山崎 真巳

Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfur-containing metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54083 Da. The deduced amino acid sequence exhibited 66-69% identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue. The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.

Random Amplified Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP) methods were used for the analysis of three different strains of Cannabis sativa that included the Mexican (drug type), Tochigishiro (fiber type) and Nara cultivated strains. Cannabis sativa samples were grouped into three chemotypes, drug type, fiber type and intermediate drug type by GC analysis. The RAPD analysis was able to distinguish the three chemotypes clearly, however, in each type, some genetic diversities was shown. The RFLP analysis showed that the genetic distance between the Tochigishiro and Nara cultivated strains was small, whereas that between the Mexican strain and the other two strains was large, and that there was no genetic diversity within each strain. These results indicated that the RAPD and RFLP analyses, using DNA as a marker, applied in combination might be useful for distinguishing between the strains having chemotypes.

Two cultivars of Perilla frutescens, red and green formas, are known to differ in anthocyanin accumulation in leaves and stems. cDNA clones encoding the enzymes involved in anthocyanin biosynthesis, chalcone synthase (CI-IS), flavanone 3-hydroylase (F3H), dihydroflavonol 4-reductase (DFR), and UDP glucose: flavonoid 3-O-glucosyltransferase (3GT), were isolated from cDNA libraries derived from the leaves of a red forma of I! frutescens by screening with partial fragments amplified by means of polymerase chain reaction (PCR) and heterologous cDNAs as probes. The deduced amino acid sequences of these four genes exhibited 40-90% identity with those reported for the corresponding gene from other unrelated species. Southern blot analysis for these genes and two other structural genes, the leucoanthocyanidin dioxygenase (LDOX, anthocyanidin synthase) and anthocyanin acyltransferase (AAT) genes, indicated that each gene comprises a small multi-gene family. More than three copies of the CHS gene are present, two copies of the other genes being present. The expression of five genes, the exception being the CHS gene, was detected only in red leaves of the red forma of P.frutescens, i.e. not in green leaves of the green forma plant. The CHS gene was expressed in both red and green leaves, but 10-fold more in red leaves than in green leaves. These results suggest that the expression of all structural genes examined is coordinately regulated in a forma-specific manner. Under weak-light conditions, the accumulation of both anthocyanin and mRNAs of biosynthetic enzymes was lower in leaves of the red forma. High-intensity white light coordinately induced the accumulation of transcripts of all six genes examined in the mature leaves of red P.frutescens.

Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants, Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SATI) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

Transgenic hairy roots of Perilla frutescens var. crispa were obtained using an Agrobacterium-Ri binaryvector system. The chimeric uidA gene encoding β-glucuronidase (GUS) flanked by the TR2′ promoter for mannopine synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching stems of young plants. The adventitious roots which arose on the infected sites were excised, and the several clones of root tissue could be maintained on hormone-free agar medium. The transgenlc states of these clones were confirmed by Southern blot hybridization. Expression of the uidA gene in the transgenic tissues was indicated by in vitro enzyme assay and histochemical staining of GUS activity. More than half of hairy roots which emerged were confirmed to be doubly transformed with TDNAs from Ri and binary Ti plasmids.

Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic RO and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15-60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.

A DNA sample, suitable for analysis of random amplified polymorphic DNA (RAPD), was isolated from commercial licorice root of four different geographical origins. The DNA isolation method involved extraction with an acidic buffer and precipitation with cetyltrimethylammonium bromide (CTAB). RAPD analysis of DNA from crude drugs and Glycyrrhiza plants was carried out by using 10-mer primers of arbitrary sequences. The results suggested the possibility that the botanical species of the commercial licorice roots can be identified by the RAPD analysis.

The genetic similarities of four species of Glycyrrhiza plants were determined by DNA analyses of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). The phylogenic trees were constructed from the genetic similarities estimated from RAPD and RFLP profiles. These results indicated that G. glabra and G. uralensis, rich in glycyrrhizin, are more closely related to each other than to G. echinata or to G. pallidiflora.

Cysteine synthase plays a key role in the sulfur assimilation pathway in plant cells. The cDNA clones encoding two isoforms of this enzyme were isolated from spinach by synthetic oligonucleotide probes. The modes of expression of these two genes differed in tissues of spinach. A heterologous expression system in Escherichia coli and transgenic tobacco was made. The application of heterologous expression to modify sulfur metabolism and to produce non-protein amino acids is discussed.

