Showing papers on "Chalcone synthase published in 2010"

Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor

Abstract: Chinese bayberry (Myrica rubra) is a fruit crop with cultivars producing fruit ranging from white (Shuijing, SJ) to red (Dongkui, DK) and dark red-purple (Biqi, BQ), as a result of different levels of anthocyanin accumulation. Genes encoding the anthocyanin biosynthesis enzymes chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), as well as MrMYB1, a R2R3 MYB transcription factor homologous to known activators of anthocyanin biosynthesis, were isolated from ripe fruit of BQ. Differences in mRNA abundance of MrF3H, MrF3′H, MrDFR1, MrANS and MrUFGT were highly correlated with differential accumulation of anthocyanins between cultivars, suggesting coordinated regulation by transcription factors. The transcript level of MrMYB1 was strongly associated with the anthocyanin content in ripe fruit of the three cultivars, as well as different anthocyanin containing tissues of BQ fruit. Fruit bagging strongly inhibited anthocyanin accumulation in fruit as well as the expression of all anthocyanin biosynthetic genes and MrMYB1. Over-expression of MrMYB1 stimulated both anthocyanin accumulation and activated an Arabidopsis-DFR promoter in tobacco (Nicotiana tabacum). MrMYB1d, an allele with a 1 bp deletion at nucleotide 30 of coding sequence, was observed in SJ and DK fruit, suggesting that a nonsense mutation of the MYB1 protein may be responsible for no or low expression of MYB1 in the white and red fruit. These results show that coordinated expression of multiple biosynthetic genes is involved in anthocyanin accumulation in Chinese bayberry fruit, and this is regulated by MrMYB1.

A SQUAMOSA MADS Box Gene Involved in the Regulation of Anthocyanin Accumulation in Bilberry Fruits

Abstract: Anthocyanins are important health-promoting phytochemicals that are abundant in many fleshy fruits. Bilberry (Vaccinium myrtillus) is one of the best sources of these compounds. Here, we report on the expression pattern and functional analysis of a SQUAMOSA-class MADS box transcription factor, VmTDR4, associated with anthocyanin biosynthesis in bilberry. Levels of VmTDR4 expression were spatially and temporally linked with color development and anthocyanin-related gene expression. Virus-induced gene silencing was used to suppress VmTDR4 expression in bilberry, resulting in substantial reduction in anthocyanin levels in fully ripe fruits. Chalcone synthase was used as a positive control in the virus-induced gene silencing experiments. Additionally, in sectors of fruit tissue in which the expression of the VmTDR4 gene was silenced, the expression of R2R3 MYB family transcription factors related to the biosynthesis of flavonoids was also altered. We conclude that VmTDR4 plays an important role in the accumulation of anthocyanins during normal ripening in bilberry, probably through direct or indirect control of transcription factors belonging to the R2R3 MYB family.

LAP5 and LAP6 Encode Anther-Specific Proteins with Similarity to Chalcone Synthase Essential for Pollen Exine Development in Arabidopsis

Abstract: Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

The Growth Reduction Associated with Repressed Lignin Biosynthesis in Arabidopsis thaliana Is Independent of Flavonoids

Abstract: Defects in phenylpropanoid biosynthesis arising from deficiency in hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) or p-coumaroyl shikimate 3′-hydroxylase (C3′H) lead to reduced lignin, hyperaccumulation of flavonoids, and growth inhibition in Arabidopsis thaliana. It was previously reported that flavonoid-mediated inhibition of auxin transport is responsible for growth reduction in HCT-RNA interference (RNAi) plants. This conclusion was based on the observation that simultaneous RNAi silencing of HCT and chalcone synthase (CHS), an enzyme essential for flavonoid biosynthesis, resulted in less severe dwarfing than silencing of HCT alone. In an attempt to extend these results using a C3′H mutant (ref8) and a CHS null mutant (tt4-2), we found that the growth phenotype of the ref8 tt4-2 double mutant, which lacks flavonoids, is indistinguishable from that of ref8. Moreover, using RNAi, we found that the relationship between HCT silencing and growth inhibition is identical in both the wild type and tt4-2. We conclude from these results that the growth inhibition observed in HCT-RNAi plants and the ref8 mutant is independent of flavonoids. Finally, we show that expression of a newly characterized gene bypassing HCT and C3′H partially restores both lignin biosynthesis and growth in HCT-RNAi plants, demonstrating that a biochemical pathway downstream of coniferaldehyde, probably lignification, is essential for normal plant growth.

Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis.

Abstract: Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies.

Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence

Abstract: Lignification of the fruit endocarp layer occurs in many angiosperms and plays a critical role in seed protection and dispersal. This process has been extensively studied with relationship to pod shatter or dehiscence in Arabidopsis. Dehiscence is controlled by a set of transcription factors that define the fruit tissue layers and whether or not they lignify. In contrast, relatively little is known about similar processes in other plants such as stone fruits which contain an extremely hard lignified endocarp or stone surrounding a single seed. Here we show that lignin deposition in peach initiates near the blossom end within the endocarp layer and proceeds in a distinct spatial-temporal pattern. Microarray studies using a developmental series from young fruits identified a sharp and transient induction of phenylpropanoid, lignin and flavonoid pathway genes concurrent with lignification and subsequent stone hardening. Quantitative polymerase chain reaction studies revealed that specific phenylpropanoid (phenylalanine ammonia-lyase and cinnamate 4-hydroxylase) and lignin (caffeoyl-CoA O-methyltransferase, peroxidase and laccase) pathway genes were induced in the endocarp layer over a 10 day time period, while two lignin genes (p-coumarate 3-hydroxylase and cinnamoyl CoA reductase) were co-regulated with flavonoid pathway genes (chalcone synthase, dihydroflavanol 4-reductase, leucoanthocyanidin dioxygen-ase and flavanone-3-hydrosylase) which were mesocarp and exocarp specific. Analysis of other fruit development expression studies revealed that flavonoid pathway induction is conserved in the related Rosaceae species apple while lignin pathway induction is not. The transcription factor expression of peach genes homologous to known endocarp determinant genes in Arabidopsis including SHATTERPROOF, SEEDSTCK and NAC SECONDARY WALL THICENING PROMOTING FACTOR 1 were found to be specifically expressed in the endocarp while the negative regulator FRUITFUL predominated in exocarp and mesocarp. Collectively, the data suggests, first, that the process of endocarp determination and differentiation in peach and Arabidopsis share common regulators and, secondly, reveals a previously unknown coordination of competing lignin and flavonoid biosynthetic pathways during early fruit development.

Differential Expression of Flavonoid Biosynthesis Genes and Accumulation of Phenolic Compounds in Common Buckwheat (Fagopyrum esculentum)

Abstract: Common buckwheat (Fagopyrum esculentum) is a short-season grain crop that is a source of rutin and other phenolic compounds. In this study, we isolated the cDNAs of 11 F. esculentum enzymes in the flavonoid biosynthesis pathway, namely, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) 1 and 2, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) 1 and 2, and anthocyanidin synthase (ANS). Quantitative real-time polymerase chain reaction analysis showed that these genes were most highly expressed in the stems and roots. However, high performance liquid chromatography analysis indicated that their flavonoid products, such as rutin and catechin, accumulated in the flowers and leaves. These results suggested that flavonoids may be transported within F. esculentum. In addition, light and dark growth conditions affected the expression levels of the biosynthesis genes and accumulation of phenolic compounds in F. esculentum sprouts.

Genome-wide analysis of the chalcone synthase superfamily genes of Physcomitrella patens

Abstract: Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of sec- ondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was infer- red from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron archi- tectures and results from phylogenetic analysis of repre- sentative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similari- ties, catalytic motifs and EST data indicated that a sur- prisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST dis- tribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.

