山崎 真巳

Anthocyanidin synthase (ANS), a member of the 2-oxo-glutarate-dependent dioxygenase family in flavonoid biosynthesis, catalyzes the conversion of leucoanthocyanidins ( e. g. 2R, 3S, 4S-cis-leucocyanidin, LCD) to flav-2-en-3,4-diols, a direct precursor of colored anthocyanidins via flavan-3,3,4-triols. The detailed oxygenation mechanism of 2R, 3S, 4S-cis-LCD to flav-2-en-3,4-diols was investigated using the density functional theory method. An initial model for the calculation was constructed from a structure obtained by a 100-ps molecular dynamics simulation of Arabidopsis ANS under physiological conditions. This model consisted of an LCD molecule as the substrate together with an iron atom, two histidine residues, an aspartic acid residue, a succinate, and an oxygen atom as ligands of the iron atom. The results of the calculation indicated that both the C-3 and C-4 positions of LCD can be oxidized, although C-4 oxidation is preferable. The C-3 oxidation required several steps to form flavan-3,3,4-triol:1) formation of Fe-III-OH and a substrate C-3 radical via hydrogen atom abstraction by Fe-IV=O, 2) formation of a C-3 ketone and a water molecule, 3) addition of OH- into the C-3 position of the ketone, and 4) addition of H+ to form flavan-3,3,4-triol. On the other hand, C-4 oxidation of 2R, 3S, 4S-cis-LCD resulted in the direct formation of 2R,3R-trans-dihydroquercetin. These results suggest that the oxidation at C-3 of LCD, a key reaction for coloring in anthocyanin biosynthesis, can be regarded as a "side reaction" from the viewpoint of quantum mechanics of enzymatic reactions. Molecular evolutional implications of ANS and related proteins are discussed in terms of reaction dynamics.

The integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fourier-transform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity, This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22 810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 overexpression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, wore induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism.

The proanthocyanidin ( PA) content was increased in seeds of pap1-D mutant of Arabidopsis thaliana, in which the expression of endogenous PAP1 gene encoding a Myb-like transcription factor was induced by activation-tagging with enhancer sequences derived from cauliflower mosaic virus 35S promoter. In contrast, the PA contents decreased in seeds of transgenic plants transformed with a PAP1 cDNA or with a PAP1 chimeric repressor, although the amount of soluble anthocyanins increased in seeds of transgenic plants over-expressing PAP1 cDNA. The enhanced radical scavenging activity was observed only in the seed extracts of pap1-D mutant, indicating that PAs are primarily responsible for radical scavenging activity in seeds. The present study suggests the feasibility of engineering a transcription factor of flavonoid biosynthesis for health beneficial plant seeds.

A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(-)-13alpha-hydroxymultiflorine/(+)-13alpha-hydroxylupanine O-tigloyltransferase, has been purified from Lupinus albus. The internal amino acid sequences were determined with protease-digested fragments of 25 and 30 kDa bands, allowing design of primers for amplification of cDNA fragments by polymerase chain reaction. Using an amplified fragment as the probe, a full-length cDNA clone was isolated. Sequence analysis revealed that the cDNA encodes a protein of 453 amino acids with a molecular mass of 51.2 kDa. Phylogenetic analysis of the deduced amino acid sequences indicated that this alkaloid acyltransferase belongs to a unique subfamily of a plant acyl-CoA-dependent acyltransferase gene family. The cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity purified and cleaved to yield the recombinant enzyme for the study of catalytic properties. The recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to (-)-13alpha-hydroxymultiflorine and (+)-13alpha-hydroxylupanine. Benzoyl-CoA could also serve efficiently as an acyl donor for these hydroxylated alkaloids. RNA blot analysis suggested that the gene was expressed in roots and hypocotyls but not in cotyledons and leaves. These results indicated that this specialized acyltransferase, isolated for the first time as tigloyltransferase from nature, is committed to control the quinolizidine alkaloid patterns in a tissue-specific manner.

Anthocyanin extracts of two blueberries, Vaccinium myrtillus (bilberry) and Vaccinium ashei (rabbiteye blueberry), and of three other berries, Ribes nigrum (black currant), Aroma melanocarpa (chokeberry), and Sambucus nigra (elderberry), were analyzed by high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization - mass spectrometry (LC/PDA/ESI-MS). Both bilberry and rabbiteye blueberry contained 15 identical anthocyanins with different distribution patterns. Black currant, chokeberry, and elderberry contained 6, 4, and 4 kinds of anthocyanins, respectively. The radical scavenging activities of these berry extracts were analyzed by using 2,2-diphenyl-1-picrylhydrazyl (DPPH). All these extracts showed potent antiradical activities.

