Toshihiko Ikenaga

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum.

Steroidal saponins are widely distributed in many plant species. Their diverse structures have resulted in a wide range of applications, including drug and medicine production. It has been suggested that the nature of the non-saccharide and oligosaccharide portions of the saponin molecule both contribute to the properties of individual saponins. Despite numerous studies on the occurrence, chemical structure, and varying pharmaceutical activities of steroidal saponins, their biosynthesis pathway is poorly understood. Glycosylation is thought to be the final step in steroidal saponin biosynthesis and it is thought to be involved in regulating the biological activities of saponins. Isolation of the glycosyltransferases that catalyze the transfer of sugar molecules to steroidal compounds will help to clarify the mechanisms that produce diverse saponins and control their activities in plants. In this study, we obtained three cDNAs encoding putative glycosyltransferases from Solanumaculeatissimum. One of the three, SaGT4A showed UDP-glucosyltransferase activity. This is the first cloned glucosyltransferase involved in steroidal saponin biosynthesis. SaGT4A catalyzes the 3-O-glucosylation of steroidal sapogenins, such as diosgenin, nuatigenin, and tigogenin. This enzyme also glucosylates steroidal alkaloids, such as solanidine, solasodine, and tomatidine. Gene expression analysis revealed that the accumulation of SaGT4A transcripts showed a unique response to wounding stress indicating the involvement of SaGT4A in plant defense system.

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

SummaryHairy 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 Gamborgs 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, Gamborgs B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100μg · 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.

Induction of furanocoumarin biosynthesis in Glehnia littoralis cell suspension cultures by elicitor treatment

Cell suspension cultures were established from Glehnia littoralis plants belonging to two different geographic strains. When the cells were treated with yeast extract, they started to produce and excrete furanocoumarins into the culture medium; a major component, bergapten, and a minor one, xanthotoxin, were detected and identified by HPLC and GC/MS. Changes in phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production after elicitor treatment were traced, showing that PAL activity increased rapidly, reached a maximum after 24 h, and then declined to the normal level after 96 h which preceded the induced bergapten production. The induced-PAL activity of the cultured cells established from an S-type plant which accumulated trace amounts of furanocoumarins was about 50% of that in the cultured cells from an N-type plant that accumulated more than 0.1% furanocoumarins in the underground parts. However, the elicited production of bergapten was about six times higher in the cell cultures from the S-type plant. Addition of the PAL inhibitor 2-aminoindan-2-phosphoric acid (AIP) at 10 microM suppressed the induction of PAL activity and furanocoumarin production.

Anthocyanin production of Glehnia littoralis callus cultures

A stable callus line that produces anthocyanins was established from callus derived from a petiole of a Glehnia littoralis seedling and subcultured in the dark. The major anthocyanin which made up about 60% of the total anthocyanins was determined as cyanidin 3-O-(6-O-(6-O-(E)-feruloyl-beta-D-glucopyranosyl) -2-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) by chemical and spectroscopic analyses. Anthocyanin contents in the cells cultured on B5 basal medium containing NAA (1 mg l-1), kinetin (0.01 mg l-1) and 3% sucrose reached 14% (dry wt basis) and the productivity has been sustained for 5 years.

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls.

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

Winter Cherry Bugs Feed on Plant Tropane Alkaloids and De-epoxidize Scopolamine to Atropine

The winter cherry bug colonizes the Duboisia leichhardtii tree, which is a rich source of scopolamine. It consumes the tropane alkaloids atropine and scopolamine. Quantitative analysis revealed that the ratio of scopolamine to atropine in the winter cherry bug (0.46) was far from that found in the leaves of the host plant (7.20). To elucidate whether the winter cherry bugs selectively excrete or decompose scopolamine, they were fed scopolamine and/or atropine together with sucrose. They took up scopolamine as well as atropine, and converted scopolamine into atropine.

Tropic acid moiety of atropine may be recycled in Duboisia

Abstract Ester derivatives of labelled tropic acid (denoted tropoyl-A and -D) were isolated as metabolites of atropine from Duboisia myoporoides seedlings supplied with [carbonyl- 14 C]atropine sulphate and were then fed to D. leichhardtii root cultures to determine whether they were metabolic end-products or whether they could be metabolized further. Both tropoyl-A and tropoyl-D were incorporated into tropane alkaloids (atropine, 6-hydroxyatropine and scopolamine). Incorporation from tropoyl-D exceeded that from tropoyl-A. This result shows that both tropoyl-A and -D are precursors of tropane alkaloids and that the tropic acid moiety is possibly recycled in plants of Duboisia .

Genetic Transformation of Duboisia Species

The genus Duboisia (family Solanaceae) is indigenous to Australia and comprises three species. Each species is located in a distinct area: Duboisia myoporoides along the eastern seaboard in Australia, D. leichhardtii in a very restricted area of Southeast Queensland known locally as the Southwest Burnett, and D. hopwoodii in Central and Western Burnett (Griffin 1967).

Steroidal saponin production in callus cultures of Solanum aculeatissimum Jacq.

Abstract Callus was induced from the epicotyl of S. aculeatissimum, and the relation between culture conditions and the production of steroidal saponins in the callus was studied. The results indicated that the callus produced the steroidal saponins aculeatiside A and B. The highest production of steroidal saponins occurred at the middle of the log phase. Optimal conditions for the production of steroidal saponins were culturing on MS basal medium supplemented with the combination of 0.05 ppm or 0.1 ppm NAA and 10 ppm BA, and fructose as a carbon source, in the dark at 25  °C. Under these optimal conditions, the callus produced 0.8% (per dry weight) steroidal saponins, or 0.32% aculeatiside A and 0.48% aculeatiside B.

Different Responses Between Anthocyanin-Producing and Non-Producing Cell Cultures of Glehnia Littoralis to Stress

Using anthocyanin-producing (Violet) and non-producing (White) cell lines of G. littoralis, effects of various stresses on cell growth, anthocyanin content, furanocoumarin induction, and PAL activity were determined. Yeast extract (YE) and free radical generators (H2O2, AAPH and X-ray) were used as potent stress inducers. Cell growth and anthocyanin content was not effected with X-ray or YE treatment in both cell lines, whereas not only the decrease of cell growth but also bleaching of anthocyanin in Violet cell cultures was observed with H2O2 and AAPH. AAPH did not cause furanocoumarin induction either in Violet or White cell cultures. X-ray irradiation, H2O2 and YE treatments induced bergapten formation in White cell cultures, whereas no stress compound was found at all in Violet cell cultures by any treatment. Increase of PAL activity in Violet cell cultures correlated with furanocoumarin production. The reasons why Violet cells did not produce furanocoumarin are discussed.