荞麦黄酮及其生物合成调控研究进展

荞麦属植物资源丰富,且富含黄酮类成分.通过文献查阅,总结了荞麦黄酮历年研究情况以及热点研究领域.荞麦黄酮研究论文最早发表于1952年,在1952—1999近五十年的时间内,荞麦黄酮的研究论文较少,年发文量少于10篇,荞麦黄酮的研究处于起步阶段.自2000年后,荞麦黄酮逐渐获得更多研究学者的关注,年度发文量逐年上升.近年...     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Detection of flavonoids in microalgae from different evolutionary lineages

Flavonoids are important secondary plant metabolites believed to be present mainly in land plants. As phenolics were detected previously in microalgae using photometric assays, we wanted to investigate the nature of these phenolics and verify whether flavonoids are present. Therefore, in this study, we used state‐of‐the‐art ultra‐high performance liquid chromatography‐two‐dimensional mass spectrometry (UHPLC‐MS/MS) technology to investigate whether microalgae also contain flavonoids. For this, representative microalgal biomass samples from divergent evolutionary lineages (Cyanobacteria, Rhodophyta, Chlorophyta, Haptophyta, Ochrophyta) were screened for a set of carefully selected precursors, intermediates, and end products of the flavonoid biosynthesis pathways. Our data unequivocally showed that microalgae contain a wide range of flavonoids and thus must possess the enzyme pool required for their biosynthesis. Further, some of the microalgae displayed an intricate flavonoid pattern that is compatible with the established basic flavonoid pathway as observed in higher plants. This implies that the flavonoid biosynthesis pathway arose much earlier in evolution compared to what is generally accepted.     

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t‐resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β‐cyclodextrins

The growing demand for t‐resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large‐scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β‐cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t‐resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t‐resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non‐bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Direct isolation of flavonoids from plants using ultra‐small anatase TiO2 nanoparticles

Surface functionalization of nanoparticles has become an important tool for in vivo delivery of bioactive agents to their target sites. Here we describe the reverse strategy, nanoharvesting, in which nanoparticles are used as a tool to isolate bioactive compounds from living cells. Anatase TiO₂ nanoparticles smaller than 20 nm form strong bonds with molecules bearing enediol and especially catechol groups. We show that these nanoparticles enter plant cells, conjugate enediol and catechol group‐rich flavonoids in situ, and exit plant cells as flavonoid‐nanoparticle conjugates. The source plant tissues remain viable after treatment. As predicted by the surface chemistry of anatase TiO₂ nanoparticles, quercetin‐based flavonoids were enriched amongst the nanoharvested flavonoid species. Nanoharvesting eliminates the use of organic solvents, allows spectral identification of the isolated compounds, and opens new avenues for use of nanomaterials for coupled isolation and testing of bioactive properties of plant‐synthesized compounds.     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H]⁺. Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS³ experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula.     

Plant Flavonoid <Emphasis Type="Italic">O</Emphasis>-Methyltransferases: Substrate Specificity and Application

Flavonoids consist of a large family of compounds, which has been estimated to be more than 10,000 compounds. The structural diversity of these compounds comes from different modification reactions. The O-methylation reaction is one of the most important modification reactions of flavonoids and the resulting O-methylated flavonoids have been shown to display new biological activities. The regioselective and substrate specific O-methylation is mediated by O-methyltranferases (OMTs). To date, 30 flavonoid OMTs (FOMTs) have been biochemically characterized from various plants. FOMTs utilize common reaction mechanisms to transfer a methyl group to the hydroxyl group of the flavonoid. Phylogenetic tree analysis along with biochemical characterization of FOMTs provides clues about their substrate specificity and regioselectivity. FOMTs can be used for the production of O-methylated flavonoids that have a particular biological activity.     

