Yoshie Kitamura

Root Growth-promoting Activity of Unsaturated Oligomeric Uronates from Alginate on Carrot and Rice Plants

The root elongation activity of unsaturated oligomeric uronates from alginate on carrot and rice plants was investigated. Unsaturated oligomeric uronates were prepared by digesting polymannuronate (PM) and polyguluronate (PG) with an alginate lyase purified from Pseudoalteromonas sp. strain No. 272. The root elongation activity was measured by elongation in length of carrot- and rice-excised root incubated in the B5-medium containing 0.8% agar in the dark. PM and PG showed no activity, but the enzymatic digestion mixtures of PG had promoting activity on roots of both plants at a final concentration of 0.5 mg/ml. The maximum activity was obtained at 0.75 mg/ml. The dependence of activity on degree of polymerization of the uronates was tested and the pentamer was most active, but the mechanism of the action of unsaturated uronates on the cells remains to be solved.

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.

Phloem Transport of Tropane and Pyridine Alkaloids in Duboisia myoporoides

Summary The phloem sap of Duboisia myoporoides R. B R . (Solanaceae) was collected from shoots treated with EDTA solution. The usefulness of the method was confirmed by using 14 C-Iabeled atropine. Analysis of phloem sap showed existence of alkaloids (scopolamine, atropine, nicotine and anabasine) and sucrose, indicating that tropane and pyridine alkaloids are transported through not only the xylem but also the phloem in D. myoporoides plants.

Conversion of Phenylalanine and Tropic Acid into Tropane Alkaloids by Duboisia leichhardtii Root Cultures

Summary 14C-labelled phenylalanine and tropic acid, which are possible precursors of the acid moiety of the tropane alkaloids, hyoscyamine and scopolamine, were fed to Duboisia leicbhardtii root cultures. 14C-phenylalanine was efficiently incorporated into tropane alkaloids and more than 50 % of the total radioactivity extractable from the tissues and the medium was recovered in the alkaloidal fraction after 1 week in culture. On the other hand, less than 1 % of 14C-tropic acid was converted to tropane alkaloids even after 3 weeks in culture. Formation of labelled hyoscyamine, scopolamine and apoatropine was confirmed by TLC-autoradiography and that of the former two was also confirmed by enzymatic hydrolysis. A change in the ratio of radioactivity of scopolamine to hyoscyamine over 3 weeks of culture was traced, indicating that the root cultures sustained the ability to convert hyoscyamine to scopolamine for at least 21 days, although biosynthesis of hyoscyamine in the roots stopped after the first 7 days in culture. A possible reason why little 14C-tropic acid was incorporated into tropane alkaloids is discussed.

Differences of atropine esterase activity between intact roots and cultured roots of various tropane alkaloid-producing plants

Abstract The tropane alkaloid distribution and atropine esterase activity in Atropa belladonna , Datura tatula , Duboisia leichhardtii , Hyoscyamus niger and Scopolia japonica were determined at various developmental stages and compared with those in their cultured roots. High atropine esterase activity was found in the roots of every tropane alkaloid- producing plant at any stage of development, though much higher activities were detected in the roots of annual and biennial herbs at the fruiting stage. In contrast to these findings, no cultured roots contained detectable amounts of atropine esterase activity in spite of their production of hyoscyamine and scopolamine.

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 105/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.

A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency

Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions.

Phenyllactic acid in Duboisia leichhardtii root cultures B feeding of phenyl[1-14C]alanine

Abstract Phenyl[1-14C]alanine which is a precursor of the acid moiety of tropane alkaloids and one of the possible intermediates—namely phenylpyruvic, phenyllactic and tropic acids—were simultaneously added to Duboisia leichhardtii root cultures in order to trap the labelled intermediates. When phenyllactic acid was added to 0.5–5 mm, incorporation of phenyl[1-14C]alanine into the organic acid fraction was much greater in comparison with the cases using phenylpyruvic acid, tropic acid or no intermediate at all. TLC-autoradiogram of the organic acid fraction obtained from phenyllactic acid feeding experiments showed the existence of a radioactive spot corresponding to phenyllactic acid. Trapping of labelled phenyllactic acid was confirmed by recrystallization, NMR and melting point.

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.