Roles In Flavonoid Biosynthesis Research Articles

Genome-wide identification and functional analysis of CYP450 genes in eggplant (Solanum melongena L.) with a focus on anthocyanin accumlation

BackgroundIn plants, Cytochrome P450 (CYP450) constitutes the largest family of metabolic enzymes and plays a crucial role in various physiological processes, including plant growth and development. Eggplants are well-known for their peel’s high concentration of anthocyanin compounds that provide significant health benefits to humans. The accumulation of anthocyanins in eggplant peels is an important process during growth and development. Therefore, it is essential to identify the CYP450 genes in eggplant (SmCYPs) and analyze their expression profiles during the period of anthocyanin accumulation in the peel.ResultsA total of 180 SmCYPs were identified in the eggplant genome and classified into eight subfamilies based on phylogenetic analysis. These SmCYPs exhibited highly conserved gene structure (exon/intron) and protein motifs, especially within their respective subgroup. Sixteen pairs of genes with collinearity were identified through gene duplication analysis. Promoter cis-acting element analysis revealed that SmCYPs are involved in various responses, including growth and development, stress responsiveness, and light responsiveness. Transcriptome data analysis revealed that all SmCYPs were expressed in various eggplant tissues, such as roots, stems, leaves, flowers, and fruits; with diverse expression patterns among members. The expression patterns of SmCYPs in eggplant peel also exhibited diversity during different stages of anthocyanin accumulation. qRT-PCR analysis demonstrated similar expression patterns for 15 selected SmCYPs as observed in the transcriptome data. Metabolomics analysis further suggested that SmCYPs are involved in the biosynthesis of secondary metabolites and metabolic pathways related to flavonoid and flavone/flavonol biosynthesis. Notably, three specific SmCYPs (SmCYP73A1/75A/98A1) play a significant role in flavonoid biosynthesis, particularly in anthocyanin synthesis in eggplant.ConclusionGenome-wide identification, phylogenetic analysis, expression profile analysis, and exploration of metabolic pathways related to SmCYPs provide valuable insights into the roles of these genes in anthocyanin accumulation in various tissues and organs, including eggplant peel. The findings from this study lay a foundation for the functional analysis of SmCYPs involvement in anthocyanin accumulation in eggplant peel, providing a molecular basis for breeders to cultivate novel varieties of eggplants with high levels of anthocyanin content.

Dynamic Changes in Flavonoids’ Accumulation Pattern in Tilia miqueliana Flowers at Different Developmental Stages Based on Widely Targeted Metabolomic Analysis

Tilia miqueliana is an endemic species belonging to the genus Tilia L. (Tiliaceae) in China, which is known for its fragrant flowers and nectar, but the dynamic changes in metabolites during its growth and development are still unclear. In this study, the metabolic profiles from T. miqueliana flowers at three developmental stages were detected by performing an ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS)-based widely targeted metabolomic analysis. A total of 1138 metabolites were detected, with 288 Differentially Accumulated Metabolites (DAMs) determined, flavonoids accounting for the largest proportion. The trend analysis showed that DAMs present seven distinctive patterns, and subclass 5 obtained the largest amount with continuously increased relative content during flower development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analysis of DAMs showed different overlap and variability in metabolic pathways, indicating different directions of flavonoids’ metabolic flux in the three developmental stages. A correlation network analysis further revealed five core metabolites that played essential roles in flavonoid biosynthesis. This research provides comprehensive insights into the exploitation and utilization of T. miqueliana as well as a scientific basis for phylogenetic studies of the genus Tilia.

