Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

Regulatory ligand binding in plant chalcone isomerase-like (CHIL) proteins.

Chalcone isomerase-like (CHIL) protein is a noncatalytic protein that enhances flavonoid content in green plants by serving as a metabolite binder and a rectifier of chalcone synthase (CHS). Rectification of CHS catalysis occurs through direct protein-protein interactions between CHIL and CHS, which alter CHS kinetics and product profiles, favoring naringenin chalcone (NC) production. These discoveries raise questions about how CHIL proteins interact structurally with metabolites and how CHIL-ligand interactions affect interactions with CHS. Using differential scanning fluorimetry on a CHIL protein from Vitis vinifera (VvCHIL), we report that positive thermostability effects are induced by the binding of NC, and negative thermostability effects are induced by the binding of naringenin. NC further causes positive changes to CHIL-CHS binding, whereas naringenin causes negative changes to VvCHIL-CHS binding. These results suggest that CHILs may act as sensors for ligand-mediated pathway feedback by influencing CHS function. The protein X-ray crystal structure of VvCHIL compared with the protein X-ray crystal structure of a CHIL from Physcomitrella patens reveals key amino acid differences at a ligand-binding site of VvCHIL that can be substituted to nullify the destabilizing effect caused by naringenin. Together, these results support a role for CHIL proteins as metabolite sensors that modulate the committed step of the flavonoid pathway.

Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus.

BACKGROUND: Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones. METHODS AND RESULTS: In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4 kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16 °C and partially purified. CONCLUSION: These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

Flavonoids are involved in salt tolerance through ROS scavenging in the halophyte Atriplex canescens.

KEY MESSAGE: The content of flavonoids could increase in A. canescens under saline conditions. Overexpression of AcCHI in transgenic A. thaliana promotes flavonoid biosynthesis, thereby functioning in the tolerance of transgenic plants to salt and osmotic stress by maintaining ROS homeostasis. Atriplex canescens is a halophytic forage shrub with excellent adaptation to saline environment. Our previous study showed that a large number of genes related to the biosynthesis of flavonoids in A. canescens were significantly up-regulated by NaCl treatments. However, it remains unclear whether flavonoids are involved in A. canescens response to salinity. In this study, we found that the accumulation of flavonoids significantly increased in either the leaves or roots of A. canescens seedling under 100 and 300 mM NaCl treatments. Correspondingly, AcCHS, AcCHI and AcF3H, which encode three key enzymes (chalcone synthases (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), respectively) of flavonoids biosynthesis, were significantly induced in the roots or leaves of A. canescens by 100 or 300 mM NaCl. Then, we generated the transgenic Arabidopsis thaliana overexpressing AcCHI and found that transgenic plants accumulated more flavonoids through enhancing the pathway of flavonoids biosynthesis. Furthermore, overexpression of AcCHI conferred salt and osmotic stress tolerance in transgenic A. thaliana. Contrasted with wild-type A. thaliana, transgenic lines grew better with greater biomass, less H2O2 content as well as lower relative plasma permeability in either salt or osmotic stress conditions. In conclusion, our results indicate that flavonoids play an important role in A. canescens response to salt stress through reactive oxygen species (ROS) scavenging and the key enzyme gene AcCHI in flavonoids biosynthesis pathway of A. canescens has the potential to improve the stress tolerance of forages and crops.

Managing enzyme promiscuity in plant specialized metabolism: A lesson from flavonoid biosynthesis: Mission of a "body double" protein clarified.

Specificities of enzymes involved in plant specialized metabolism, including flavonoid biosynthesis, are generally promiscuous. This enzyme promiscuity has served as an evolutionary basis for new enzyme functions and metabolic pathways in land plants adapting to environmental challenges. This phenomenon may lead, however, to inefficiency in specialized metabolism and adversely affect metabolite-mediated plant survival. How plants manage enzyme promiscuity for efficient specialized metabolism is, thus, an open question. Recent studies of flavonoid biosynthesis addressing this issue have revealed a conserved strategy, namely, a homolog of chalcone isomerase with no catalytic activity binds to chalcone synthase, a key flavonoid pathway enzyme, to narrow (or rectify) the enzyme's highly promiscuous product specificity. Reducing promiscuity via specific protein-protein interactions among metabolic enzymes and proteins may be a solution adopted by land plants to achieve efficient operation of specialized metabolism, while the intrinsic promiscuity of enzymes has likely been retained incidentally.

