Showing papers on "Chalcone synthase published in 2009"
TL;DR: Observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonolsmight act as auxin transport regulators during nodulation in M. truncatula.
Abstract: Flavonoids play critical roles in legume-rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti, flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non-nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula.
234 citations
TL;DR: Production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.
Abstract: Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.
135 citations
TL;DR: Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid‐producing tissues of C. sativa, also exhibited olivetol‐producing activity, suggesting that the native OLS is functionally expressed in these tissues.
135 citations
TL;DR: It is shown that anthocyanins accumulate in both wild type and egl3, but not in gl3 loss-of-function mutants when depleted of nitrogen, and low expression of DFR appears to be the bottleneck preventing Anthocyanin synthesis in the gl3 mutant.
Abstract: The bHLH transcription factors EGL3 (ENHANCER OF GLABRA3) and its close homologue GL3 (GLABRA3) are important regulators of the anthocyanin pathway in Arabidopsis thaliana, and together with TTG1 (a WD40 repeat protein) and MYB transcription factors regulate specific genes in the pathway. In response to nitrogen depletion, the MYB genes PAP1/PAP2 (production of anthocyanin pigment 1/2) and GL3 are strongly induced, and anthocyanin synthesis is activated in seedlings and rosette stage plants. In this study we show that anthocyanins accumulate in both wild type and egl3, but not in gl3 loss-of-function mutants when depleted of nitrogen. Several structural genes of flavonoid metabolism including CHS (chalcone synthase), FLS1 (flavonol synthase 1) and ANS (anthocyanidin synthase) were induced in response to nitrogen depletion in wild type as well as in the egl3 and gl3 mutants. Strikingly, in the gl3 mutant DFR (dihydroflavonol-4-reductase) transcript level was only 2% of the levels in wild type or egl3 mutant. Hence, low expression of DFR appears to be the bottleneck preventing anthocyanin synthesis in the gl3 mutant. The specific effect on DFR, but not ANS is compatible with involvement of the MYBL2 inhibitor.
129 citations
TL;DR: Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
Abstract: RNA isolated from the glands of a D 9 -tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
126 citations
TL;DR: Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions showed that the amount of 73 proteins consistently changed in 50 μg/mL chitosan‐treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses.
Abstract: Polyphenols, including stilbenes and flavonoids, are an essential part of human diet and constitute one of the most abundant and ubiquitous groups of plant secondary metabolites, and their level is inducible by stress, fungal attack or biotic and abiotic elicitors. Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions, showed that the amount of 73 proteins consistently changed in 50 microg/mL chitosan-treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses. In particular, de-novo synthesis and/or accumulation of stilbene synthase proteins were promoted by chitosan which also stimulated trans-resveratrol endogenous accumulation and decreased its release into the culture medium. No influence was shown on cis-resveratrol. There was no effect on the accumulation of total resveratrol mono-glucosides (trans- and cis-piceid and trans- and cis-resveratroloside). Throughout the observation period the upregulation of phenylalanine ammonia lyase, chalcone synthase, chalcone-flavanone isomerase (CHI) transcript expression levels well correlated with CHI protein amount and with the accumulation of anthocyanins. Chitosan treatment strongly increased the expression of eleven proteins of the pathogenesis related protein-10 family, as well as their mRNA levels.
110 citations
TL;DR: The hypothesis that the dihydrochalcone core structure is formed by the common CHS underpins the absence of phloridzin in the closely related pear, which is based on the lack of ability to catalyze the first step in the phlorin biosynthesis.
102 citations
TL;DR: This is the first report on the heterologous expression of plant phenylpropanoid biosynthetic pathways in Streptomyces genus.
69 citations
TL;DR: Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus, and the changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification.
Abstract: The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria x ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.
59 citations
TL;DR: The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves, which included genes of phenylalanine ammonia-lyase 1 (PAL1) and flavanone 3-hydroxylase (F3H; EC 1.1.3.11).
55 citations
TL;DR: Results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.
Abstract: Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.
TL;DR: RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant, and type III PKS genes studied so far contained only one intron at a conserved site in flowering plants.
Abstract: A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS “gatekeepers” Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.
TL;DR: A suppression subtractive hybridization analysis approach was undertaken to identify genes involved in cold acclimation of tea (Camellia sinensis O. Kuntze), a broad-leaved, woody evergreen species, and predicted proteins encoded by the remaining four transcripts were putatively involved in primary and secondary metabolism.
