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.

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.

New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida.

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called "darkly-veined" petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All "darkly-veined" genotypes have the identical mutation in the AN2 gene, but express a different functional paralog - ANTHOCYANIN-4 (AN4) - abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis - CHALCONE ISOMERASE-A (CHI-A) - the plant responded directly by upregulating of another paralogs - DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI-B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the "darkly-veined" phenotypes as a result of the breeding process under human selection, and CHI-B is a backup for CHI-A acitvity in some floral tissues.

Cloning, Identification, and Functional Analysis of the Chalcone Isomerase Gene from Astragalus sinicus.

Astragalus sinicus is an important winter-growing cover crop. It is widely utilized, not only as a cover crop for its benefits in fertilizing the soil but also as a landscape ground cover plant. Anthocyanins are involved in the pigmentation of plants in leaves and flowers, which is a crucial characteristic trait for A. sinicus. The formation of anthocyanins depends significantly on the enzyme chalcone isomerase (CHI). However, research on the CHI gene of A. sinicus remains unexplored. The rapid amplification of cDNA ends (RACE) approach was used in this research to clone the CHI sequence from A. sinicus (AsiCHI). The expression profiles of the AsiCHI gene in multiple tissues of A. sinicus were subsequently examined by qRT-PCR (Quantitative Real-Time PCR). Furthermore, the function of the AsiCHI was identified by the performance of ectopic expression in Arabidopsis (Arabidopsis thaliana). The outcomes revealed that the full-length cDNA of the AsiCHI gene (GeneBank: OQ870547) measured 972 bp in length and included an open reading frame of 660 bp. The encoded protein contains 219 amino acids with a molecular weight of 24.14 kDa and a theoretical isoelectric point of 5.11. In addition, the remarkable similarity between the AsiCHI protein and the CHI proteins of other Astragalus species was demonstrated by the sequence alignment and phylogenetic analysis. Moreover, the highest expression level of AsiCHI was observed in leaves and showed a positive correlation with anthocyanin content. The functional analysis further revealed that the overexpression of AsiCHI enhanced the anthocyanidin accumulation in the transgenic lines. This study provided a better understanding of AsiCHI and elucidated its role in anthocyanin production.

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.

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.

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.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

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.

Exogenous γ-aminobutyric acid strengthens phenylpropanoid and nitrogen metabolism to enhance the contents of flavonoids, amino acids, and the derivatives in edamame.

γ-aminobutyric acid (GABA) has been reported to improve stress resistance in plants. Nonetheless, little is known about the effects of GABA on the nutritional quality and regulatory mechanisms of edamame. Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and the effects of GABA on the nutrient content of edamame seeds through physiological and metabolomic analyses. Exogenous GABA increased endogenous GABA metabolism and GABA transaminase activity and enhanced the oxoglutarate content, which entered into nitrogen metabolism and increased the activity and expression of nitrogen metabolism-related enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous GABA induced the metabolism of flavonoids, including total flavonoids, anthocyanins, 6''-o-malonyglycitin, glycitin, ononin, cyanin, and ginkgetin, by increasing the activity and expression of flavonoid biosynthetic enzymes. This is the first study to reveal that GABA effectively improves the nutritional quality of edamame through the accumulation of AAs, bioactive peptides, isoflavones, anthocyanins, sugars, and organic acids.

Genome-Wide Classification and Evolutionary Analysis Reveal Diverged Patterns of Chalcone Isomerase in Plants.