Flavonoid accumulation in Arabidopsis thaliana root galls caused by the obligate biotrophic pathogen Plasmodiophora brassicae

Abstract: SUMMARY Three different flavonoids—naringenin, quercetin and kaempferol—accumulate in root galls of Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. In addition, high-performance liquid chromatography and thin layer chromatography analysis indicated that these flavonoids and their glycosides were induced in galls rather than in healthy roots. The transcripts of selected genes involved in the biosynthesis of flavonoids were up-regulated during the time course of the disease. Some, such as chalcone synthase and chalcone isomerase, were up-regulated at both times investigated in this study, whereas up-regulation was observed only at later times for others, such as a flavonol synthase-like gene. Plants with mutations in different flavonoid biosynthesis genes were slightly more tolerant to clubroot at low infection pressure. However, flavonoid treatment of either leaves or roots did not reduce gall development. The possibility that flavonoids might influence auxin levels by regulating auxin transport or auxin degradation in roots was investigated by measuring auxin levels and response in roots of flavonoid-deficient mutants and the wild-type after inoculation with P. brassicae, as well as the antioxidative potential of flavonoids in the peroxidase-catalysed degradation of indole-3-acetic acid. In addition, the auxin transport rate from the shoots to the roots was measured in infected wild-type or flavonoid mutant plants compared with controls. In conclusion, our results indicate a role of flavonoids in the modulation of auxin efflux in root galls.

Molecular dissection of the pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum.

Abstract: 3-Deoxyanthocyanidins are the unique phytoalexins synthesized by sorghum in response to fungal inoculation. They are structurally related to anthocyanins but the final steps of their pathogen-inducible biosynthesis are not fully understood. We have identified new flavonoid structural genes from the recently completed sorghum BTx623 genome sequence. The biochemical functions of the different expressed sorghum genes were established in planta by complementation in the appropriate Arabidopsis transparent testa mutants. There is a family of nine chalcone synthase genes which are all inducible by fungal inoculation in sorghum seedlings. Specific dihydroflavonol 4-reductase (DFR) genes responsive to conditions which stimulated anthocyanin accumulation (SbDFR1) or 3-deoxyanthocyanidin production (SbDFR3) were identified. Recombinant SbDFR1 and SbDFR3 were found to function as typical DFRs by accepting dihydroflavonol substrates. On the other hand, both DFRs showed substantially lower but detectable NADPH-dependent activities toward flavanones. Reduction of flavanones to flavan-4-ols is a reaction step required for 3-deoxyanthocyanidin production. Flavanone 3-hydroxylase (F3H) converts flavanones to dihydroflavonols for anthocyanin biosynthesis. In sorghum seedlings, expression of two F3H genes was either absent or strongly suppressed during the accumulation of 3-deoxyanthocyanidins. Under such conditions, most flavanones are expected to be reduced by the pathogen-induced SbDFR3 for the formation of flavan-4-ols. Our work also revealed that 3-deoxyanthocyanidin accumulation and SbDFR3 expression were induced by methyl jasmonate treatment in sorghum roots but the stimulation effects were antagonized by salicylic acid.

Differential expression of CHS7 and CHS8 genes in soybean.

Abstract: Chalcone synthase (CHS) catalyzes the first reaction specific for flavonoid and isoflavonoid biosynthesis. The soybean genome consists of nine copies of CHS genes (CHS1-CHS9) and a duplicate copy of CHS1. Even though the soybean CHS gene family members share a high degree of sequence similarity, they play different roles during plant development or in response to environmental stimuli. Our previous work on the comparison of a global gene expression in two soybean cultivars that differ in the level of total isoflavonoid accumulation has denoted the involvement of CHS7 and CHS8 genes in isoflavonoid synthesis. We have extended our effort to understand expression patterns of these two genes in soybean and in transgenic Arabidopsis. Promoter regions of CHS7 and CHS8 genes were isolated and in silico analysis performed to investigate potential transcription factor binding sites (TFBSs). The TFBSs were verified by DNase I footprint analysis. Some unique and several common TFBSs were identified in CHS7 and CHS8 promoters. We cloned beta-glucuronidase (GUS) under CHS7 and CHS8 promoters and monitored the tissue-specific GUS expression in transformed Arabidopsis. Differential GUS activity was observed in young leaves, roots, and mature pod walls of transgenic CHS7 promoter-GUS and CHS8 promoter-GUS plants. The tissue-specific expression patterns of CHS7 and CHS8 genes were determined in soybean by quantitative RT-PCR. Both CHS7 and CHS8 genes were expressed at higher levels in roots; however, overall expression pattern of these genes varied in different tissues. The results suggest that the structural diversity within CHS7 and CHS8 promoters may lead into differential activation of these genes by different inducers as well as developmental stage- and tissue-specific differences in gene expression.