Abstract: A suppression subtractive hybridization analysis approach was undertaken to identify genes involved in cold acclimation of tea (Camellia sinensis (L.) O. Kuntze), a broad-leaved, woody evergreen species. A total of ten complementary DNA (cDNA) clones, induced by low temperature in tea leaves, were identified. These transcripts could be classified into three functional groups. Three transcripts encoded transcriptional factors. Among these, Cs-COR375 contained a putative open reading frame encoding a zinc-finger protein with two EPF-type zinc fingers, while Cs-COR041 and Cs-COR178 were corresponded to CBF-like genes. The predicted products of Cs-COR044, Cs-COR196, and Cs-COR213 cDNAs were cell structure proteins. Cs-COR044 encoded a putative 86 amino acid protein consisting of a hydrophobic N-terminal half and a hydrophilic C-terminal half relatively rich in glycine and proline. The protein was predicted to be exported to the apoplast and likely to be localized in the cell wall. Cs-COR196 and Cs-COR213 encoded for two putative early light-induced proteins, a group of nuclear-encoded thylakoid membrane proteins accumulating in response to various stresses. The predicted proteins encoded by the remaining four transcripts were putatively involved in primary and secondary metabolism. The product encoded by Cs-COR018 was β-amylase, involved in cold acclimation of various plant species. Cs-COR126 coded for chalcone synthase, while Cs-COR054 and Cs-COR113 coded for two different flavonol synthases.
TL;DR: Malonyl-CoA synthetase (MCS) was used in conjunction with chalcone synthase (CHS), thereby allowing efficient use of synthetic starter molecules and malonate as extender and resulted in up to a 30-fold higher yield of pyrone CHS products.
TL;DR: It might be concluded that the global regulatory steps of the flavonoid pathway are conserved amongst V. vinifera, M. rotundifolia and V. aestivalis.
TL;DR: In an effort to alter the types and levels of flavanone neohesperidosides in citrus, an Agrobacterium-mediated genetic transformation approach was employed and transgenic plants with decreased leaf naringin levels were obtained.
Abstract: Citrus species accumulate large quantities of flavanone glycosides in their leaves and fruit. The physiological role(s) of these compounds in citrus plants are unknown, but they have been documented to benefit human health upon consumption. Flavanone rutinosides are tasteless, whereas flavanone neohesperidosides, such as naringin, give a bitter taste to fruit and fruit juice products, reducing their palatability. In an effort to alter the types and levels of flavanone neohesperidosides in citrus, an Agrobacterium-mediated genetic transformation approach was employed. Citrus paradisi Macf. (grapefruit) epicotyl stem segments were transformed with sense (S) and antisense (AS) constructs of the target genes chalcone synthase (CHS) and chalcone isomerase (CHI), whose products catalyze the first two steps in the flavonoid biosynthetic pathway. Transformation with each of the individual constructs led to a different and unpredictable combination of viability, phenotypic change, transgene steady-state expression and alteration in flavonoid content in the resulting transgenic plants. These qualities were consistent within the transgenic plants obtained using any particular construct. Transgenic plants with decreased leaf naringin levels were obtained, particularly when the CHS-AS constructs were employed.
TL;DR: Functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, chalcone sythase (CHS) and BAS activity, and it is possible that such compounds are also produced in that plant, albeit in low concentrations.
Abstract: Benzalacetone synthase (BAS) is a member of the plant-specific type III PKS superfamily that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzalacetone. In our recent work (Ma et al. in Planta 229(3):457-469, 2008), a three-intron type III PKS gene (PcPKS2) was isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed this recombinant PcPKS2 to be a BAS. In this study, another three-intron type III PKS gene (PcPKS1) and its corresponding cDNA were isolated from P. cuspidatum. Sequence and phylogenetic analyses demonstrated that PcPKS1 is a chalcone sythase (CHS). However, functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, CHS and BAS activity. DNA gel blot analysis indicated that there are two to four CHS copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS1 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS1 transcripts in leaves were inducible by pathogen infection and wounding. BAS is thought to play a crucial role in the construction of the C(6)-C(4) moiety found in a variety of phenylbutanoids, yet so far phenylbutanoids have not been isolated from P. cuspidatum. However, since PcPKS1 and PcPKS2 (Ma et al. in Planta 229(3):457-469, 2008) have been identified in P. cuspidatum, it is possible that such compounds are also produced in that plant, albeit in low concentrations.