Camptothecin-derived compounds are widely used for clinical treatment of human cancer. They are synthesized from natural camptothecin, which is obtained by extraction from intact plants. For the feasible production of camptothecin, tissue cultures of Ophiorrhiza liukiuensis and O. kuroiwai, an interspecies hybrid of O. liukiuensis and O. pumila, have been investigated. The aseptic plants and hairy roots of O. liukiuensis and O. kuroiwai were established in addition to the previously-established O. pumila. The camptothecin production by O. kuroiwai was better than that by O. liukiuensis. 10-Methoxycamptothecin was accumulated in tissue cultures of O. liukiuensis and O. kuroiwai but not in O. pumila. Methyl jasmonic acid slightly enhanced the production of camptothecin in the O. liukiuensis hairy roots. These results indicate that the tissue cultures of O. liukiuensis and O. kuroiwai would be the feasible ways for production of camptothecin and related alkaloids.

Camptothecin derivatives are clinically used as anti-tumor alkaloids that are currently obtained by extraction from intact plants. Seeking for the alternative sources for commercial production and for fundamental study, cell and tissue cultures have been investigated. In the present study, we developed a method for regeneration of Ophiorrhiza pumila plant from hairy roots transformed with Agrobacterium rhizogenes. The regeneration frequency was over 83%. Integration of a rol B gene from T-DNA of A. rhizogenes was confirmed by polymerase chain reaction in both of the hairy roots and the regenerated plants. The transformed plants accumulated camptothecin in amounts of 66-111% compared with that in the wild-type plants.

Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-C-13]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-C-13]Glc. The in vivo study followed incorporation of [1-C-13]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by C-13 nuclear magnetic resonance spectroscopy. The C-13-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-D-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in O-18(2)/(OH2)-O-16, O-16(2)/(OH2)-O-18, and O-18(2)/(OH2)-O-18 atmospheres. In the O-18(2)/(OH2)-O-16 atmosphere close to complete incorporation of a single O-18 label was observed in the dihydroflavonol products (e.g. (2R, 3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R, 3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15834. Southern blot analysis of the established roots revealed that only the T-L-DNA was integrated into the plant genome without incorporation of the T-R-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degreesC under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.

In Perilla frutescens, the expression of all structural anthocyanin genes is coordinately regulated by light in form-specific manner in aerial vegetative tissues. To find the regulatory factors for entire anthocyanin pathway in dicot plants, the red form specifically expressed genes were profiled by mRNA differential display. As a result, a novel bHLH factor gene Myc-F3G1 was isolated. The deduced amino acid sequence of MYC-F3G1 exhibited high homology with that of petunia and, which directly regulates the expression of anthocyanin structural genes. The expression of Myc-F3G was detected specifically in red form but not in green form and induced by light induction. These expression patterns are similar to those of structural anthocyanin genes and Myb-P1, a light-inducible Myb gene. In yeast two-hybrid systems, MYC-F3G1 interacted with another bHLH factor MYC-RP and a WD40 repeat protein PFWD but not with a light-inducible Myb factor Myc-P1. These results suggest that a protein complex including MYC-F3G1, MYC-RP, Myb-P1 and PFWD regulates the expression of anthocyanin biosynthetic genes in P frutescens. These findings shall provide valuable information for genetic engineering of entire anthocyanin pathway in dicot plants. (C) 2003 Elsevier Science B.V. All rights reserved.

This review describes biochemistry, molecular biology and regulation of anthocyanin biosynthesis, with particular emphasis on mechanistic features and late steps of anthocyanin biosynthesis including glycosylation and vacuolar sequestration. The literature from 1997 to the beginning of 2002 is reviewed, and 163 references are cited.

Camptothecin derivatives are clinically used antitumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA). We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin. The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH: cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.

We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed. (C) 2003 Elsevier Science Ltd. All rights reserved.

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression. (C) 2003 Elsevier Science Ltd. All rights reserved.