Genetic Channelization Mechanism of Four Chalcone Isomerase Homologous Genes for Synergistic Resistance to Fusarium wilt in Gossypium barbadense L.

Duplication events occur very frequently during plant evolution. The genes in the duplicated pathway or network can evolve new functions through neofunctionalization and subfunctionalization. Flavonoids are secondary metabolites involved in plant development and defense. Our previous transcriptomic analysis of F6 recombinant inbred lines (RILs) and the parent lines after Fusarium oxysporum f. sp. vasinfectum (Fov) infection showed that CHI genes have important functions in cotton. However, there are few reports on the possible neofunctionalization differences of CHI family paralogous genes involved in Fusarium wilt resistance in cotton. In this study, the resistance to Fusarium wilt, expression of metabolic pathway-related genes, metabolite content, endogenous hormone content, reactive oxygen species (ROS) content and subcellular localization of four paralogous CHI family genes in cotton were investigated. The results show that the four paralogous CHI family genes may play a synergistic role in Fusarium wilt resistance. These results revealed a genetic channelization mechanism that can regulate the metabolic flux homeostasis of flavonoids under the mediation of endogenous salicylic acid (SA) and methyl jasmonate (MeJA) via the four paralogous CHI genes, thereby achieving disease resistance. Our study provides a theoretical basis for studying the evolutionary patterns of homologous plant genes and using homologous genes for molecular breeding.

Yeast-based system for in vivo evaluation of alleles of the anthocyanin production pathway.

Plants produce anthocyanins to incite the pollination and seed dispersion performed by pigment-attracted animals. These natural blue-to-red-coloured pigments can be used as food colourants and antioxidants. For this purpose, microbial bioproduction of anthocyanins has become of industrial interest in recent years. 20 new alleles of anthocyanin production pathway genes were extracted and characterised for protein expression level and stability using a developed single-PCR product gene-entry system for tagged protein synthesis in yeast S. cerevisiae. Enzymatic activities of these proteins in the episomally complemented in vivo systems were compared by HPLC-MS analysis. Results show that the codon optimisation of the anthocyanin pathway genes is not essential for the effective heterologous expression in yeast. Elevating the cellular abundance of CHS and F3H enzymes can increase anthocyanidin production from supplemented precursors. New alleles VmF3Hv1 and VuCHS were shown to have the best performance in the analysed system. System complementation with flavonoid 3',5'-hydroxylase substantially increases total anthocyanidin production. The described single-entry yeast episomal complementation system is a convenient and rapid tool for the complex evaluation of new alleles in vivo.

[Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro].

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Elusive partners: a review of the auxiliary proteins guiding metabolic flux in flavonoid biosynthesis.

Flavonoids are specialized metabolites widely distributed across the plant kingdom. They are involved in the growth and survival of plants, conferring the ability to filter ultra-violet rays, conduct symbiotic partnerships, and respond to stress. While many branches of flavonoid biosynthesis have been resolved, recent discoveries suggest missing auxiliary components. These overlooked elements can guide metabolic flux, enhance production, mediate stereoselectivity, transport intermediates, and exert regulatory functions. This review describes several families of auxiliary proteins from across the plant kingdom, including examples from specialized metabolism. In flavonoid biosynthesis, we discuss the example of chalcone isomerase-like (CHIL) proteins and their non-catalytic role. CHILs mediate the cyclization of tetraketides, forming the chalcone scaffold by interacting with chalcone synthase (CHS). Loss of CHIL activity leads to derailment of the CHS-catalyzed reaction and a loss of pigmentation in fruits and flowers. Similarly, members of the pathogenesis-related 10 (PR10) protein family have been found to differentially bind flavonoid intermediates, guiding the composition of anthocyanins. This role comes within a larger body of PR10 involvement in specialized metabolism, from outright catalysis (e.g., (S)-norcoclaurine synthesis) to controlling stereochemistry (e.g., enhancing cis-trans cyclization in catnip). Both CHILs and PR10s hail from larger families of ligand-binding proteins with a spectrum of activity, complicating the characterization of their enigmatic roles. Strategies for the discovery of auxiliary proteins are discussed, as well as mechanistic models for their function. Targeting such unanticipated components will be crucial in manipulating plants or engineering microbial systems for natural product synthesis.