TL;DR: The results demonstrate that the level of flavonoids in lemon balm can be increased significantly and that plants such as Lemon balm could potentially be used to prevent diseases that have been purported to be caused by free radical damage.
Abstract: Medicinal plants are widely used in traditional medicine because plant secondary metabolites have been shown to benefit a broad spectrum of health conditions. Lemon balm, Melissa officinalis L., a member of the mint family, is native to Europe and is well known for its ability to reduce stress and anxiety, promote sleep, and ease pain and discomfort associated with digestion. In various plant species, strong anthocyanin induction is triggered by sucrose, but not by other sugars or osmotic stress; however, the mechanisms that induce anthocyanin accumulation in lemon balm leaves in response to sucrose and phytohormones remain unclear. In this study, we investigated the mechanisms that lead to increased levels of flavonoids in lemon balm plants. We observed that sucrose significantly increases the level of flavonoids in lemon balm plants and that sucrose induction appears to be mediated by the phytohormones abscisic acid and ethylene. We also identified delphinidin as the anthocyanidin that is primarily enriched in leaves grown in high-sucrose medium. Finally, we observed that reactive oxygen species levels are positively correlated with sucrose-mediated anthocyanin accumulation. Taken together, our results demonstrate that the level of flavonoids in lemon balm can be increased significantly and that plants such as lemon balm could potentially be used to prevent diseases that have been purported to be caused by free radical damage. Chemical abbreviations used: ABA, (+)-cis, transabscissic acid; ACC, 1-aminocyclopropane-carboxylic acid; CHI, chalcone isomerase; CHS, chalcone synthase; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; GA, gibberellic acid; IAA, indole-3-acetic acid.
01 Jan 2009
TL;DR: To study different structural genes expression pattern in anthocyanin biosynthesis pathway of cineraria with different flower color, internal segments of five structural genes,chalcone synthase(CHS),chalcone isomerase (CHI),flavanone 3-hydroxylase(F3H),flavonoid 3'-hydroxyase( F3'H) and dihydroflav onol 4-reductase(DFR), were isolated from c
Abstract: To study different structural genes expression pattern in anthocyanin biosynthesis pathway of cineraria with different flower color,internal segments of five structural genes,chalcone synthase(CHS),chalcone isomerase(CHI),flavanone 3-hydroxylase(F3H),flavonoid 3'-hydroxylase(F3'H)and dihydroflavonol 4-reductase(DFR),were isolated from cineraria petals using RT-PCR.Using semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR),the expression patterns of CHS,CHI,F3H,F3'H,DFR,flavonoid 3',5'-hydroxylase(F3'5'H)and anthocyanin 3-O-glucoside-6"-O-malonyltransferase(3MaT)in five developmental stages of cineraria with different flower colors were determined for the first time.The results indicated that there is no CHS transcript in white flowers.In red flowers,F3'H mRNA was expressed at high level.But no F3'5'H transcript was found.F3'5'H was highly expressed in Stage I but no F3'H mRNA was observed in blue flowers.And the transcripts of F3'H and F3'5'H were found in purple petals.In addition,these genes were expressed at high level in early stage and underwent moderate decreases in expression.Interestingly,genes expression level increased again in stage Ⅳ.However,in stage V,there is little expression level could be detected in petals.
TL;DR: RNA gel blot analysis in different tissues indicated that endogenous CHS siRNAs are accumulated only in the seed coat, supporting the suggestion that CHS PTGS is seed coat-specific.
Abstract: Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The inhibition of seed coat pigmentation is due to naturally occurring posttranscriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. In the present study, RNA gel blot analysis in different tissues indicated that endogenous CHS siRNAs are accumulated only in the seed coat, supporting the suggestion that CHS PTGS is seed coat-specific. The probe region of a CHS gene was divided into several parts, each of which was used for RNA gel blot analysis in the seed coat. Interestingly, endogenous CHS siRNAs were only detected with probes corresponding to the exon 2 region. The results were confirmed by deep sequencing analysis of CHS siRNAs in the seed coat, i.e., CHS siRNAs were predominantly derived from the 3′-half of exon 2 of the CHS gene. Mapping of CHS siRNA sequences on CHS genes and an inverted repeat region of the CHS pseudogene (pseudoCHS3) suggested that primary CHS siRNAs may be generated from double-stranded RNA of pseudoCHS3, and subsequently secondary CHS siRNAs may be produced according to the two-hit model, which has been proposed to explain siRNA amplification by the RNA-dependent RNA polymerase.