The WD-repeat proteins are found in eukaryotes and play an important role in the regulation of a wide variety of cellular functions such as signal transduction, transcription, and proliferation. In this study, we have isolated a cDNA encoding a novel WD-repeat protein, PFWD, from the anthocyanin-pigmented leaves of Perilla frutescens using AN11 cDNA from Petunia hybrida as the probe. The C-terminal region of PFWD contains a WD repeat that is highly conserved in homologous proteins from a variety of organisms that do not produce anthocyanin such as yeast, nematodes and mammals. Transgenic Arabidopsis plants overexpressing PFWD exhibited phenotypic changes including enhancement of anthocyanin production and reduced viability. A study of the interaction between PFWD and anthocyanin regulatory proteins using a yeast two-hybrid system showed strong interaction between PFWD and MYC-RP, a MYC-like protein from P. frutescens. PFWD fusion proteins transiently expressed in onion epidermal cells were localized in the cytosol under both dark and light conditions. However, co-expression of PFWD and MYC-RP fusion proteins resulted in nuclear localization of PFWD. We propose a model of genetic regulation in which the PFWD protein acts in signal transduction process in a variety of pathways through protein interaction with MYC proteins.

This review deals with the application of differential display to investigate gene expression in plants. A substantial articles reports the isolation and profiling of various genes expressed in cells using this technique. Genes involved in physiological events, stress responses, signal transduction and secondary metabolism have been isolated and characterized. Some of the isolated genes encode transcription factors, membrane proteins and rare enzymes that were previously difficult to purify. These results suggest that differential display is a powerful tool used to investigate the rare genes involved in the plant life cycle without using information from proteins.

Although substantial progress has been made on the molecular genetics of anthocyanin biosynthesis, the biochemistry of some components, such as anthocyanidin synthase, are not fully understood. To explore anthocyanin formation in more detail, and in particular, the late-stage of the biosynthetic pathway, Perilla frutescens (Labiatae) was chosen as a model plant. Two chemo-varietal forms exist in P. frutescens, the pigmented red form and, in striking contrast, the non-pigmented green form, which contains only a trace amount of anthocyanin in the leaves and stems. Using this plant, we investigated the biochemical characteristics of anthocyanidin synthase and two anthocyanin glycosyltransferases, and in addtion we used this plant to investigate the expression and regulation of flavonoid biosynthesis genes. P. frutescens represents a good model plant for investigating anthocyanin biosynthesis. Further exploitation of this model system will require the establishment of a suitable transformation system for P. frutescens. Future work will be directed towards further characterization of the chemo-varietal forms and investigating their evolution from the ancestral form.

A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.

A molecular biological approach was applied to the study of diversity and regulation of secondary metabolism in medicinal plants at various levels. For the inter-species diversity, RFLP (restriction fragment length polymorphysm) and RAPD (random amplified polymorphic DNA) analyses of genomic DNA were performed on the plants, belonging to the same genus or family and containing related compounds. Phylogenetic trees of lupin alkaloid containing plants and other medicinal plants, based on RFLP and/or RAPD profiles, showed the relationship between the diversities in genomes and secondary metabolisms. The chemotypes regarding anthocyanin production in <i>Perilla frutescens</i> var. <i>crispa</i>, were subjected to the study on intra-species diversity. The structural genes and the regulatory genes involved in anthocyanin biosynthesis were isolated and their expression in red and green forms was determined by Northern blot analysis. The expression of all structural genes examined was co-ordinately regulated in form-specific manner and by light illumination. The anthocyanin production was enhanced in transgenic plants over-expressing <i>Myc</i> homologue genes from perilla. These results suggested that a protein complex including bHLH factors might regulate the expression of a series of structural genes. Additionally, cDNAs coding anthocyanin 5-<i>O</i>-glucosyltransferase and anthocyanidin synthase were isolated and characterized using recombinant proteins for the first time. In conclusion, it was indicated that the molecular biological techniques are powerful tools for the investigation of diversity and regulation of and for the genetic engineering of secondary metabolism in medicinal plants.<br>

Cysteine synthase and alliinase (alliin-lyase) are terminal enzymes responsible for synthesis of cysteine and degradation of alliin (S-alk(en)yl-cysteine sulfoxide), respectively, in Allium plants. We determined the intercellular localization of cysteine synthase and alliinase using antibodies against these proteins. Cysteine synthase was predominantly localized in bundle sheath and phloem cells of three Allium plants, A. tuberosum, A. cepa and A. sativum. Lower amounts could be detected in mesophyll cells. Alliinase was almost exclusively localized in bundle sheath cells of the three Allium species. These results suggest the importance of bundle sheath cells for both synthesis and degradation of sulfurcontaining compounds in Allium. plants.