Cloning and molecular characterization of R2R3-MYB and bHLH-MYC transcription factors from Citrus sinensis

Abstract: Members of the MYB and MYC family regulate the biosynthesis of phenylpropanoids in several plant species. Two sequences, called CsMYB8 and CsMYC2, were identified from Citrus sinensis, and both the cDNA and the genomic clones were isolated and characterized from the flesh of common and blood oranges. Analysis by real-time polymerase chain reaction showed that the expression pattern of CsMYC2 is generally higher in rind than in flesh and in blood oranges than in common ones. In contrast, no significant difference in expression was observed for CsMYB8. The expression pattern of the structural genes chalcone synthase, anthocyanidin synthase, and UDP-glucose-flavonoid 3-O-glucosyltransferase, which code for three enzymes involved in the anthocyanin biosynthetic pathway, was also analyzed and correlated with CsMYC2, in flesh, rind, and leaf of the common and blood oranges, and in leaf of Citrus limon cultivars (characterized by anthocyanin absence or variable content). Surprisingly, CsMYC2 is highly expressed in the leaf and expression is correlated with UFGT expression in this organ. These results suggest that CsMYC2 is involved in the regulation of the flavonoid biosynthetic pathway in Citrus.

Structure and Function of the Chalcone Synthase Superfamily of Plant Type III Polyketide Synthases

Abstract: Covering: 2002 to 2009 This review covers recent advances in structure and function studies on the chalcone synthase (CHS) superfamily of plant type III polyketide synthases (PKSs), which catalyze iterative decarboxylative condensations of malonyl unit with a CoA-linked starter molecule to produce structurally diverse, pharmaceutically important plant secondary metabolites. The functional diversity and catalytic potential of the type III PKSs are remarkable. Studies on the enzymes are now progressing rapidly; recent crystallographic and site-directed mutagenesis studies have revealed intimate structural details of the enzyme reactions, which enabled the structure-based and precursor-directed engineered biosynthesis of unnatural novel polyketides. The literature of type III PKSs of plant origin published over the last eight years will be reviewed, and is intended to compliment the coverage of the literature by Austin and Noel in Nat. Prod. Rep., 2003, 20, 79–110.

Photosynthetic redox imbalance influences flavonoid biosynthesis in Lemna gibba

Abstract: Plants accumulate flavonoids in response to a myriad of environmental challenges, especially when exposed to ultraviolet (UV) radiation or situations causing oxidative stress. However, the origin and nature of the signal triggering their accumulation remain obscure. In this study, a group of flavonoids belonging to the flavone class was identified in Lemna gibba (duckweed). These flavones accumulated upon exposure to UV radiation, low temperature, copper and the photosynthetic electron transport (PET) inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and 1,2-dihydroxyanthraquinone (DHATQ). All of these stressors were also shown to promote PET chain (PETC) reduction; however, in the co-presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or a light regime that oxidized the PETC, flavonoid accumulation ceased. Chloroplast-derived reactive oxygen species (ROS) were not associated with all of the stress conditions that promoted both PETC reduction and flavonoid synthesis, indicating that ROS were not a strict requisite for flavonoid accumulation. Transcripts for the flavonoid biosynthetic genes, chalcone synthase (CHS) and chalcone isomerase, were similarly responsive to the PETC redox state, as were a panel of transcripts revealed by differential display PCR. Collectively, these results provide evidence that PETC redox status is one of the factors affecting flavonoid biosynthesis.