The Impact of Exogenous Sodium Selenite Treatment on the Nutritional Value and Active Constituents of Pueraria lobata

Moderate amounts of Se can promote crop growth, enhance stress resistance, increase yield, and improve nutritional quality. In the present study, kudzu seedlings were used as experimental materials, and their physiological indicators, antioxidant activity, nutritional components, and flavonoid content were measured after being treated with Na2SeO3 hydroponics. Transcriptome sequencing analysis was used to reveal the relevant genes involved in regulating the effects of exogenous Se on the content of Se-compounds and flavonoids in kudzu. The results indicated that treatment with 20 mg/L Na2SeO3 significantly increased stem and root lengths, dry and fresh weight, lateral root development, and chlorophyll b content. However, at higher concentrations (30–40 mg/L), lateral root abundance and chlorophyll levels decreased. Na2SeO3 treatment also augmented the antioxidant capacity and enhanced the content of major nutrients in kudzu seedlings. The total Se content in kudzu escalated with increasing Na2SeO3 concentration, with selenomethionine emerging as the primary organic-Se species. After treatment with Na2SeO3, the content of puerarin in both aboveground and underground parts decreased, while the content of total flavonoids increased. Daidzin increased in the roots. Differential expression gene analysis revealed that genes such as TRXB2, SYM, MMT1, and METE were involved in Se uptake and transformation in kudzu, while bZIP43 and WRKY47 played a role in flavonoid biosynthesis.

CcCHIL, a type IV chalcone isomerase that can improve (2S)-naringenin production in Saccharomyces cerevisiae

(2S)-Naringenin, a crucial precursor in plant flavonoid synthesis, holds significance in diverse biological processes and potential therapeutic applications for human diseases. The economically valuable Citrus reticulata 'Chachi' citrus cultivar, renowned for its flavonoid-rich peel, was subjected to integrated transcriptomic and metabolomic analysis in this study to reveal patterns of flavonoid accumulation. The analysis revealed a strong correlation between the expression pattern of the gene encoding type IV chalcone isomerase (chalcone isomerase like, CHIL), CcCHIL, and the accumulation of flavonoids in C. reticulata 'Chachi' peel. Previous studies have demonstrated that type IV chalcone isomerase can enhance flavonoid accumulation in citrus and improve the catalytic efficiency of chalcone synthase in vitro, thereby increasing the titer of (2S)-naringenin. In this study, we constructed a genetically engineered yeast strain capable of de novo synthesis of (2S)-naringenin. We found that CcCHIL can increase (2S)-naringenin production in engineered yeast by 13.60 %. Subsequently, we conducted experiments using CHILs from various species and found that GmCHIL from Glycine max can increase (2S)-naringenin production in engineered yeast by 45.35 %. Sequence alignment, molecular docking predictions and site-directed mutagenesis showed that Asn151 is one of the key sites for CHIL improving (2S)-naringenin production. Our study unveiled CcCHIL's crucial role in flavonoid biosynthesis in C. reticulata 'Chachi' peel and paving the way for future synthesis of intricate and economically valuable flavonoids.

Widely targeted LC-MS/MS approach provides insights into variations in bioactive flavonoid compounds and their antioxidant activities in green, red, and purple sugarcane

Sugarcane produces enormous secondary metabolites that have significant nutritive and pharmacological value. However, flavonoid diversity, their molecular mechanism, and the transcription factor involved in the regulation of flavonoids are mostly unknown in sugarcane. This study revealed a total of 162 flavonoids, encompassing various subclasses such as flavones, flavonoid carbonosides, flavonols, dihydroflavones, dihydroflavonols, chalcones, flavanols, and isoflavones. Among the different color varieties, the purple rind of YT71-210 exhibited the maximum level of flavonoids (19.26 mg/mL) with significantly higher antiradical activities, followed by the red rind of ROC22 (16.83 mg/mL). Conversely, the green rind of YT93-159 displayed the lowest level of flavonoids (6.14 mg/mL). Furthermore, our transcriptome analysis identified candidate MYB transcription factors that are potentially associated with flavonoid biosynthesis. Notably, correlation analysis revealed a strong association between one candidate MYB gene and the flavonoid compounds, further supporting its role in flavonoid biosynthesis. To conclude, our study provides a comprehensive understanding of the flavonoid composition and the identification of candidate MYB transcription factors associated offers valuable insights into the genetic regulation of flavonoids.