Flavonoids as a class of important secondary metabolites are widely present in land plants, and chalcone isomerase (CHI) is the key rate-limiting enzyme that participates in catalyzing the stereospecific isomerization of chalcones to yield their corresponding flavanones. However, the phylogenetic dynamics and functional divergence of CHI family genes during the evolutionary path of green plants remains poorly understood. Here, a total of 122 CHI genes were identified by performing a genome-wide survey of 15 representative green plants from the most ancestral basal plant chlorophyte algae to higher angiosperm plants. Phylogenetic, orthologous groups (OG) classification, and genome structure analysis showed that the CHI family genes have evolved into four distinct types (types I-IV) containing eight OGs after gene duplication, and further studies indicated type III CHIs consist of three subfamilies (FAP1, FAP2, and FAP3). The phylogeny showed FAP3 CHIs as an ancestral out-group positioned on the outer layers of the main branch, followed by type IV CHIs, which are placed in an evolutionary intermediate between FAP3 CHIs and bona fide CHIs (including type I and type II). The results imply a potential intrinsic evolutionary connection between CHIs existing in the green plants. The amino acid substitutions occurring in several residues have potentially affected the functional divergence between CHI proteins. This is supported by the analysis of transcriptional divergence and cis-acting element analysis. Evolutionary dynamics analyses revealed that the differences in the total number of CHI family genes in each plant are primarily attributed to the lineage-specific expansion by natural selective forces. The current studies provide a deeper understanding of the phylogenetic relationships and functional diversification of CHI family genes in green plants, which will guide further investigation on molecular characteristics and biological functions of CHIs.

Integrative analysis of transcriptome and metabolome reveals the effect of DNA methylation of chalcone isomerase gene in promoter region on Lithocarpus polystachyus Rehd flavonoids.

Metabolite biosynthesis is regulated by gene expression, which is altered by DNA methylation in the promoter region. Chalcone isomerase (CHI) gene encodes a key enzyme in the Lithocarpus polystachyus Rehd flavonoid pathway, and the expression of L. polystachyus CHI (LpCHI) is closely related to the synthesis of flavonoid metabolites. In this study, we analyzed the DNA methylation site of the LpCHI promoter and its effect on gene expression and metabolite accumulation. The proportions of three types of LpCHI promoter DNA methylation are 7.5%, 68.75%, 18.75%, determined by bisulfite sequencing. Transcriptome sequencing shows that LpCHI is strongly up-regulated in LpCHI promoter methylation Type A but down-regulated in LpCHI promoter methylation Type B and Type C. The expression of LpCHI shows no significant difference between Type B and Type C. Moreover, nine kinds of differentially expressed transcription factors (DETFs) bind to seven CpG-sites of the LpCHI promoter region to regulate LpCHI expression. The results of metabolomics show that differentially accumulated flavonoids are higher in LpCHI promoter methylation Type A than in LpCHI promoter methylation Type B and Type C. Additionally, a positive correlation was found between the LpCHI expression and flavonoids accumulation. These results show that the effect of CpG site-specificity on gene transcription is great than that of overall promoter DNA methylation on gene transcription. The mechanisms of flavonoid genes regulating metabolite accumulation are further revealed.

AfCHIL, a Type IV Chalcone Isomerase, Enhances the Biosynthesis of Naringenin in Metabolic Engineering.

Naringenin is an essential precursor for all flavonoids, and effectively promoting naringenin production is crucial in metabolic engineering. The interaction between plant metabolic enzymes ensures metabolic flux. The effect can effectively improve the natural product synthesis of engineering microbial systems. In this study, chalcone isomerase genes in Allium fistulosum have been identified. The expression of AfCHIL is closely related to the accumulation of anthocyanins, and the expression of AfCHIL and AfCHS was highly synchronized. Yeast two-hybrid and firefly luciferase complementation imaging assay further confirmed AfCHIL physically interacted with AfCHS/AfCHI. The bioconversion experiment confirmed that AfCHIL reduced the derailment produced by AfCHS and increased the yield of naringenin. In addition, a system of biosynthesis naringenin involved in AfCHS was constructed, and these results suggested that the potential function between CHS with CHIL advanced naringenin production effectively. In conclusion, this study illustrated the function of AfCHIs in Allium fistulosum and provided new insight into improving the synthesis efficiency of naringenin.

Virtual screening and docking analysis of novel ligands for selective enhancement of tea (Camellia sinensis) flavonoids.