A structure-based mechanism for benzalacetone synthase from Rheum palmatum

Abstract: Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 A resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 A resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase's coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

Cloning and Molecular Analysis of HlbZip1 and HlbZip2 Transcription Factors Putatively Involved in the Regulation of the Lupulin Metabolome in Hop (Humulus lupulus L.)

Abstract: Hop (Humulus lupulus L.), the essential source of beer flavor is of interest from a medicinal perspective in view of its high content in health-beneficial terpenophenolics including prenylflavonoids. The dissection of biosynthetic pathway(s) of these compounds in lupulin glands, as well as its regulation by transcription factors (TFs), is important for efficient biotechnological manipulation of the hop metabolome. TFs of the bZIP class were preselected from the hop transcriptome using a cDNA-AFLP approach and cloned from a cDNA library based on glandular tissue-enriched hop cones. The cloned TFs HlbZIP1A and HlbZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively, and both are similar to the group A3 bZIP TFs according to the composition of characteristic domains. While HlbZIP1A is rather neutral (pI 6.42), HlbZIP2 is strongly basic (pI 8.51). A truncated variant of HlbZIP1 (HlbZIP1B), which is strongly basic but lacks the leucine zipper domain, has also been cloned from hop. Similar to the previously cloned HlMyb3 from hop, both bZIP TFs show a highly specific expression in lupulin glands, although low expression was observed also in other tissues including roots and immature pollen. Comparative functional analyses of HlbZip1A, HlbZip2, and subvariants of HlMyb3 were performed in a transient expression system using Nicotiana benthamiana leaf coinfiltration with Agrobacterium tumefaciens strains bearing hop TFs and selected promoters fused to the GUS reference gene. Both hop bZIP TFs and HlMyb3 mainly activated the promoters of chalcone synthase chs_H1 and the newly cloned O-methyl transferase 1 genes, while the response of the valerophenone synthase promoter to the cloned hop TFs was very low. These analyses also showed that the cloned bZIP TFs are not strictly G-box-specific. HPLC analysis of secondary metabolites in infiltrated Petunia hybrida showed that both hop bZIP TFs interfere with the accumulation and the composition of flavonol glycosides, phenolic acids, and anthocyanins, suggesting the possibility of coregulating flavonoid biosynthetic pathways in hop glandular tissue.

Expression for caffeine biosynthesis and related enzymes in Camellia sinensis.

Abstract: Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid that is present in high concentrations in the tea plant Camellia sinensis. Caffeine synthase (CS, EC 2.1.1.160) catalyzes the S-adenosyl-L-methionine-dependent N-3- and N-1-methylation of the purine base to form caffeine, the last step in the purine alkaloid biosynthetic pathway. We studied the expression profile of the tea caffeine synthase (TCS) gene in developing leaves and flowers by means of northern blot analysis, and compared it with those of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), and S-adenosyl-L-methionine synthase (SAMS, EC 2.5.1.6). The amount of TCS transcripts was highest in young leaves and declined markedly during leaf development, whereas it remained constant throughout the development of the flower. Environmental stresses other than heavy metal stress and plant hormone treatments had no effect on the expression of TCS genes, unlike the other three genes. Drought stress suppressed TCS gene expression in leaves, and the expression pattern mirrored that of the dehydrin gene. The amounts of TCS transcripts increased slightly on supply of a nitrogen source. We discuss the regulation of TCS gene expression.

Response of the phenylpropanoid pathway to Venturia inaequalis infection in maturing fruit of ‘Braeburn’ apple

Abstract: SummaryThis study investigated the activities of key enzymes in the phenylpropanoid pathway and the accumulation of phenolic products in apple peel in response to infection with Venturia inaequalis [(Cooke) G. Wint.]. We compared healthy apple peel with apple peel showing symptomatic scab lesions, and with peel tissue from 1 – 2 mm around the scab lesions in fruit 1 month before maturity [140 d after full bloom (DAFB)] and at physiological maturity (175 DAFB). Infection with V. inaequalis enhanced the synthesis of some phenolic compounds. Compared to healthy peel, scab lesion tissue had ≤ 3.1-times higher hydroxycinnamic acid content, ≤ 1.3-times higher dihydrochalcone content, and ≤ 3.9-times higher flavan-3-ol content. Scab lesions showed slightly higher phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, flavonol synthase, and dihydroflavonol 4-reductase activities. The total amount of phenolics remained relatively stable between the two sampling dates, except for epicatechin and caffei...

