Unraveling the mechanism of flower color variation in Brassica napus by integrated metabolome and transcriptome analyses.

Brassica napus is one of the most important oil crops in the world. Breeding oilseed rape with colorful flowers can greatly enhance the ornamental value of B. napus and thus improve the economic benefits of planting. As water-soluble flavonoid secondary metabolites, anthocyanins are very important for the synthesis and accumulation of pigments in the petals of plants, giving them a wide range of bright colors. Despite the documentation of over 60 distinct flower shades in B. napus, the intricacies underlying flower color variation remain elusive. Particularly, the mechanisms driving color development across varying flower color backgrounds necessitate further comprehensive investigation. This research undertook a comprehensive exploration through the integration of transcriptome and metabolome analyses to pinpoint pivotal genes and metabolites underpinning an array of flower colors, including beige, beige-red, yellow, orange-red, deep orange-red, white, light-purple, and purple. First, we used a two-way BLAST search to find 275 genes in the reference genome of B. napus Darmor v10 that were involved in making anthocyanins. The subsequent scrutiny of RNA-seq outcomes underscored notable upregulation in the structural genes F3H and UGT, alongside the MYB75, GL3, and TTG1 transcriptional regulators within petals, showing anthocyanin accumulation. By synergizing this data with a weighted gene co-expression network analysis, we identified CHS, F3H, MYB75, MYB12, and MYB111 as the key players driving anthocyanin synthesis in beige-red, orange-red, deep orange-red, light-purple, and purple petals. By integrating transcriptome and weighted gene co-expression network analysis findings with anthocyanin metabolism data, it is hypothesized that the upregulation of MYB75, which, in turn, enhances F3H expression, plays a pivotal role in the development of pigmented oilseed rape flowers. These findings help to understand the transcriptional regulation of anthocyanin biosynthesis in B. napus and provide valuable genetic resources for breeding B. napus varieties with novel flower colors.

A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network.

Genes of metabolic pathways are individually or collectively regulated, often via unclear mechanisms. The anthocyanin pathway, well known for its regulation by the MYB/bHLH/WDR (MBW) complex but less well understood in its connections to MYC2, BBX21, SPL9, PIF3, and HY5, is investigated here for its direct links to the regulators. We show that MYC2 can activate the structural genes of the anthocyanin pathway but also suppress them (except F3'H) in both Arabidopsis and Oryza when a local MBW complex is present. BBX21 or SPL9 can activate all or part of the structural genes, respectively, but the effects can be largely overwritten by the local MBW complex. HY5 primarily influences expressions of the early genes (CHS, CHI, and F3H). TF-TF relationships can be complex here: PIF3, BBX21, or SPL9 can mildly activate MYC2; MYC2 physically interacts with the bHLH (GL3) of the MBW complex and/or competes with strong actions of BBX21 to lessen a stimulus to the anthocyanin pathway. The dual role of MYC2 in regulating the anthocyanin pathway and a similar role of BBX21 in regulating BAN reveal a network-level mechanism, in which pathways are modulated locally and competing interactions between modulators may tone down strong environmental signals before they reach the network.

Flower color polymorphism of a wild Iris on the Qinghai-Tibet plateau.

BACKGROUND: Flower color plays a crucial role in attracting pollinators and facilitating environmental adaptation. Investigating the causes of flower color polymorphism and understanding their potential effects on both ecology and genetics can enhance our understanding of flower color polymorphism in wild plant. RESULTS: In this study, we examined the differences of potential male and female fitness between purple- and yellow- flower individuals in Iris potaninii on the Qinghai-Tibet Plateau, and screened key genes and positively selective genes involved in flower color change. Our results showed that yellow flower exhibited a higher pollen-to-ovule ratio. Yellow flowers were derived from purple flowers due to the loss of anthocyanins, and F3H could be an essential gene affecting flower color variation though expression regulation and sequence polymorphism in this species. Furthermore, our findings suggest that genes positively selected in yellow-flowered I. potaninii might be involved in nucleotide excision repair and plant-pathogen interactions. CONCLUSIONS: These results suggest that F3H induces the flower color variation of Iris potaninii, and the subsequent ecological and additive positive selection on yellow flowers may further enhance plant adaptations to alpine environments.

Identification of Flavanone 3-Hydroxylase Gene Family in Strawberry and Expression Analysis of Fruit at Different Coloring Stages.