TL;DR: The results suggested that the engineered biosynthesis of plant polyketide synthases by combination of the structure-based and the precursor-directed approach would lead to further production of chemically and structurally divergent unnatural novel polyketides.
Abstract: Pentaketide chromone synthase (PCS) and octaketide synthase (OKS) are novel plant-specific type III polyketide synthases (PKSs) obtained from Aloe arborescens. Recombinant PCS expressed in Escherichia coli catalyzes iterative condensations of five molecules of malonyl-CoA to produce a pentaketide 5,7-dihydroxy-2-methylchromone, while recombinant OKS carries out sequential condensations of eight molecules of malonyl-CoA to yield octaketides SEK4 and SEK4b, the longest polyketides produced by the structurally simple type III PKS. The amino acid sequences of PCS and OKS are 91% identical, sharing 50–60% identity with those of other chalcone synthase (CHS) superfamily type III PKSs of plant origin. One of the most characteristic features is that the conserved active-site Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed mutagenesis and X-ray crystallographic analyses demonstrated that the chemically inert single residue lining the active-site cavity controls the polyketide chain length and the product specificity depending on the steric bulk of the side chain. On the basis of the crystal structures, an F80A/Y82A/M207G triple mutant of the pentaketide-producing PCS was constructed and shown to catalyze condensations of nine molecules of malonyl-CoA to produce an unnatural novel nonaketide naphthopyrone, whereas an N222G mutant of the octaketides-producing OKS yielded a decaketide benzophenone SEK15 from ten molecules of malonyl-CoA. On the other hand, the type III PKSs exhibited broad substrate specificities and catalytic potential. OKS accepted p-coumaroyl-CoA as a starter substrate to produce an unnatural novel C19 hexaketide stilbene and a C21 heptaketide chalcone. Remarkably, the C21 chalcone-forming activity was dramatically increased in the structure-guided OKS N222G mutant. In addition, OKS N222G mutant also yielded unnatural novel polyketides from phenylacetyl-CoA and benzoyl-CoA as a starter substrate. These results suggested that the engineered biosynthesis of plant polyketides by combination of the structure-based and the precursor-directed approach would lead to further production of chemically and structurally divergent unnatural novel polyketides.
TL;DR: Comparative and bioinformatic analyses revealed that the deduced protein of CsCHS-bo was highly homologous to CHS from other plant species, and was in a subgroup with CHS of Ruta Palmatum.
Abstract: Chalcone synthase (CHS) is involved in the biosynthesis of anthocyanin. In this study, a full-length DNA of CHS gene (named as CsCHS-bo) was cloned from the blood orange, Citrus sinensis (L.) Osbeck cv. Ruby. The gene was 1,512 bp in size containing an open reading frame (1,176 bp) encoding 391 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of CsCHS-bo was highly homologous to CHS from other plant species. The protein of CsCHS-bo had four CHS-specific conserved motifs and a CHS-family signature sequence GFGPG. Phylogenetic analysis indicated that the protein of CsCHS-bo was in a subgroup with CHS of Ruta Palmatum. The CsCHS-bo was localized to the chromosomes 2p, 4p and 6p by an improved fluorescence in situ hybridization technique, indicating that at least three copies of CsCHS-bo were present in the genome.
TL;DR: Exposure to UV-B radiation has a significant effect on gene expression in rice and may activate secondary metabolic pathways, in turn increase the synthesis of UV-protective substances (flavonoids) and induce the transcription of the relevant mRNAs for repairing the damaged DNA.
Abstract: The differences in gene expression between two rice genotypes—the ultraviolet-B (UV-B)-resistant cultivar Lemont and the UV-B-sensitive cultivar Dular—in response to irradiation with UV-B were studied. Suppression subtractive hybridization and real-time quantitative reverse transcriptase polymerase chain reaction were used in the analysis. The results showed that enhanced UV-B radiation could induce differential expression of the genes related to defense response. The expression of genes encoding HECT domain-containing protein, thylakoid-bound ascorbate peroxidase, receptor-like protein kinase, Rad6 (ubiquitin carrier protein), phenylalanine ammonia-lyase, 4-coumarate: CoA ligase and chalcone synthase was up-regulated to different extents in the two rice cultivars: The cultivar Lemont exhibited a higher increase in the gene expression compared with the cultivar Dular. A large difference was found in the expression pattern of the universal stress protein (USP): showing down-regulation in Dular but up-regulation in Lemont. Hence exposure to UV-B radiation has a significant effect on gene expression in rice. In response to UV exposure, a molecular response is initially induced in different UV-B-resistant rice cultivars. This response may activate secondary metabolic pathways, in turn increase the synthesis of UV-protective substances (flavonoids) to prevent UV-B damage and induce the transcription of the relevant mRNAs for repairing the damaged DNA.