Flavonoid Metabolism in Tetrastigma hemsleyanum Diels et Gilg Based on Metabolome Analysis and Transcriptome Sequencing.

Tetrastigma hemsleyanum Diels et Gilg, known as a "plant antibiotic", possesses several attractive properties including anti-inflammatory, anti-tumor, and antioxidant effects, with its efficacy being attributed to flavonoids. However, the flavonoid biosynthesis of T. hemsleyanum has rarely been studied. In this study, we investigated the flavonoid metabolism of T. hemsleyanum through metabolome analysis and transcriptome sequencing. The metabolomic results showed differences in the flavonoids of the leaves and root tubers of T. hemsleyanum. A total of 22 flavonoids was detected, and the concentrations of most flavonoids in the leaves were higher than those in the root tubers. Transcriptome analysis revealed that differentially expressed genes (DEGs) in the leaves and root tubers were enriched in photosynthesis-antenna proteins. Pearson correlation analysis indicated that the expression levels of chalcone isomerase (CHI) and UDP-glycose flavonoid glycosyltransferase (UFGT) were highly correlated with the concentrations of most flavonoids. Further, this study found that the photosynthesis-antenna proteins essentially contributed to the difference in the flavonoids in T. hemsleyanum. The gene expressions and concentrations of the total flavonoids of leaves and root tubers in Hangzhou, Jinhua, Lishui, and Taizhou in Zhejiang Province, China, showed that CHI (CL6715.Contig1_All, Unigene19431_All, CL921.Contig4_All) and UFGT (CL11556.Contig3_All, CL11775.Contig1_All) were the potential key genes of accumulation of most flavonoids in T. hemsleyanum.

[Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides].

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

Overexpression of chalcone isomerase in apple reduces phloridzin accumulation and increases susceptibility to herbivory by two-spotted mites.

Apples (Malus spp.) accumulate significant quantities of the dihydrochalcone glycoside, phloridzin, whilst pears (Pyrus spp.) do not. To explain this difference, we hypothesized that a metabolic bottleneck in the phenylpropanoid pathway might exist in apple. Expression analysis indicated that transcript levels of early phenylpropanoid pathway genes in apple and pear leaves were similar, except for chalcone isomerase (CHI), which was much lower in apple. Apples also showed very low CHI activity compared with pear. To relieve the bottleneck at CHI, transgenic apple plants overexpressing the Arabidopsis AtCHI gene were produced. Unlike other transgenic apples where phenylpropanoid flux was manipulated, AtCHI overexpression (CHIox) plants were phenotypically indistinguishable from wild-type, except for an increase in red pigmentation in expanding leaves. CHIox plants accumulated slightly increased levels of flavanols and flavan-3-ols in the leaves, but the major change was a 2.8- to 19-fold drop in phloridzin concentrations compared with wild-type. The impact of these phytochemical changes on insect preference was studied using a two-choice leaf assay with the polyphagous apple pest, the two-spotted spider mite (Tetranychus urticae Koch). Transgenic CHIox leaves were more susceptible to herbivory, an effect that could be reversed (complemented) by application of phloridzin to transgenic leaves. Taken together, these findings shed new light on phenylpropanoid biosynthesis in apple and suggest a new physiological role for phloridzin as an antifeedant in leaves.

Three AP2/ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus.

Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)-naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference-induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration-responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB-RAV transcriptional complex regulating flavonoid production.

Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.

Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.

Adaptive Evolution of Chalcone Isomerase Superfamily in Fagaceae.

Chalcone Isomerase (CHI) catalyzes the biosynthesis of flavonoids and secondary metabolism in plants. Currently, there is no systematic analysis of CHIs gene family in Fagaceae which is available. In this study, twenty-two CHI proteins were identified in five species of the Fagaceae family. The CHI superfamily in Fagaceae can be classified into three subfamilies and five groups using phylogenetic analysis, analysis of physicochemical properties, and structural prediction. Results indicated that serine (Ser) and isoleucine (Ile) residues determine the substrate preferred by active Type I Fagaceae CHI, and the chalcone isomerase-like (CHIL) of Fagaceae had active site residues. Adaptive analysis of CHIs showed that CHIs are subject to selection pressure. The active CHI gene of Fagaceae was located in the cytoplasm, and it had the typical gene structure of CHI and contains four exons. All the twenty-two identified CHIs had the conserved domain motif 3, and the different groups had their own structural characteristics. In the process of fatty acid binding protein (FAP) evolution to CHIL and CHI, the physical and chemical properties of proteins also had significant differences in addition to changes in protein functions.

Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

Identification and evolutionary analysis of chalcone isomerase-fold proteins in ferns.

The distribution of type I and II chalcone isomerases (CHIs) in plants is highly family specific. We have previously reported that ancient land plants, such as the liverworts and Selaginella moellendorffii, harbor type II CHIs. To better understand the function and evolution of CHI-fold proteins, transcriptomic data obtained from 52 pteridophyte species were subjected to sequence alignment and phylogenetic analysis. The residues determining type I/II CHI identity in the pteridophyte CHIs were identical to those of type I CHIs. The enzymatic characterization of a sample of 24 CHIs, representing all the key pteridophyte lineages, demonstrated that 19 of them were type I enzymes and that five exhibited some type II activity due to an amino acid mutation. Two pteridophyte chalcone synthases (CHSs) were also characterized, and a type IV CHI (CHIL) was demonstrated to interact physically with CHSs and CHI, and to increase CHS activity by decreasing derailment products, thus enhancing flavonoid production. These findings suggest that the emergence of type I CHIs may have coincided with the divergence of the pteridophytes. This study deepens our understanding of the molecular mechanism of CHIL as an enhancer in the flavonoid biosynthesis pathway.

Integrating molecular characterization and metabolites profile revealed CtCHI1's significant role in Carthamus tinctorius L.

BACKGROUND: As a traditional Chinese herb, safflower (Carthamus tinctorius L.) is valued for its florets to prevent cardiovascular and cerebrovascular diseases. Basing on previous chemical analysis, the main active compounds are flavonoids in its florets. Although flavonoid biosynthetic pathway has been well-documented in many model species, unique biosynthetic pathway remains to be explored in safflower. Of note, as an important class of transitional enzymes, chalcone isomerase (CHI) has not been characterized in safflower. RESULTS: According to our previous research, CHIs were identified in a safflower transcriptome library built by our lab. To characterize CHI in safflower, a CHI gene named CtCHI1 was identified. A multiple sequences alignment and phylogenetic tree demonstrate that CtCHI1 shares 92% amino acid identity and close relationship with CHI to Saussurea medusa. Additionally, subcellular localization analysis indicated CtCHI1-GFP fusion protein was mainly in the cell nucleus. Further, we purified CtCHI1 protein from E. coli which can effectively catalyze isomerization of 2',4',4,6'-tetrahydroxychalcone into naringenin in vitro. Via genetic engineer technology, we successfully obtained transgenic tobacco and safflower lines. In transgenic tobacco, overexpression of CtCHI1 significantly inhibited main secondary metabolites accumulation, including quercetin (~ 79.63% for ovx-5 line) and anthocyanins (~ 64.55% for ovx-15 line). As shown in transgenic safflower, overexpression of CtCHI1 resulted in upstream genes CtPAL3 and CtC4H1 increasing dramatically (up to ~ 3.9fold) while Ct4CL3, CtF3H and CtDFR2 were inhibited. Also, comparing the whole metabolomics database by PCA and PLS-DA between transgenic and control group, 788 potential differential metabolites were marked and most of them displayed up-regulated trends. In parallel, some isolated secondary metabolites, such as hydroxysafflor yellow A (HSYA), rutin, kaempferol-3-O-ß-rutinoside and dihydrokaempferol, accumulated in transgenic safflower plants. CONCLUSIONS: In this study, we found that CtCHI1 is an active, functional, catalytic protein. Moreover, CtCHI1 can negatively and competitively regulate anthocyanins and quercetin pathway branches in tobacco. By contrast, CtCHI1 can positively regulate flavonol and chalcone metabolic flow in safflower. This research provides some clues to understand CHI's differential biochemical functional characterization involving in flavonoid pathway. More molecular mechanisms of CHI remain to be explored in the near future.

[Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum].

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced ß-sandwich fold. A large ß-sheet( ß4-ß11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning ß4,ß5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

[Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju'].

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using ß-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.