Analysis of R2R3-MYB Repressor Subfamily Identifies FcMYB57 as a Negative Regulator of Anthocyanin Biosynthesis in Ficus carica L. Fruit

Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries. Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig production. R2R3-MYB transcription factors have promotive or repressive regulatory roles in plant anthocyanin biosynthesis. In this study, 113 R2R3-MYB genes were identified in Ficus carica, 3 of which were assigned to the S4 subfamily of flavonoid-biosynthesis repressors. FcMYB57 was further recruited as a candidate anthocyanin-biosynthesis repressor based on its sequence features and expression, which was significantly negatively correlated with that of anthocyanin-biosynthesis structural genes. Transient overexpression of FcMYB57 in strawberry totally inhibited fruit pigmentation and significantly increased fruit firmness. The metabolomic analysis confirmed a significant reduction in the contents of cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside, as well as other flavonoids, and transmission electron microscopy revealed an increment in cell-wall thickness. Transcriptome analysis showed downregulation of anthocyanin-biosynthesis structural genes and upregulation of genes related to xylan synthesis. Yeast one-hybrid and dual luciferase assays demonstrated a negative regulatory effect of FcMYB57 on the promoter of FcUFGT3 (UDP glucose-flavonoid 3-O-glcosyl-transferase). Yeast two-hybrid assay showed that FcMYB57 does not interact with FcbHLH42, 3, 14, MYC2, or FcTTG1, all of which have a previously identified or predicted role in flavonoid biosynthesis, however, interaction was detected with FcTPL (Topless), suggesting that FcMYB57 serves as an active repressor of anthocyanin biosynthesis. This is the first identification of an anthocyanin-biosynthesis repressor in fig, with a possible role in fig fruit quality.

Role of 6mA in the Regulation of Metabolic Biosynthesis in Sorghum Callus.

Plant cell culture technology helps to obtain natural plant-derived metabolites. The callus of sorghum, a prominent cereal crop, possesses various metabolites with potential health benefits. However, the epigenetic mechanism regulating metabolic biosynthetic capabilities in sorghum remains unknown. Therefore, we conducted N6-methyladenine (6mA) methylome analysis using transcriptome profiling and metabolome analysis to investigate the role of 6mA alterations in two calluses having different biosynthetic capacities, which were derived from immature sorghum embryos. Our findings indicate that the 6mA upregulation within gene bodies is crucial in transcriptional activity potentially mediated by the DNA demethylase SbALKBH1. Furthermore, 6mA was significantly enriched in genes involved in the biosynthesis of flavonoids and isoflavonoids. This could serve as a novel source of bioactive compounds for human health. Thus, 6mA could play an essential role in flavonoid biosynthesis in the sorghum callus.

Multi-omics integration analysis reveals the molecular mechanisms of drought adaptation in homologous tetraploid alfalfa(Medicago sativa 'Xinjiang-Daye').

Drought stress is a predominant abiotic factor leading to decreased alfalfa yield. Genomic ploidy differences contribute to varying adaptation mechanisms of different alfalfa cultivars to drought conditions. This study employed a multi-omics approach to characterize the molecular basis of drought tolerance in a tetraploid variant of alfalfa (Medicago sativa, Xinjiang-Daye). Under drought treatment, a total of 4446 genes, 859 proteins, and 524 metabolites showed significant differences in abundance. Integrative analysis of the multi-omics data revealed that regulatory modules involved in flavonoid biosynthesis, plant hormone signalling transduction, linoleic acid metabolism, and amino acid biosynthesis play crucial roles in alfalfa adaptation to drought stress. The severity of drought led to the substantial accumulation of flavonoids, plant hormones, free fatty acids, amino acids, and their derivatives in the leaves. Genes such as PAL, 4CL, CHI, CHS, PP2C, ARF_3, and AHP_4 play pivotal regulatory roles in flavonoid biosynthesis and hormone signalling pathways. Differential expression of the LOX gene emerged as a key factor in the elevated levels of free fatty acids. Upregulation of P5CS_1 and GOT1/2 contributed significantly to the accumulation of Pro and Phe contents. ERF19 emerged as a principal positive regulator governing the synthesis of the aforementioned compounds. Furthermore, observations suggest that Xinjiang-Daye alfalfa may exhibit widespread post-transcriptional regulatory mechanisms in adapting to drought stress. The study findings unveil the critical mechanisms by which Xinjiang-Daye alfalfa adapts to drought stress, offering novel insights for the improvement of alfalfa germplasm resources.

