Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888 bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57–85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

Plants produce an enormous variety of natural products with highly diverse structures. These products are commonly termed "secondary metabolites" in contrast to the "primary metabolites" which are essential for plant growth and development. Secondary metabolites were formerly regarded as "waste products" without physiological function for the plant. With the emergence of the field of chemical ecology about 30 years ago, it became evident, however, that these natural products fulfill important functions in the interaction between plants and their biotic and abiotic environment. They can serve, for example, as defense compounds against herbivores and pathogens, as flower pigments that attract pollinators, or as hormones or signal molecules. In addition to their physiological function in plants, natural products also have a strong impact on human culture and have been used throughout human history as condiments, pigments, and pharmaceuticals.This chapter provides an overview about the diversity of secondary metabolites in plants, faceted cultural history. The compounds are classified into four different groups according to their biosynthetic origin: alkaloids, phenylpropanoids, polyketides, and terpenoids. Since more than 200,000 structures of natural products from plants are known, only selected groups and compounds are presented.

Differential screening by PCR-select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin-accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid-derived metabolism. The cDNAs encoding glutathione S-transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST-like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2S)-naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla.

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51–67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.

Plumbago indica L. contains naphthoquinones that are derived from six acetate units. To characterize the enzyme catalyzing the first step in the biosynthesis of these metabolites, a cDNA encoding a type III polyketide synthase (PKS) was isolated from roots of P. indica. The translated polypeptide shared 47-60% identical residues with PKSs from other plant species. Recombinant P. indica PKS expressed in Escherichia coli accepted acetyl-CoA as starter and carried out five decarboxylative condensations with malonyl coenzyme A (-CoA). The resulting hexaketide was not folded into a naphthalene derivative. Instead, an alpha-pyrone, 6-(2',4'-dihydroxy-6'-methylphenyl)-4-hydroxy-2-pyrone, was produced. In addition, formation of alpha-pyrones with linear keto side chains derived from three to six acetate units was observed. As phenylpyrones could not be detected in P. indica roots, we propose that the novel PKS is involved in the biosynthesis of naphthoquinones, and additional cofactors are probably required for the biosynthesis of these secondary metabolites in vivo.

Sorghum SbSTS1 was the first example of a stilbene synthase gene in monocots. Previously, we demonstrated that the gene was involved in defense responses. To examine its biochemical function in planta, SbSTS1 was overexpressed in transgenic Arabidopsis. Metabolite analysis revealed that cis-resveratrol glucoside (piceid) accumulated as the major stilbene in the transgenic lines. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode, up to 580 microg g(-1) FW of cis-piceid were detected in 2-week-old plants, which represent a convenient source of the cis-isomers for pharmacological investigations. Our results also suggested the presence of unknown stilbene isomerase activities in Arabidopsis.

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

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.

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 an1, 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 Myb-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.

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.

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthraniloyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4-Coumaroyl-CoA was the preferred starter substrate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloyl-CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N-methylanthraniloyl-CoA as the starter substrate, while the conversion of cinnamoyl-CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mutagenesis.

Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of M r 42 681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70–75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four‐step purification protocol, and the affinities to N‐methylanthraniloyl‐CoA and malonyl‐CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44±3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81±4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.