TL;DR: The findings of the study highlight the necessity of investigating the molecular response of a plant to a pathogen by studying not only an individual gene of the phenylpropanoid pathway but several genes and their gene homologs.
Abstract: The impact of Rhizoctonia solani on gene expression of 14 genes and gene homologs encoding key enzymes leading to isoflavone synthesis in the phenylprapanoid pathway was quantified in soybean (Glycine max) seedlings using quantitative reverse transcription polymerase chain reaction. This method allowed accurate quantitative evaluation of expression ratios of genes encoding phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone reductase (CHR), and isoflavone synthase (IFS) in 1-, 2-, and 3-week-old soybean seedlings infected with R. solani and in seedlings that were not infected. Rhizoctonia solani inoculations affected expression ratios of 4 of 14 genes. Compared with control seedlings, a substantial upregulation of CHS 5, CHS 6, and IFS 2 transcript ratios by 34%, 208%, and 226% was observed, respectively. Ratios of the gene encoding CHR were markedly upregulated in infected seedlings at 1 and 2 weeks postinfection with a substantial downregulation 3 weeks after i...
TL;DR: Results demonstrate that CHS family from S. baicalensis has the typical molecular structure and function of chalcone synthase, compared with the experimental data for Medicago sativa CHS protein.
Abstract: Prediction and analysis of molecular structure and biochemical function are of theoretical guiding significance for gene discovery and application, and considered as one of the central problem of computational biology. Here, some characteristic features of chalcone synthase (CHS) family from Scutellaria baicalensis were described via bioinformatic analysis, and showed as following: the nucleic acid sequences and amino acid sequences of three chs member genes shared high similarity in the molecular structures and physicochemical properties; SbCHS proteins were localized to the cytosol, and possessed a good hydrophobic nature, with lacking any transmembrane topological structure. The phylogram analysis suggested that they were a group genes with significant functional association and genetic conservation. The secondary structures of the SbCHSs were mainly composed of α-helixes and random coils, and the tertiary structures contained malonyl CoA linkers, besides, each of CHS-A and CHS-B with N-glycosylation motif included. Taken together, these results demonstrate that CHS family from S. baicalensis has the typical molecular structure and function of chalcone synthase, compared with the experimental data for Medicago sativa CHS protein.
TL;DR: The results indicate that the members of rice CHS superfamily probably diverged into four branches, and that two conserved chs genes in one distant branch might play some functions in fertility.
Abstract: Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme in the biosynthesis of flavonoids, which plays an important role in flower pigmentation and protection against UV, plant-microbe interactions, and plant fertility. In many plants, genes encoding CHS constitute a multigene family, wherein sequence and functional divergence occurred repeatedly. Since the genome of rice (Oryza sativa) has been completely sequenced, many genes possessing typical CHS domains were assumed to be chs genes, although the sequence and functional divergence of this large gene family has not as yet been investigated. In this study, all putative CHS members from O. sativa were analyzed by the phylogenetic methods. Our results indicate that the members of rice CHS superfamily probably diverged into four branches. Members of each branch may perform specific functions. Two conserved chs genes clustered with chs genes from other monocotyledon and dicotyledon species are believed to encode true CHSs responsible for the biosynthesis of flavonoids and anthocyanins. Two chs genes in one distant branch might play some functions in fertility. Several other putative chs genes were clustered together, and the function of this branch could not be predicted. Many tentative chs genes were clustered together with fatty acid synthase (FAS) genes. These genes may belong to the fas gene family.
TL;DR: The silencing of CHS genes produces a highly visible phenotype, lack of color in the seed coat or flower, that facilitated the discovery of anthocyanin pigments.