Genome-wide identification, comparative analysis and functional roles in flavonoid biosynthesis of cytochrome P450 superfamily in pear (Pyrus spp.)

BackgroundThe cytochrome P450 (CYP) superfamily is the largest enzyme metabolism family in plants identified to date, and it is involved in many biological processes, including secondary metabolite biosynthesis, hormone metabolism and stress resistance. However, the P450 gene superfamily has not been well studied in pear (Pyrus spp.).ResultsHere, the comprehensive identification and a comparative analysis of P450 superfamily members were conducted in cultivated and wild pear genomes. In total, 338, 299 and 419 P450 genes were identified in Chinese white pear, European pear and the wild pear, respectively. Based on the phylogenetic analyses, pear P450 genes were divided into ten clans, comprising 48 families. The motif and gene structure analyses further supported this classification. The expansion of the pear P450 gene family was attributed to whole-genome and single-gene duplication events. Several P450 gene clusters were detected, which have resulted from tandem and proximal duplications. Purifying selection was the major force imposed on the long-term evolution of P450 genes. Gene dosage balance, subfunctionalization and neofunctionalization jointly drove the retention and functional diversification of P450 gene pairs. Based on the association analysis between transcriptome expression profiles and flavonoid content during fruit development, three candidate genes were identified as being closely associated with the flavonoid biosynthesis, and the expression of one gene was further verified using qRT-PCR and its function was validated through transient transformation in pear fruit.ConclusionsThe study results provide insights into the evolution and biological functions of P450 genes in pear.

FaMYB5 Interacts with FaBBX24 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Strawberry (Fragaria × ananassa).

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3'H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5-FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit.

Omics Analysis Unveils the Pathway Involved in the Anthocyanin Biosynthesis in Tomato Seedling and Fruits.

The purple tomato variety 'Indigo Rose' (InR) is favored due to its bright appearance, abundant anthocyanins and outstanding antioxidant capacity. SlHY5 is associated with anthocyanin biosynthesis in 'Indigo Rose' plants. However, residual anthocyanins still present in Slhy5 seedlings and fruit peel indicated there was an anthocyanin induction pathway that is independent of HY5 in plants. The molecular mechanism of anthocyanins formation in 'Indigo Rose' and Slhy5 mutants is unclear. In this study, we performed omics analysis to clarify the regulatory network underlying anthocyanin biosynthesis in seedling and fruit peel of 'Indigo Rose' and Slhy5 mutant. Results showed that the total amount of anthocyanins in both seedling and fruit of InR was significantly higher than those in the Slhy5 mutant, and most genes associated with anthocyanin biosynthesis exhibited higher expression levels in InR, suggesting that SlHY5 play pivotal roles in flavonoid biosynthesis both in tomato seedlings and fruit. Yeast two-hybrid (Y2H) results revealed that SlBBX24 physically interacts with SlAN2-like and SlAN2, while SlWRKY44 could interact with SlAN11 protein. Unexpectedly, both SlPIF1 and SlPIF3 were found to interact with SlBBX24, SlAN1 and SlJAF13 by yeast two-hybrid assay. Suppression of SlBBX24 by virus-induced gene silencing (VIGS) retarded the purple coloration of the fruit peel, indicating an important role of SlBBX24 in the regulation of anthocyanin accumulation. These results deepen the understanding of purple color formation in tomato seedlings and fruits in an HY5-dependent or independent manner via excavating the genes involved in anthocyanin biosynthesis based on omics analysis.

Ant13 Encodes Regulatory Factor WD40 Controlling Anthocyanin and Proanthocyanidin Synthesis in Barley (Hordeum vulgare L.)

Flavonoid compounds like anthocyanins and proanthocyanidins are important plant secondary metabolites having wide biological activities for humans. In this study, the molecular function of the Ant13 locus, which is one of the key loci governing flavonoid synthesis in barley, was determined. It was found that Ant13 encodes a WD40-type regulatory protein, which is required for transcriptional activation of a set of structural genes encoding enzymes of flavonoid biosynthesis at the leaf sheath base (colored by anthocyanins) and in grains (which accumulate proanthocyanidins). Besides its role in flavonoid biosynthesis, pleiotropic effects of this gene in plant growth were revealed. The mutants deficient in the Ant13 locus showed similar germination rates but a decreased rate of root and shoot growth and yield-related parameters in comparison to the parental cultivars. This is the seventh Ant locus (among 30) for which molecular functions in flavonoid biosynthesis regulation have been determined.