Flavour of tea is mainly contributed by a group of polyphenols - flavonoids. However, the content of flavonoid fluctuates seasonally and is found to be higher in the first flush of tea, when compared to the second flush. This disparity in the flavonoid content, and hence taste, incurs heavy economic losses to the tea plantation industry each harvest season. For our present study, four key product-specific enzymes (PAL, FNS, FLS and ANS) of the tea-specific flavonoid pathway were selected to perform molecular docking studies with specific virtually screened allosteric modulators. Results of docking analyses showed Naringenin, 2-Morpholin-4-ium-4-ylethanesulfonate, 6-C-Glucosylquercetin, 2-Oxoglutaric acid, 3,5,7,3',4'-pentahydroxyflavone to be capable of improving the spontaneity of the enzyme-substrate reactions in terms of docking score, RMSD values, and non-covalent interactions (H-bond,hydrophobic interaction, Π-stacking, salt bridge, etc.). Further, the evolutionary relationship of tea flavonoid pathway enzymes was constructed and compared with related taxa. The codon usage-based of tea flavonoid biosynthetic genes indicated the non-biasness of their nucleotide composition. Overall this study will provide a direction towards putative ligand-dependent enhancement of flavonoid content, irrespective of seasonal variation.

[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.

Identification of Chalcone Isomerase Family Genes and Roles of CnCHI4 in Flavonoid Metabolism in Camellia nitidissima.

Camellia nitidissima is a woody plant with high ornamental value, and its golden-yellow flowers are rich in a variety of bioactive substances, especially flavonoids, that are beneficial to human health. Chalcone isomerases (CHIs) are key enzymes in the flavonoid biosynthesis pathway; however, there is a scarcity of information regarding the CHI family genes of C. nitidissima. In this study, seven CHI genes of C. nitidissima were identified and divided into three subfamilies by phylogenetic analysis. The results of multiple sequence alignment revealed that, unlike CnCHI1/5/6/7, CnCHI2/3/4 are bona fide CHIs that contain all the active site and critical catalytic residues. Analysis of the expression patterns of CnCHIs and the total flavonoid content of the flowers at different developmental stages revealed that CnCHI4 might play an essential role in the flavonoid biosynthesis pathway of C. nitidissima. CnCHI4 overexpression significantly increased flavonoid production in Nicotiana tabacum and C. nitidissima. The results of the dual-luciferase reporter assay and yeast one-hybrid system revealed that CnMYB7 was the key transcription factor that governed the transcription of CnCHI4. The study provides a comprehensive understanding of the CHI family genes of C. nitidissima and performed a preliminary analysis of their functions and regulatory mechanisms.

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 / 中国中药杂志

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.

Biochemical and Molecular Characterization of the Rice Chalcone Isomerase Family.

Chalcone isomerase (CHI) is a key enzyme in flavonoid biosynthesis. In plants, CHIs occur in multigene families, and they are divided into four types, types I-IV. Type I and II CHIs are bona fide CHIs with CHI activity, and type III and IV CHIs are non-catalytic members with different functions. Rice contains seven CHI family genes (OsCHIs). Molecular analysis suggested that OsCHI3 is a type I CHI, and the other OsCHIs were classified into types III and IV. To elucidate their biochemical functions, OsCHI1, OsCHI3, OsCHI6, and OsCHI7 were expressed in Escherichia coli, and the recombinant OsCHI proteins were purified. An activity assay of recombinant OsCHIs showed that OsCHI3 catalyzed the isomerization of naringenin chalcone and isoliquiritigenin, whereas the other recombinant OsCHIs had no CHI activity. OsCHI3 also exhibited a strong preference to naringenin chalcone compared to isoliquiritigenin, which agrees well with the catalytic properties of type I CHIs. These results ascertain OsCHI3 to be a bona fide CHI in rice. OsCHI3 and the other OsCHIs were expressed constitutively throughout the rice growth period and different tissues. OsCHI3 expression was induced immediately in response to ultra-violet (UV) stress, suggesting its involvement in the biosynthesis of sakuranetin, a flavonoid phytoalexin in rice.