Abstract: Chalcone synthase (CHS) is required for the biosynthesis of anthocyanin pigments that give color to various plant tissues, such as the flower and seed coat. The silencing of CHS genes produces a highly visible phenotype, lack of color in the seed coat or flower, that facilitated the discovery of
TL;DR: The increasing expression of GmmyBJ6 in soybean cultivar Zhongdou 27, induced by UV-B radiation, drought, and high-salt treatment, indicated that GmMYBJ 6 was associated with response to abiotic stresses.
Abstract: MYB transcription factor is one of the largest families in plants, which plays an important role in regulating plant development and physiological metabolism. In this study, the expression and function of the new MYB transcription factor gene GmMYBJ6 (GenBank No. DQ902863), isolated from soybean (Glycine max L.), were characterized. The expression pattern of GmMYBJ6 in different organs was examined using Northern blotting analysis. The expression of GmMYBJ6 was detected only in the leaves. The transcriptional activation ability of GmMYBJ6 protein was confirmed by the yeast assay system and the activity of beta-galactosidase was 28.48 U/mL. The green fluorescent protein expression vector p163-GFP-GmMYBJ6 was constructed and transformed into the epidermal cells of onion via particle bombardmental method. The results of instantaneous expression showed that GmMYBJ6 proteins were localized in cell nucleus. Semi-quantitative RT-PCR analysis indicated that GmMYBJ6 improved the expression of certain flavonoid biosynthetic genes, such as PAL (Phenylalanine ammonia lyase), C4H (cinnamate-4-hydroxylase), 4CL (4-coumaroyl-CoA ligase), CHS (Chalcone synthase), CHI (Chalcone isomerase), F3H (Flavanone 3-hydroxylase), and FLS (Flavonol synthase), resulting an increase of the total flavonoid levels in positive tobacco transformants. Additionally, the increasing expression of GmMYBJ6 in soybean cultivar Zhongdou 27, induced by UV-B radiation, drought, and high-salt treatment, indicated that GmMYBJ6 was associated with response to abiotic stresses.
TL;DR: A growing number of functionally divergent chalcone synthase (CHS) superfamily type III polyketide synthases (PKSs) have been cloned and characterized as mentioned in this paper.
Abstract: A growing number of functionally divergent the chalcone synthase (CHS) superfamily type III polyketide synthases (PKSs) have been cloned and characterized, which include recently obtained pentaketide chromone synthase (PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens). Recombinant PCS expressed in Escherichia coli catalyzes iterative condensations of five molecules of malonyl-CoA to produce a pentaketide, 5,7-dihydroxy-2-methylchromone, while OKS carries out sequential condensations of eight molecules of malonyl-CoA to yield aromatic octaketides, SEK4 and SEK4b, the longest polyketides generated by the structurally simple type III PKS. The two enzymes share 91% amino acid sequence identity, maintaining most of the active-site residues of CHS including the Cys-His-Asn catalytic triad. One of the most characteristic features is that the conserved Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed mutagenesis and X-ray crystallographic studies clearly demonstrated that the chemically inert single residue lining the active-site cavity controls the polyketide chain length and the product specificity depending on the steric bulk of the side chain. Finally, on the basis of the crystal structures of both wild-type and M207G-mutant PCS, a triple mutant PCS F80A/Y82A/M207G was constructed and shown to catalyze condensations of nine molecules of malonyl-CoA to produce a novel nonaketide naphthopyrone with a fused tricyclic ring system. Structure-based engineering of the type III PKS superfamily enzymes would thus lead to further production of chemically and structurally divergent unnatural novel polyketides.
Journal Article•
TL;DR: In this article, two flax lines overexpressing genes encoding three essential enzymes of the flavonoid synthesis pathway: chalcone synthase (CHS), chchcone isomerase (ChI), and dihydroflavonol reductase (DFR) were generated.
Abstract: Two flax lines overexpressing genes encoding three essential enzymes of the flavonoid synthesis pathway: chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR), were generated. Phenolic compounds were extracted from the flaxseed lines and the control sample (Linola) using 80% methanol. Then the complex of phenolic compounds present in the crude extract was hydrolysed at basic conditions. The crude and hydrolysed extracts were analysed using an RP-HPLC-DAD method. The chromatograms of the crude extracts of the control sample and transgenic lines were the same. The finger print analysis of the hydrolysed extracts shows the influence of the expression of genes encoding three above mentioned enzymes on the proportion between secoisolariciresinol diglucoside (SDG) and phenolic acids derivatives forming the phenolic complex in flaxseed.