In Perilla frutescens, there are two varietal forms of anthocyanin accumulation, i.e. red and green forms. The cDNA clones encoding flavone synthase II (FSII) and flavonoid 3'-hydroxylase (F3'H), two cytochrome P450s that are involved in the biosynthesis of flavones and anthocyanins, were isolated from P. frutescens. The FSH cDNA encoded a 57.1 kDa protein designated as CYP93B6, and the F3'H cDNA encoded 57.5 kDa protein designated as CYP75B4. Recombinant CYP93B6 expressed in yeast converted flavanones to flavones with K-m values of 8.8-11.9 muM. Recombinant CYP75B4 catalyzed 3'-hydroxylation of flavanones to the corresponding compounds with K-m values of 18-20 muM. The CYP93B6 transcript accumulated to an equal level in leaves of both red and green forms of P frutescens, in agreement with the accumulation pattern of flavones in the leaves. However, the CVP75B4 transcript was predominantly expressed in the red form of P. frutescens, and its expression was induced by light in conjunction with other transcripts of biosynthetic enzymes of anthocyanin. These results indicate that gene expression of a set of anthocyanin biosynthetic enzymes including F3'H is regulated coordinately only in the red form of P. frutescens but not in the green form, whilst FSH gene expression is controlled in a similar manner in red and green forms of P. frutescens.

In the conversion from colorless leucoanthocyanidin to colored anthocyanidin 5-glucoside, at least two enzymes, anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (3-GT), are postulated to be involved. Despite the importance of this reaction sequence for coloring in anthocyanin biosynthesis, the biochemical reaction mechanism has not been clarified, and the possible involvement of a dehydratase has not been excluded. Here we show that recombinant ANSs from several model plant species, snapdragon, petunia, torenia, and maize, catalyze the formation of anthocyanidin in vitro through a 2-oxoglutarate-dependent oxidation of leucoanthocyanidin, Crude extracts of Escherichia coli, expressing recombinant ANSs from these plant species, and purified recombinant enzymes of petunia and maize catalyzed the formation of anthocyanidin in the presence of ferrous ion, 8-oxoglutarate, and ascorbate, The in vitro formation of colored cyanidin 5-glucoside from leucocyanidin, via a cyanidin intermediate, was demonstrated using petunia ANS and 3-GT, The entire reaction sequence did not require any additional dehydratase but was dependent on moderate acidic pH conditions following the enzymatic steps. The present study indicated that the in vivo cytosolic reaction sequence involves an ANS-catalyzed 8-oxogiutarate-dependent conversion of leucoanthocyanidin (flavan3,4-cis-diol) to 3-flaven-2,3-diol (pseudobase), most probably through 2,3-desaturation and isomerization, followed by glucosylation at the C-3 position by 3-GT.

Camptothecin derivatives are used clinically as anti-tumor alkaloids. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but transformed plant cell cultures may be an alternative source of production. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) transformed by Agrobacterium rhizogenes strain 15834. This hairy root culture grew well, increasing by 16-fold during 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of the cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amounts. Camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20) that absorbed camptothecin. Camptothecin was easily recovered from the resin. Our method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture.

The bitter and sweet forms of a plant species differing with alkaloid contents may provide a model system for investigation of alkaloid biosynthesis at a molecular level. The pattern and concentration of quinolizidine alkaloids were determined by capillary GC-R IS in bitter and sweet plants of Lupinus angustifolius Bitter plant contained lupanine, 13 alpha-hydroxylupanine. angustifoline, alpha-isolupanine, tetrahydrorhombifoline, and ester-derivatives of 13 alpha-hydroxylupanine. In contrast. no alkaloid was detected in sweet plant, The enzymatic activity of acyltransferase for formation of 13 alpha-tigloyloxylupanine was similar or even higher in the cell-free extracts of sweet plant than that in bitter plant. These results suggest that the biosynthetic step(s) of ring closure forming the initial cyclic alkaloid, lupanine, from cadaverine is presumably blocked in sweet plant, and that the later steps for modification of the cyclized alkaloids are not altered. We hypothesized that the gene(s) encoding enzyme(s) for ring-closure step might be repressed in sweet plant, and that the expression might take place only in bitter plant. To isolate the genes specifically expressed in bitter plant, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis,vas carried out. However, no bitter-specific gene tvas isolated, suggesting that alkaloid biosynthesis in sweet plant may be down-regulated at a post-transcriptional level.

The cDNA clone, TAN1, encoding anthocyanidin synthase (ANS) was isolated by screening of a cDNA library constructed from flower petals of Torenia fournieri cv. Summer Wave Blue using a heterologous probe. Nucleotide sequence analysis revealed that it contains an open reading frame encoding a polypeptide of 376 amino acids. The postulated amino acid sequence shows 48 % to 72 % identities with those of previously reported ANSs. Southern blot analysis suggested that there is a single copy of ANS gene in the genome of T. fournieri. Northern blot analysis indicated that ANS gene is expressed in flower bud but not in leaf or mature flower.

