De novo sequencing and analysis of Lophophora williamsii transcriptome, and searching for putative genes involved in mescaline biosynthesis.

BACKGROUND: Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites. RESULTS: In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR. CONCLUSIONS: High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

A tolerance gene for prenylated flavonoid encodes a 26S proteasome regulatory subunit in Sophora flavescens.

Yeast functional screening with a Sophora flavescens cDNA library was performed to identify the genes involved in the tolerant mechanism to the self-producing prenylated flavonoid sophoraflavanone G (SFG). One cDNA, which conferred SFG tolerance, encoded a regulatory particle triple-A ATPase 2 (SfRPT2), a member of the 26S proteasome subunit. The yeast transformant of SfRPT2 showed reduced SFG accumulation in the cells.

Prenylation of flavonoids by biotransformation of yeast expressing plant membrane-bound prenyltransferase SfN8DT-1.

Prenylated flavonoids are natural products that exhibit diverse biological effects and often represent the active components of various medicinal plants. This study demonstrated the production of prenylated naringenin by biotransformation using transgenic yeast expressing naringenin 8-dimethylallyltransferase, a membrane-bound enzyme, without feeding of prenyl donors. This method provides the possibility of generating prenylated flavonoids that occur rarely in nature.

Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids.

Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L-1 (0.35 µM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.

Genetic diversity, population structure, and conservation of Sophora moorcroftiana (Fabaceae), a shrub endemic to the Tibetan Plateau.

Sophora moorcroftiana is a perennial leguminous low shrub endemic to the middle reaches of Yarlung Zangbo River in Tibet. It is an important species to fix sand dunes and to avoid the formation of shifting sands; therefore, its progressive over-exploitation may enhance land desertification. The levels and distribution of genetic variability of this species were evaluated from 10 natural populations at 24 loci encoding 13 enzymes, using allozyme analysis by starch gel electrophoresis. Data obtained revealed moderate levels of genetic variation within populations (Pp=27.5%, Ap=1.5, Hep=0.122) and a considerable divergence among populations (FST=0.199). Significant positive correlations (r2=0.49, p<0.05; r2=0.46, p<0.05) were found between elevation and both mean number of alleles per locus (A) and gene diversity (He) in the studied populations of S. moorcroftiana. Lower genetic diversity in lower elevation populations might be due to the negative effects of human pressures and habitat fragmentation, to adaptation to high altitudes as a consequence of a peripatric speciation process, or to directional gene flow along the river basin from the source populations located in the west at higher altitudes. The evaluation of the degree of threat has led to the inclusion of this species in the category of EN ("endangered"), and conservation strategies for this endemic species are discussed on the basis of these findings.

Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes.

BACKGROUND: Nuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene. RESULTS: We isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event. CONCLUSION: This is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events.

8-dimethylallylnaringenin 2'-hydroxylase, the crucial cytochrome P450 mono-oxygenase for lavandulylated flavanone formation in Sophora flavescens cultured cells.

8-dimethylallylnaringenin (8-DMAN) 2'-hydroxylase, which is indispensable for the formation of a lavandulylated flavanone, sophoraflavanone G, was detected in cell suspension cultures of Sophora flavescens. The enzyme catalyzes the 2'-hydroxylation of 8-DMAN to leachianone G, and is tightly bound to the membrane. It required NADPH and molecular oxygen as cofactors, and was inhibited by several cytochrome P450 inhibitors such as carbon monoxide and cytochrome c, indicating that the reaction is mediated by a cytochrome P450 monooxygenase. The optimum pH of 8-DMAN 2'-hydroxylase was 8.5, and the enzyme hydroxylated only 8-DMAN. Apparent Km values for 8-DMAN and NADPH of the enzyme were 55 and 34 microM, respectively.

Isolation and characterization of a conserved domain in the eremophyte H+-PPase family.

H(+)-translocating inorganic pyrophosphatases (H(+)-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H(+)-PPase could be an early-originated protein that participated in many important biochemical and physiological processes. In this study we cloned 14 novel sequences from 7 eremophytes: Sophora alopecuroid (Sa), Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi), Suaeda salsa (Ss), Suaeda rigida (Sr), Halostachys caspica (Hc), and Karelinia caspia (Kc). These novel sequences included 6 ORFs and 8 fragments, and they were identified as H(+)-PPases based on the typical conserved domains. Besides the identified domains, sequence alignment showed that there still were two novel conserved motifs. A phylogenetic tree was constructed, including the 14 novel H(+)-PPase amino acid sequences and the other 34 identified H(+)-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these 48 H(+)-PPases were classified into two groups: type I and type II H(+)-PPase. The novel 14 eremophyte H(+)-PPases were classified into the type I H(+)-PPase. The 3D structures of these H(+)-PPase proteins were predicted, which suggested that all type I H(+)-PPases from higher plants and algae were homodimers, while other type I H(+)-PPases from bacteria and protozoans and all type II H(+)-PPases were monomers. The 3D structures of these novel H(+)-PPases were homodimers except for SaVP3, which was a monomer. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H(+)-PPase family.

Attenuation of ERK/RSK2-driven NFκB gene expression and cancer cell proliferation by kurarinone, a lavandulyl flavanone isolated from Sophora flavescens ait. roots.

We have analyzed in molecular detail how kurarinone, a lavandulyl flavanone isolated from Sophora flavescens, suppresses nuclear factor-κB (NFκB)-driven interleukin-6 (IL6) expression and cancer cell growth. Interleukin-6 (IL6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes responsivity to hormones and to chemotherapeutics. Our results in estrogen-unresponsive fibroblasts, ribosomal S6 kinase 2 kinase (RSK2) knockout cells, and estrogen receptor (ER)-deficient breast tumor cells show that kurarinone can inhibit tumor cell proliferation and selectively block nuclear NFκB transactivation of specific target genes such as IL6, cyclin D1, SOD2 but not TNFAIP2. This occurs via attenuation of extracellular signal-regulated protein (ERK) and RSK2 kinase pathways and inhibition of S6 kinase ribosomal protein (S6RP) and histone H3 S10 phosphorylation. As constitutive NFκB and RSK2 activity are important hallmarks of human cancers, including hematopoietic malignancies and solid tumors, prenylated flavanones represent an attractive class of natural inhibitors of the ERK/RSK2 signaling pathway for cancer therapy.

Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens.

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.

Characterization of leachianone G 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme for the formation of the lavandulyl group of sophoraflavanone G in Sophora flavescens Ait. cell suspension cultures.

Leachianone G (LG) 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2" position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2'-hydroxy function in LG was indispensable to the activity. The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 microm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2"-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2'-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.