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

A 43 Bp-Deletion in the F3'H Gene Reducing Anthocyanins Is Responsible for Keeping Buds Green at Low Temperatures in Broccoli.

Most broccoli cultivars or accessions exhibit green buds under appropriate growth conditions, which turn purple at cold temperatures. However, certain cultivars consistently maintain green buds both during normal growth and at cold temperatures. In this study, we used BSA-seq (bulked segregation analysis-sequencing), along with fine mapping and transcriptome analysis to identify a candidate gene (flavonoid 3'-hydroxylase, F3'H) responsible for reducing anthocyanin accumulation in the mutant GS and HX-16 broccoli (Brassica oleracea L. var. italica), which could retain green buds even at low temperatures. A 43-bp deletion was detected in the coding sequence (CDS) of the F3'H gene in HX-16 and the mutant GS, which significantly decreased F3'H expression and the accumulation of cyanidin and delphinidin in the mutant GS. Furthermore, the expression of F3'H was upregulated at low temperatures in the wild line PS. Our results demonstrated the efficacy of utilizing the 43-bp InDel (Insertion-Deletion) in predicting whether buds in B. oleracea L. will turn purple or remain green at cold temperatures across forty-two germplasm materials. This study provides critical genetic and molecular insights for the molecular breeding of B. oleracea and sheds light on the molecular mechanisms underlying the effect of low temperatures on bud color in broccoli.

A novel transcriptional repressor complex MYB22-TOPLESS-HDAC1 promotes rice resistance to brown planthopper by repressing F3'H expression.

The brown planthopper (BPH) is the most destructive pest of rice. The MYB transcription factors are vital for rice immunity, but most are activators. Although MYB22 positively regulates rice resistance to BPH and has an EAR motif associated with active repression, it remains unclear whether it is a transcriptional repressor affecting rice-BPH interaction. Genetic analyses revealed that MYB22 regulates rice resistance to BPH via its EAR motif. Several biochemical experiments (e.g. transient transcription assay, Y2H, LCA, and BiFC) indicated that MYB22 is a transcriptional repressor that interacts with the corepressor TOPLESS via its EAR motif and recruits HDAC1 to form a tripartite complex. Flavonoid-3'-hydroxylase (F3'H) is a flavonoid biosynthesis pathway-related gene that negatively regulates rice resistance to BPH. Based on a bioinformatics analysis and the results of EMSA and transient transcription assays, MYB22 can bind directly to the F3'H promoter and repress gene expression along with TOPLESS and HDAC1. We revealed a transcriptional regulatory mechanism influencing the rice-BPH interaction that differs from previously reported mechanisms. Specifically, MYB22-TOPLESS-HDAC1 is a novel transcriptional repressor complex with components that synergistically and positively regulate rice resistance to BPH through the transcriptional repression of F3'H.

The molecular docking and molecular dynamics study of flavonol synthase and flavonoid 3'-monooxygenase enzymes involved for the enrichment of kaempferol.

Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, antidiabetic activities etc. It has been reported in many vegetables, fruits, herbs and medicinal plants. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols. Whereas flavonoid 3'-monooxygenase (F3'H, EC 1.14.14.82) catalyses the hydroxylation of dihydroflavonol, and flavonol. FLS is involved in the synthesis of the kaempferol whereas F3'H causes degradation of kaempferol. The present study aimed to analyse the binding affinity, stability and activating activity of enzyme FLS as well as inhibitory activity of enzyme F3'H involved in the enrichment of the kaempferol using the in-silico approaches. Computational study for physico-chemical properties, conserved domain identification, 3-D structure prediction and its validation, conservation analysis, molecular docking followed by molecular dynamics analysis of FLS and F3'H, protein-activator (FLS-LIG Complex) and protein-inhibitor (F3'H-LIG Complex) complexes have been performed. Other structural analyses like root mean square fluctuation (RMSF), root mean square deviation (RMSD), surface area solvent accessibility (SASA), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA), Poisson-Boltzmann analysis (MM_PBSA) and the dynamic cross correlation map (DCCM) analysis to explore the structural, functional and thermodynamic stability of the proteins and the complexes were also studied. The molecular docking result showed that FLS binds strongly with the activator ascorbate (CID _54670067) while F3'H binds with the inhibitor ketoconazole (CID_456201). The most powerful inhibitor (ketoconazole for F3'H) and activator (ascorbate for FLS) is determined by computing the thermodynamic binding free energy through MM_PBSA analysis. The current work provides wide-ranging structural and functional information about FLS and F3'H enzymes showing detailed molecular mechanism of kaempferol biosynthesis and its degradation and hence kaempferol enrichment. Finding of the present work opens up new possibilities for future research towards enrichment of kaempferol by using activator (ascorbate) for FLS and inhibitor (ketoconazole) for F3'H as well as for its large-scale production using in vitro approaches.Communicated by Ramaswamy H. Sarma.