The restriction fragment length polymorphisms (RFLPs) in genomic DNA were detected among six species of Lupinus plants using rice DNA coding for ribosomal RNA (rDNA) as a probe. Additionally, the fragment patterns were compared between alkaloid-rich 'bitter' forms and alkaloid-poor 'sweet' forms of L. albus and L. luteus. The hybridizing patterns for several enzymes were distinguished among these species and between bitter form and sweet form of L. albus. The phylogenic tree constructed from RFLP profiles was related with the pattern of alkaloid production, indicating the usefulness of RFLP for DNA characterization of medicinal plants.

Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda-gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.

Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3' end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R0 tissues and in backcrossed F1 progeny with a non-transformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.

Agrobacterium-Ti/Ri plasmids are natural gene vectors, by which a number of attempts have been made in genetic engineering of secondary metabolism in pharmaceutically important plants in the last few years. Opines are biosynthesized by transformed crown galls and hairy roots integrated with T-DNAs of Ti/Ri plasmids. These opines are classified into five families according to their structures and biogenesis. The production of opines is a natural example of genetic engineering of the biosynthetic machinery of plant cells for the benefit of the bacterial pathogen. One recent advance in transgenic technology of potential value to pharmacognosy is an application of transgenic organ cultures such as hairy roots and shooty teratomas to over-production and biotransformation of secondary metabolites. The hairy roots induced by Ri plasmid of Agrobacterium rhizogenes have been proved to be an efficient means of producing secondary metabolites that are normally biosynthesized in roots of differentiated plants. So far the specific metabolites produced by hairy root cultures and/or plants regenerated from hairy roots of 63 species have been analyzed and reported. As an alternative means of producing metabolites normally produced in leaves of plants, the shooty teratomas incited by the tumor-forming Ti plasmid or a shooty mutant of Agrobacterium tumefaciens have been used for the de novo biosynthesis and biotransformation of some specific secondary products. A second and more direct way to manipulate secondary pathways is performed by transferring and expressing specifically modified genes into medicinal plant cells with Agrobacterium vector systems. The genes encoding neomycin phosphotransferase and beta-glucuronidase have been used as model genes under the transcriptional control of appropriate promoters. Recently some specific genes that can eventually modify the fluxes of secondary metabolism have been integrated and expressed in medicinal plant cells. Future prospects are also discussed.

Transgenic hairy roots and shooty teratomas of Atropa belladonna L., Nicotiana tabacum L. and Solanum tuberosum L. were obtained with Agrobacterium Ti and Ri plasmids. The hairy roots of A. belladonna, N. tabacum and S. tuberosum accumulated tropane alkaloids, nicotine alkaloids and steroidal alkaloids, respectively. The shooty teratomas of these plant species failed to produce these alkaloids. However, the shooty teratomas had the abilities to store and metabolize the alkaloids. These results indicate that the biosynthesis and metabolism of these solanaceous alkaloids are regulated in correlation to tissue differentiation of the plants.

Transgenic tobacco (Nicotiana tabacum L.), licorice (Glycyrrhiza uralensis Fisher) and foxglove (Digitalis purpurea L.) were obtained with binary vector systems based on a disarmed Agrobacterium tumefaciens and on a virulent A. rhizogenes. The chimeric neomycin phosphotransferase II (NPT-II) gene (kan) and the beta-glucuronidase (GUS) gene (uidA) were under the control of the TR1' and 2' promoters, respectively, on a binary vector, pGSGlucl. Tissue-specific expression of the chimeric TR2'-uidA gene was studied using regenerants of N. tabacum from transformed callus and hairy roots, and also using the transformed roots of G. uralensis and D. purpurea. In all these transformed tissues, the phloem and surrounding tissues were stained with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide, showing the specific expression of uidA. The enzymatic assays of NPT-II and GUS indicated that the expression of both TR1'-kan and TR2'-uidA were coordinately enhanced by wounding and by the addition of plant growth regulators. These results indicate that the gene expression by the dual TR promoters is regulated in the several plant species studied in a tissue-specific manner and enhanced by physiological stresses.

Green callus and multiple shoots were induced from the epicotyl segments of Sophora flavescens var. angustifolia (Leguminosae) on the Murashige and Skoog (MS) agar medium in the presence of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA). The green callus produced matrine as the only detectable alkaloid. The concentrations of matrine in the callus were positively correlated to the amounts of chlorophyll in the cells. No alkaloid was produced in the non-green callus. The multiple shoots accumulated not only matrine but also 5,6-dehydrolupanine and anagyrine. These results suggest that the production of lupin alkaloids in S. flavescens var. angustifolia is related to the tissue differentiation, in particular, to the formation of chloroplasts and that the biosyntheses of matrine-type and anagyrine-type alkaloids are differently regulated in different developmental stages of the cells.