Rejuvenation increases leaf biomass and flavonoid accumulation in Ginkgo biloba.

Rejuvenation refers to the transition from an adult state to a juvenile state. Trunk truncation at the base of the tree can result in tree rejuvenation. However, little is known about the association of rejuvenation with leaf biomass and flavonoid accumulation. The results of this study showed that, compared with control leaves, leaves of renewed Ginkgo biloba shoots were larger, thicker, and more lobed and had higher fresh/dry weights and chlorophyll contents. The leaf biomass per hectare of rejuvenated trees was twofold higher than that of the untruncated controls. Moreover, we observed a marked increase in the accumulation of flavonol glycosides via metabolomic analysis and detected upregulated expression of genes involved in flavonoid biosynthesis, including CHS, FLS, F3'H, DFR, and LAR. Overexpression of GbCHS in ginkgo calli confirmed that GbCHS plays an important role in flavonoid biosynthesis. Interestingly, the contents of gibberellins significantly increased in the rejuvenated leaves. Moreover, exogenous gibberellin treatment significantly increased GbCHS expression and flavonoid contents. Our findings show that truncation can stimulate tree rejuvenation by altering hormone levels, representing an effective and feasible approach for enhancing the biomass and flavonoid content of G. biloba leaves.

The expansion and diversity of theCYP75gene family in Vitaceae

The CYP75 gene family plays an important role in flavonoid biosynthesis in plants. Little is known about the evolution of the gene family within the grape family. Here, we extracted the CYP75 genes from transcriptome data of 15 grape species and 36 representative genomes from other plants to explore the evolutionary history of the CYP75 gene family in Vitaceae. The structure of the CYP75 protein sequences is highly conserved with the variation mainly occurring in the N terminal and the middle region. The evolutionary analyses suggested classifying the CYP75 gene family into three groups in Vitaceae, namely Vitaceae A1, Vitaceae A2 and Vitaceae B. The Vitaceae A1 and A2 belong to the CYP75A subfamily and the Vitaceae B belongs to the CYP75B subfamily. Within the Vitaceae A1, most Vitaceae taxa present only one copy of the CYP75A protein sequence except for Vitis vinifera with a high number of sequences, which might have originated through recent gene duplications after its split from the other species. Vitaceae A2 contain only CYP75A sequences from Vitaceae sister to one from Camellia sinensis, probably representing a relict lineage. The CYP75B proteins were found to be dominated in Vitaceae and other angiosperms. Our results provide important insights into understanding the evolutionary history of the CYP75 gene family in Vitaceae and other angiosperms.

NF-Y plays essential roles in flavonoid biosynthesis by modulating histone modifications in tomato.

NF-Y transcription factors are reported to play diverse roles in a wide range of biological processes in plants. However, only a few active NF-Y complexes are known in plants and the precise functions of NF-Y complexes in flavonoid biosynthesis have not been determined. Using various molecular, genetic and biochemical approaches, we found that NF-YB8a, NF-YB8b and NF-YB8c - a NF-YB subgroup - can interact with a specific subgroup of NF-YC and then recruit either of two distinct NF-YAs to form NF-Y complexes that bind the CCAAT element in the CHS1 promoter. Furthermore, suppressing the expression of particular NF-YB genes increased the levels of H3K27me3 at the CHS1 locus and significantly suppressed the expression of CHS1 during tomato fruit ripening, which led to the development of pink-coloured fruit with colourless peels. Altogether, by demonstrating that NF-Y transcription factors play essential roles in flavonoid biosynthesis and by providing significant molecular insight into the regulatory mechanisms that drive the development of pink-coloured tomato fruit, we provide a major advance to our fundamental knowledge and information that has considerable practical value for horticulture.

