentration of Botrytis.

Biotransformation of 21-O-acetyl-deoxycorticosterone by cell suspension cultures of Digitalis lanata (strain W.1.4).

Cell cultures of Digitalis species are known to accept exogenous substrates for biotransformation reactions. We here report the biotransformation of 21-O-acetyl-deoxycorticosterone (1) by cell suspension cultures of Digitalis lanata strain W.1.4. Nine derivatives of 1 were obtained and their chemical structures determined by spectroscopic methods. 2ß-Hydroxylation and C-21-glucosylation of the steroidal nucleus were described for the first time in suspension-cultured plant cells. Steroid 5α- and 5ß-reduction products were also observed. Among the compounds isolated and structures elucidated were 2ß,3ß,21-trihydroxy-4-pregnen-20-one, 2ß,3α,21-trihydroxy-4-pregnen-20-one and 3ß,21-dihydroxy-5α-pregnan-20-one-3ß-O-ß-glucoside.

Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea.

BACKGROUND: Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome. RESULTS: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other. CONCLUSIONS: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.