Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Rice myo-inositol-3-phosphate synthase 2 (RINO2) alleviates heat injury-induced impairment in pollen germination and tube growth by modulating Ca2+ signaling and actin filament cytoskeleton.

Low phytic acid (lpa) crop is considered as an effective strategy to improve crop nutritional quality, but a substantial decrease in phytic acid (PA) usually has negative effect on agronomic performance and its response to environment adversities. Myo-inositol-3-phosphate synthase (MIPS) is the rate-limiting enzyme in PA biosynthesis pathway, and regarded as the prime target for engineering lpa crop. In this paper, the rice MIPS gene (RINO2) knockout mutants and its wild type were performed to investigate the genotype-dependent alteration in the heat injury-induced spikelet fertility and its underlying mechanism for rice plants being imposed to heat stress at anthesis. Results indicated that RINO2 knockout significantly enhanced the susceptibility of rice spikelet fertility to heat injury, due to the severely exacerbated obstacles in pollen germination and pollen tube growth in pistil for RINO2 knockout under high temperature (HT) at anthesis. The loss of RINO2 function caused a marked reduction in inositol and phosphatidylinositol derivative concentrations in the HT-stressed pollen grains, which resulted in the strikingly lower content of phosphatidylinositol 4,5-diphosphate (PI (4,5) P2) in germinating pollen grain and pollen tube. The insufficient supply of PI (4,5) P2 in the HT-stressed pollen grains disrupted normal Ca2+ gradient in the apical region of pollen tubes and actin filament cytoskeleton in growing pollen tubes. The severely repressed biosynthesis of PI (4,5) P2 was among the regulatory switch steps leading to the impaired pollen germination and deformed pollen tube growth for the HT-stressed pollens of RINO2 knockout mutants.

Carotenoid biosynthesis genes LcLCYB, LcLCYE, and LcBCH from wolfberry confer increased carotenoid content and improved salt tolerance in tobacco.

Carotenoids play essential roles in plant growth and development and provide plants with a tolerance to a series of abiotic stresses. In this study, the function and biological significance of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase, which are responsible for the modification of the tetraterpene skeleton procedure, were isolated from Lycium chinense and analyzed. The overexpression of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase promoted the accumulation of total carotenoids and photosynthesis enhancement, reactive oxygen species scavenging activity, and proline content of tobacco seedlings after exposure to the salt stress. Furthermore, the expression of the carotenoid biosynthesis genes and stress-related genes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase, and pyrroline-5-carboxylate reductase) were detected and showed increased gene expression level, which were strongly associated with the carotenoid content and reactive oxygen species scavenging activity. After exposure to salt stress, the endogenous abscisic acid content was significantly increased and much higher than those in control plants. This research contributes to the development of new breeding aimed at obtaining stronger salt tolerance plants with increased total carotenoids and vitamin A content.

Chemical diversity in angiosperms - monoterpene synthases control complex reactions that provide the precursors for ecologically and commercially important monoterpenoids.

Monoterpene synthases (MTSs) catalyze the first committed step in the biosynthesis of monoterpenoids, a class of specialized metabolites with particularly high chemical diversity in angiosperms. In addition to accomplishing a rate enhancement, these enzymes manage the formation and turnover of highly reactive carbocation intermediates formed from a prenyl diphosphate substrate. At each step along the reaction path, a cationic intermediate can be subject to cyclization, migration of a proton, hydride, or alkyl group, or quenching to terminate the sequence. However, enzymatic control of ligand folding, stabilization of specific intermediates, and defined quenching chemistry can maintain the specificity for forming a signature product. This review article will discuss our current understanding of how angiosperm MTSs control the reaction environment. Such knowledge allows inferences about the origin and regulation of chemical diversity, which is pertinent for appreciating the role of monoterpenoids in plant ecology but also for aiding commercial efforts that harness the accumulation of these specialized metabolites for the food, cosmetic, and pharmaceutical industries.

Functional Characterization of a CruP-Like Isomerase in Dunaliella.

Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.

Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

Mutational Analysis of (+)-Limonene Synthase.

The monoterpene limonene is produced by the enzyme limonene synthase in one of the simplest terpene cyclization reactions. The enzyme can use linalyl diphosphate (LPP) and neryl diphosphate (NPP) as substrates in addition to the naturally occurring substrate geranyl diphosphate (GPP), but the relationship among the three alternative substrates is not well understood. We explored the (+)-limonene synthase ((+)-LS) reaction using site-directed mutagenesis with the three different substrates (GPP, NPP, and LPP) to tease out details of the mechanism. In total, 23 amino acid positions in the active site of (+)-LS were targeted for mutation. In all cases, substitution with Ala resulted in a significant loss of enzyme activity using GPP or NPP as the substrate, but the mutations fell into two groups depending on the effect of using LPP as a substrate: group 1 mutations resulted in the loss of activity with all three substrates (GPP, NPP, and LPP); group 2 mutations resulted in loss of activity with GPP and NPP, but retained near-WT activity with LPP as a substrate. Importantly, mutations resulting in loss of activity with LPP but retention of activity with GPP and NPP were never observed. These data, in combination with the substrate order of reactivity for the WT enzyme (LPP > NPP > GPP), are consistent with a role for LPP as an intermediate in the (+)-LS reaction using either GPP or NPP as a substrate.