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

SummaryFlavanones 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.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level

BackgroundLeguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness.ResultsRhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants.ConclusionsAlfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

Identification of the bHLH gene family in Dracaena cambodiana reveals candidate genes involved in flavonoid biosynthesis

The basic helix-loop-helix (bHLH) transcription factors (TFs) regulate multiple biological processes by altering the expression of target genes. However, the bHLH superfamily genes in the dragon tree (Dracaena cambodiana Pierre ex Gagnep) have not been characterized, and their role in flavonoid biosynthesis remain unclear. Here, 62 bHLH genes in D. cambodiana were identified, and these genes were classified into 19 subfamilies based on a phylogenetic comparison with proteins from Arabidopsis. Transcriptomic analysis revealed that 8 DcbHLHs were up-regulated when exposed to the inducer of dragon’s blood and ultraviolet radiation b (UVB). Interaction network analysis revealed that 3 bHLHs (DcbHLH2, DcbHLH5 and DcbHLH56) are likely involved in flavonoid biosynthesis. In addition, DcbHLH5 and flavonoids biosynthetic genes were co-expressed in response to the inducer and UVB exposure. Furthermore, DcbHLH5 could bind to the promoters of flavonoid biosynthesis genes and activate their expression. This study suggests that DcbHLH proteins maybe involved in flavonoid biosynthesis.

Transcriptome Sequencing and Metabolism Analysis Reveals the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulbs

Onion (Allium cepa L.) is an important bulbous vegetable crop that possesses important properties related to health as well as extraordinary colors. Naturally white onion bulbs were used in this study to reveal the complex metabolic mechanisms that underlie phenotypic traits, especially bulb pigmentation. Six libraries (three dark-red and three white) were constructed and analyzed to elucidate differences in cyanidin (Cy) metabolism between dark-red and white onion bulbs. Libraries were screened using RNA-sequencing (RNA-seq) to reveal the differentially expressed genes (DEGs) involved in anthocyanin biosynthesis at the transcriptional level. Comparison with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database shows that a total of 27 unigenes participate in onion anthocyanin biosynthesis and 16 DEGs perform critical roles in flavonoid biosynthesis. Expression patterns of color-related flavonoid compounds associated with the onion anthocyanin biosynthesis pathway (ABP) show that flavonoid 3′,5′-hydroxylase (F3′5′H) and dihydroflavonol 4-reductase (DFR) genes play crucial roles in the biosynthesis of dark-red bulbs, the expression levels of flavonol synthase (FLS) and DFR genes may act to block blue pigmentation, and the loss of Cy from white onion bulbs might explain multibranching in the synthesis of this compound. Positive variation in the F3′5′H/F3′H ratio also affects onion bulb color diversity. The transcriptome presented here provides a basis for future onion molecular breeding based on variations in the diversity of ornamental plant pigmentation.

The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples.

In plants, flavonoids play critical roles in resistance to biotic and abiotic stresses, and contribute substantially to the quality, flavor, and nutritional quality of many fruit crops. In apple (Malus×domestica), inbreeding has resulted in severe decreases in the genetic diversity and flavonoid content. Over the last decade, we have focused on the genetic improvement of apple using wild red-fleshed apple resources (Malus sieversii f. niedzwetzkyana). Here, we found that the MYB transcription factors (TFs) involved in the synthesis of proanthocyanidins can be classified into TT2 and PA1 types. We characterized a PA1-type MYB transcription factor, MdMYBPA1, from red-fleshed apple and identified its role in flavonoid biosynthesis using overexpression and knockdown-expression transgenes in apple calli. We explored the relationship between TT2- and PA1-type MYB TFs, and found that MdMYB9/11/12 bind the MdMYBPA1 promoter. In addition, MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production. In binding analyses, MdbHLH33 directly bound to the low-temperature-responsive (LTR) cis-element of the MdMYBPA1 promoter and promotes its activity. In addition, the calli expressing both MdMYBPA1 and MdbHLH33, which together form a complex, produced more anthocyanin under low temperature. Our findings shed light on the essential roles of PA1-type TFs in the metabolic network of proanthocyanidin and anthocyanin synthesis in plants. Studies on red-fleshed wild apple are beneficial for breeding new apple varieties with high flavonoid contents.