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.

The Ethylene Response Factor ERF5 Regulates Anthocyanin Biosynthesis in 'Zijin' Mulberry Fruits by Interacting with MYBA and F3H Genes.

Ethylene promotes ripening in fruits as well as the biosynthesis of anthocyanins in plants. However, the question of which ethylene response factors (ERFs) interact with the genes along the anthocyanin biosynthesis pathway is yet to be answered. Herein, we conduct an integrated analysis of transcriptomes and metabolome on fruits of two mulberry genotypes ('Zijin', ZJ, and 'Dashi', DS, with high and low anthocyanin abundance, respectively) at different post-flowering stages. In total, 1035 upregulated genes were identified in ZJ and DS, including MYBA in the MBW complex and anthocyanin related genes such as F3H. A KEGG analysis suggested that flavonoid biosynthesis and plant hormone signaling transduction pathways were significantly enriched in the upregulated gene list. In particular, among 103 ERF genes, the expression of ERF5 showed the most positive correlation with the anthocyanin change pattern across both genotypes and in the post-flowering stages, with a Pearson correlation coefficient (PCC) of 0.93. Electrophoresis mobility shift assay (EMSA) and luciferase assay suggested that ERF5 binds to the promoter regions of MYBA and F3H and transcriptionally activates their gene expression. We elucidated a potential mechanism by which ethylene enhances anthocyanin accumulation in mulberry fruits and highlighted the importance of the ERF5 gene in controlling the anthocyanin content in mulberry species. This knowledge could be used for engineering purposes in future mulberry breeding programs.

Melatonin enhances stress tolerance in pigeon pea by promoting flavonoid enrichment, particularly luteolin in response to salt stress.

Melatonin improves plant resistance to multiple stresses by participating in the biosynthesis of metabolites. Flavonoids are an important family of plant secondary metabolites and are widely recognized to be involved in resistance; however, the crosstalk between melatonin and flavonoid is largely unknown. We found that the resistance of pigeon pea (Cajanus cajan) to salt, drought, and heat stresses were significantly enhanced by pre-treatment with melatonin. Combined transcriptome and LC-ESI-MS/MS metabolomics analyses showed that melatonin significantly induced the enrichment of flavonoids and mediated the reprogramming of biosynthetic pathway genes. The highest fold-increase in expression in response to melatonin treatment was observed for the CcF3´H family, which encodes an enzyme that catalyses the biosynthesis of luteolin, and the transcription factor CcPCL1 directly bonded to the CcF3´H-5 promoter to enhance its expression. In addition, salt stress also induced the expression of CcPCL1 and CcF3´H-5, and their overexpression in transgenic plants greatly enhanced salt tolerance by promoting the biosynthesis of luteolin. Overall, our results indicated that pre-treatment of pigeon pea with melatonin promoted luteolin biosynthesis through the CcPCL1 and CcF3´H-5 pathways, resulting in salt tolerance. Our study shows that melatonin enhances plant tolerance to multiple stresses by mediating flavonoid biosynthesis, providing new avenues for studying the crosstalk between melatonin and flavonoids.

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.

Anthocyanin Biosynthesis and a Regulatory Network of Different-Colored Wheat Grains Revealed by Multiomics Analysis.

Colored wheat has always been a popular research area because of its high performance in the field and significant medical uses. Progress has been made mapping the genes of purple or blue grains; however, the reason why different grain colors form in wheat is not well understood. We created wheat lines with different grain colors (purple and blue) using the white grain cultivar Xiaoyan22 and located the candidate region related to the purple and blue grains in chromosome 2A, 2B, and 4D, 2A, respectively, by the bulked segregant RNA-seq. The transcriptomic and metabolomic analyses of the three grains at different developmental stages indicated that the upregulation of flavonoid 3'-hydroxylase/flavonoid 3',5'hydroxylase 2 and TaMYC1/TaMYC4 was important for the formation of purple/blue grains. The blue TaMYC4 had 16 nonsynonymous single nucleotide variants verified by Sanger sequencing and possessed a different splicing mode in the bHLH_MYC_N domain compared with the reference database. Targeted high-performance liquid chromatography-mass spectrometry/mass spectrometry analysis of anthocyanins found that the purple and blue grains contained more pelargonidin, cyanidin, and delphinidin, respectively. This study provides a comprehensive understanding of the different color formations of wheat grains and useful information about genetic improvements in wheat and other crops.