Delila protein is a member of MYC-like transcriptional activator family, which regulates the expression of structural genes in the biosynthesis of anthocyanin in snapdragon. We have mutated several amino acid of Delila to elucidate their function using transcriptional activation by yeast onehybrid system. Among several site - directed mutations, the replacement of alanine-161 with 18 different amino acids resulted in severe decrease in the transcriptional activation of the yeast GALl promoter Delila fused to the yeast GAL4 DNA - binding domain. This lack of induction indicates the importance of alanine-161 for transcriptional activation by Delila protein. Although the mutational change at residue 161 from alanine to aspartic acid exhibited no activity, the deletion of N - terminus (1 136 amino acids) of this mutant resulted in the recovery of activity. These results suggest that alanine161 located outside the activation domain in Delila plays a critical role for its transcriptional activation in yeast.

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1-115 amino acids deleted) of Myc-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.

UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa, Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida, To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT, Several biochemical properties (optimum pH, K-m values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs, Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P, frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P, frutescens.

Anthocyanidin synthase (ANS), an enzyme of the biosynthetic pathway to anthocyanin, has been postulated to catalyze the reaction(s) from the colorless leucoanthocyanidins to the colored anthocyanidins. Although cDNAs have been isolated that encode putative ANS, which exhibits significant similarities in amino acid sequence with members of a family of 2-oxoglutarate-dependent oxygenases, no biochemical evidence has been presented which identifies the actual reaction that is catalyzed by ANS. Here we show that anthocyanidins are formed in vitro through 2-oxoglutarate-dependent oxidation of leucoanthocyanidins catalyzed by the recombinant ANS and subsequent acid treatment. A cDNA encoding ANS was isolated from red and green formas of Perilla frutescens by differential display of mRNA. Recombinant ANS tagged with maltose-binding-protein (MBP) was purified, and the formation of anthocyanidins from leucoanthocyanidins was detected by the ANS-catalyzed reaction in the presence of ferrous ion, 2-oxoglutarate and ascorbate, being followed by acidification with HCl. Equimolar stoichiometry was confirmed for anthocyanidin formation and liberation of CO2 from 2-oxoglutarate. The presumptive two-copy gene of ANS was expressed in leaves and stems of the red forma of P. frutescens but not in the green forma plant. This corresponds to the accumulation pattern of anthocyanin. The mechanism of the reaction catalyzed by ANS is discussed in relation to the molecular evolution of a fa mi ly of 2-oxoglutarate-dependent oxygenases.

Angelica acutiloba, a medicinal plant used as a natural medicine Touki, was clonally propagated through axillary buds in vitro. No substantial differences were found in the random amplified polymorphic DNA (RAPD) pattern between the original A. acutiloba and the plant propagated in vitro, suggesting no changes in the DNA sequences and structure during in vitro propagation. The genetic similarities of several Angelica plants were investigated by restriction fragment length polymorphism (RFLP) and RAPD analyses. The RFLP and RAPD patterns of A. sinensis Diels were substantially different from those of A. acutiloba. Using ten different restriction enzymes, no RFLP was observed in the varieties of A. acutiloba. By RAPD analysis, A. acutiloba varieties can be classified into two major subgroups, i.e., A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyamae Hikino. The varieties of A. acutiloba Kitagawa in Japan and Angelica spp. in northeast China exhibited a very close genetic relationship.

Serine biosynthesis in plants proceeds by two pathways; a photorespiratory pathway which is associated with photorespiration and a pathway from phosphoglycerate. A cDNA encoding plastidic phosphoserine aminotransferase (PSAT) which catalyzes the formation of phosphoserine from phosphohydroxypyruvate has been isolated from Arabidopsis thaliana. Genomic DNA blot analysis indicated that this enzyme is most probably encoded by a single gene and is mapped on the lower arm of chromosome 4. The deduced protein contains an N-terminal extension exhibiting the general features of a plastidic transit peptide, which was confirmed by subcellular organelle localization using GFP (green flourescence protein). Northern analysis indicated preferential expression of PSAT in roots of light-grown plants, supporting the idea that the phosphorylated pathway may play an important role in supplying the serine requirement of plants in non-green tissues. In situ hybridization analysis of PSAT revealed that the gene is generally expressed in all types of cells with a significantly higher amount in the meristem tissue of root tips.

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.