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

Abstract: A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

Isolation and characterization of chalcone synthase gene isolated from Dendrobium Sonia Earsakul.

Abstract: To isolate and characterize chalcone synthase gene in anthocyanin biosynthetic pathway during flower development of Dendrobium Sonia Earsakul. The gene was isolated from floral tissues of the orchid by reverse transcriptase polymerase chain reaction. Characterization of the gene considered to its relatedness to chalcone synthase gene in other orchid plants elucidated by construction of a neighbor-joining phylogenetic tree. Gene expression pattern related to flower development and pigmentation was investigated by relative quantification real time polymerase chain reaction. A complete coding sequence was obtained and sequence analysis revealed that the gene of Dendrobium Sonia Earsakul consisted of 1,188 bp. Blast analysis and multiple alignments showed that the chalcone synthase gene of Dendrobium Sonia Earsakul shares high homology to chalcone synthase gene of Dendrobium genus particularly Dendrobium hybrid Uniwai prince. Phylogenetic tree revealed that chalcone synthase of Dendrobium genus are highly conserved. The chalcone synthase gene of Dendrobium Sonia Earsakul was highly expressed in young flower bud with no pigmentation and the expression was sharply decreased when young flower bud started accumulation of pigments. Expression of chalcone synthase gene was then maintained at the same level until young bud developed into fully opened flowers.

Stimulation of Defense Reactions in Medicago truncatula by Antagonistic Lipopeptides from Paenibacillus sp. Strain B2

Abstract: With the aim of obtaining new strategies to control plant diseases, we investigated the ability of antagonistic lipopolypeptides (paenimyxin) from Paenibacillus sp. strain B2 to elicit hydrogen peroxide (H2O2) production and several defense-related genes in the model legume Medicago truncatula. For this purpose, M. truncatula cell suspensions were used and a pathosystem between M. truncatula and Fusarium acuminatum was established. In M. truncatula cell cultures, the induction of H2O2 reached a maximum 20 min after elicitation with paenimyxin, whereas concentrations higher than 20 μM inhibited H2O2 induction and this was correlated with a lethal effect. In plant roots incubated with different concentrations of paenimyxin for 24 h before inoculation with F. acuminatum, paenimyxin at a low concentration (ca. 1 μM) had a protective effect and suppressed 95% of the necrotic symptoms, whereas a concentration higher than 10 μM had an inhibitory effect on plant growth. Gene responses were quantified in M. truncatula by semiquantitative reverse transcription-PCR (RT-PCR). Genes involved in the biosynthesis of phytoalexins (phenylalanine ammonia-lyase, chalcone synthase, chalcone reductase), antifungal activity (pathogenesis-related proteins, chitinase), or cell wall (invertase) were highly upregulated in roots or cells after paenimyxin treatment. The mechanisms potentially involved in plant protection are discussed.

Defence genes and antioxidant enzymes in stage-dependent resistance to rice neck blast

Abstract: The rice variety Jiajing3768 is susceptible to neck blast at the booting stage (BS) but resistant at the full heading stage (FHS). This variety was used to analyze the inducible expression of defence genes and activities of antioxidant enzymes in the necks, at both the BS and FHS, after inoculation with Magnaporthe oryzae. We found that defence genes PR1b (pathogenesis-related class 1b), PBZ1 (probenazole-inducible gene), PAL (phenylalanine ammonia-lyase), and CHS (chalcone synthase) may play roles in the resistance difference to neck blast between the BS and FHS in Jiajing3768. No significant differences were observed between the BS and FHS in the inducible expression of PR1a, PR4, and JIOsPR10 (jasmonic acid induced PR 10). The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participated in the stage-dependent resistance difference in Jiajing3768, and that oxidative damage is lower in the necks at the FHS than at the BS.