Elicitation with methyl jasmonate combined with cultivation in the Plantform™ temporary immersion bioreactor highly increases the accumulation of selected centellosides and phenolics in Centella asiatica (L.) Urban shoot culture

Centella asiatica (L.) Urban is an important pharmacopoeial plant used not only in medicine but also in cosmetology. C. asiatica agitated shoot cultures were established to study the influence of ethephon, methyl jasmonate, L ‐phenylalanine (Eth 50 µM, MeJa 50 µM, L‐Phe 2.4 g/L of medium, respectively; seven variants of the supplementation) on the accumulation of secondary metabolites: the main centellosides (asiaticoside and madecassoside) and selected phenolic acids, and flavonoids in the biomass. Microshoots were harvested two and six days after the supplementation. Secondary metabolites were analyzed in methanolic extracts by UPLC‐MS/MS (centellosides) and by HPLC‐DAD (phenolics). In comparison with the reference cultures, the concentrations of individual secondary metabolites increased as follows: centellosides up to 5.6‐fold (asiaticoside), phenolic acids up to 122‐fold (p‐coumaric acid) and flavonoids up to 22.4‐fold (kaempherol). The highest production increase of individual compounds was observed for different variants of supplementation. Variant C (50 µM MeJa), the most optimal for centellosides and flavonoid accumulation, was selected for the experiment with bioreactors. Bioreactor Plantform?, compared to RITA^® system and agitated cultures, appeared to be the most advantageous for secondary metabolites production in C. asiatica shoot cultures. The phenolic acid, flavonoid, centelloside, and total secondary metabolite productivity in Plantform? system is 1.8‐fold, 1.7‐fold, 2.8‐fold, 2.1‐fold, respectively, higher than in MeJa elicitated agitated cultures, and 4.3‐fold, 7.3‐fold, 12.2‐fold, 7.2‐fold, respectively, higher than in control agitated cultures.     

The control of red colour by a family of MYB transcription factors in octoploid strawberry (Fragaria × ananassa) fruits

Octoploid strawberry (Fragaria × ananassa Duch.) is a model plant for research and one of the most important non‐climacteric fruit crops throughout the world. The associations between regulatory networks and metabolite composition were explored for one of the most critical agricultural properties in octoploid strawberry, fruit colour. Differences in the levels of flavonoids are due to the differences in the expression of structural and regulatory genes involved in flavonoid biosynthesis. The molecular mechanisms underlying differences in fruit colour were compared between red and white octoploid strawberry varieties. FaMYB genes had combinatorial effects in determining the red colour of fruit through the regulation of flavonoid biosynthesis in response to the increase in endogenous ABA at the final stage of fruit development. Analysis of alleles of FaMYB10 and FaMYB1 in red and white strawberry varieties led to the discovery of a white‐specific variant allele of FaMYB10, FaMYB10‐2. Its coding sequence possessed an ACTTATAC insertion in the genomic region encoding the C‐terminus of the protein. This insertion introduced a predicted premature termination codon, which suggested the loss of intact FaMYB10 protein playing a critical role in the loss of red colour in white octoploid strawberry.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

In vitro Antioxidant and Anti‐inflammatory Activities of Extracts from Nicotiana tabacum L. (Solanaceae) Leafy Galls Induced by Rhodococcus fascians

Plant galls are widely distributed, and their extracts are used in traditional medicine worldwide. Traditional remedies containing extracts of plant galls in China, India and some African countries have effective in the treatment of various pathologies. To open a new promising procedure for screening bioactive compounds from plant galls, standardized plant materials were generated in vitro and used for phytochemical and biological investigations. Methanol aqueous chloroform and hexane extracts of Nicotiana tabacum leafy galls induced by Rhodococcus fascians were used to evaluate phenolic and flavonoid contents, and to investigate antioxidant activity by 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging and ferric reducing antioxidant/power assays and anti‐inflammatory activity by the lipoxygenase inhibition assay. Infection by R. fascians modifies significantly the phytochemical profile of N. tabacum as well as its biological properties. The total polyphenolic content was increased (120–307%), and that of flavonoids was reduced (20–42.5%). Consequently, antioxidant and anti‐inflammatory activities of non‐infected tobacco extracts are significantly modified compared to plants treated with leafy gall extracts. This shows that infection by R. fascians favoured the production of anti‐inflammatory and antioxidant compounds in N. tabacum. The study indicates the benefit of plant galls used in traditional medicines against various pathologies.