Monoterpene synthases contribute to the volatile production in tana (Zanthoxylum ailanthoides) through indigenous cultivation practices.

Tana (Zanthoxylum ailanthoides), a perennial deciduous species in the Rutaceae family, possesses leaves with a unique fragrance that indigenous peoples incorporate into their traditional cuisine. In Kalibuan, the cultivated tana trees were pruned repeatedly to maintain a shorter height, which led to the growth of new leaves that were spicier and pricklier. Tana leaves contain a range of volatile terpenoids, and the pungent aroma may arise from the presence of monoterpenoids. To gain insight into the biosynthetic pathway, five candidate monoterpene synthase genes were cloned and characterized using a purified recombinant protein assay. The main product of Za_mTPS1, Za_mTPS2, and Za_mTPS5 is sabinene, geraniol, and (E)-ß-ocimene, respectively. The main product of Za_mTPS3 and Za_mTPS4 is linalool. Real-time PCR analysis revealed that Za_mTPS1 and Za_mTPS5 are expressed at higher levels in prickly leaves of cultivated tana, suggesting that they may contribute to the distinctive aroma of this plant.

Functional Characterization of Lycopene ß- and ε-Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60.

Lutein is a high-value carotenoid with many human health benefits. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently ß-cyclize both ends of lycopene to produce ß-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into ß-monocyclic γ-carotene and ultimately produced α-carotene with a ß-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

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.

Screening of Structurally Distinct Lycopene ß-Cyclases for Production of the Cyclic C40 Carotenoids ß-Carotene and Astaxanthin by Corynebacterium glutamicum.

Lycopene ß-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of ß-carotene and derived carotenoids. It catalyzes isomerase reactions to form ß-carotene from lycopene by ß-cyclization of both of its ψ-ends. Lycopene ß-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene ß-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene ß-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene ß-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene ß-cyclases were screened in the lycopene-overproducing Corynebacterium glutamicum strain for ß-carotene and astaxanthin production. This systematic screening facilitates the identification of new enzymes for carotenoid production. Higher astaxanthin production and less reduction of total carotenoids were achieved with the cytosolic lycopene ß-cyclase CrtL from Synechococcus elongatus and the membrane-bound heterodimeric lycopene ß-cyclase CrtYcd from Brevibacterium linens.

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.

Structural Basis for (2R,3R)-Taxifolin Binding and Reaction Products to the Bacterial Chalcone Isomerase of Eubacterium ramulus.

The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by a reverse Michael addition. The overall fold and the constitution of the active site of the enzyme completely differ from the well-characterised chalcone isomerase of plants. For (+)-taxifolin, CHI catalyses the intramolecular ring contraction to alphitonin. In this study, Fwe perform crystal structure analyses of CHI and its active site mutant His33Ala in the presence of the substrate taxifolin at 2.15 and 2.8 Å resolution, respectively. The inactive enzyme binds the substrate (+)-taxifolin as well defined, whereas the electron density maps of the native CHI show a superposition of substrate, product alphitonin, and most probably also the reaction intermediate taxifolin chalcone. Evidently, His33 mediates the stereospecific acid-base reaction by abstracting a proton from the flavonoid scaffold. The stereospecificity of the product is discussed.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

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.

Flavonoid Biosynthesis Genes in Triticum aestivum L.: Methylation Patterns in Cis-Regulatory Regions of the Duplicated CHI and F3H Genes.

Flavonoids are a diverse group of secondary plant metabolites that play an important role in the regulation of plant development and protection against stressors. The biosynthesis of flavonoids occurs through the activity of several enzymes, including chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H). A functional divergence between some copies of the structural TaCHI and TaF3H genes was previously shown in the allohexaploid bread wheat Triticum aestivum L. (BBAADD genome). We hypothesized that the specific nature of TaCHI and TaF3H expression may be induced by the methylation of the promoter. It was found that the predicted position of CpG islands in the promoter regions of the analyzed genes and the actual location of methylation sites did not match. We found for the first time that differences in the methylation status could affect the expression of TaCHI copies, but not the expression of TaF3Hs. At the same time, we revealed significant differences in the structure of the promoters of only the TaF3H genes, while the TaCHI promoters were highly homologous. We assume that the promoter structure in TaF3Hs primarily affects the change in the nature of gene expression. The data obtained are important for understanding the mechanisms that regulate the synthesis of flavonoids in allopolyploid wheat and show that differences in the structure of promoters have a key effect on gene expression.