Tal2b targets and activates the expression of OsF3H03g to hijack OsUGT74H4 and synergistically interfere with rice immunity.

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited. Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control. Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g , which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice. OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.

Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3'-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3'-hydroxylase BrF3'H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3'H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3'H protein catalyzes the 3'-hydroxylation of a wide range of 4'-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3'H among those tested. Transient expression of BrF3'H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3'H in planta. As the first functional characterization of BrF3'H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

Flavonoid 3'-hydroxylase of Camellia nitidissima Chi. promotes the synthesis of polyphenols better than flavonoids.

Camellia nitidissima Chi. is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3'-hydroxylase (F3'H) plays an important role in the synthesis of flavonoids, polyphenols and anthocyanins. We used PCR amplification, quantitative PCR, High-performance liquid chromatography, subcellular localization, and agrobacterium-mediated leaf disk method to study the the function of CnF3'H. The full length of CnF3'H was 1859 bp (GenBank code: HQ290518.1), with an open reading frame of 1577 bp, and encoded 518 amino acid. A phylogenetic tree analysis showed that CnF3'H was closely related to Camellia sinensis L. and C. sinensis cultivar Zhonghuang. CnF3'H was expressed in flowers, leaves, fruits, sepals, petals and stamens of C. nitidissima, and during the flowering process the expression level in flower decreased initially and then increased. CnF3'H expression was significantly positive correlated with polyphenol contents in C. nitidissima. A CnF3'H-EGFP expression vector was constructed to do the subcellular localization, we found that CnF3'H was obviously localized in the nuclear envelope and cytomembrane. In transgenic tobacco flowers, the total polyphenol content and various polyphenol constituents were significantly increased with high CnF3'H expression level, while total flavonoid contents and some flavonol constituents were increased slightly. These findings suggest that CnF3'H promotes the synthesis of polyphenols better than flavonoids.

Tree peony variegated flowers show a small insertion in the F3'H gene of the acyanic flower parts.

BACKGROUND: The tree peony (Paeonia suffruticosa Andr.) cultivar 'Er Qiao' is appreciated for its unstable variegated flower coloration, with cyanic and acyanic flowers appearing on different branches of the same plant and occasionally in a single flower or petal. However, the variegation mechanism is still unclear. RESULTS: In this study, we found significantly higher contents and more diverse sets of anthocyanins in the cyanic petals than in the acyanic petals. Comparative transcriptome analysis between the two flower types revealed 477 differentially expressed genes (DEGs). Quantitative real-time PCR results verified that the transcript levels of the flavonol synthase (FLS) gene were significantly increased in the acyanic petals. Furthermore, we found that a GCGGCG insertion at 246 bp in the flavonoid 3'-hydroxylase (F3'H) gene-coding region constitutes a duplication of the 241-245 bp section and was consistently found only in acyanic flowers. Sequence alignment of the F3'H gene from different plant species indicated that only the acyanic petals of 'Er Qiao' contained the GCGGCG insertion. The transformation of Arabidopsis tt7-1 lines demonstrated that the ectopic expression of F3'H-cyanic, but not F3'H-acyanic, could complement the colors in the hypocotyl and seed coat. CONCLUSION: In summary, we found that an indel in F3'H and the upregulation of FLS drastically reduced the anthocyanin content in acyanic petals. Our results provide molecular candidates for a better understanding of the variegation mechanisms in tree peony.

Biochemical and genetic polymorphism of Bromopsis inermis populations under chronic radiation exposure.

MAIN CONCLUSION: For the subsequent assessment of the genetic mechanisms responsible for the resistance of plants to chronic irradiation, the analysis of RAPD-cDNA with the subsequent isolation, cloning, and sequencing of expressed polymorphic sequences is a promising technique. A study was conducted on Bromopsis inermis populations that have been growing for a long time in the EURT area. Using RAPD primers, we studied the genetic spectra of plants. In analysing the UPGMA algorithm, we identified two well-distinguishable clusters with a high level of bootstrap support (> 85%): background samples hit the first, and impact samples hit the second. Our data indicate a decrease in diversity in the most polluted population, as well as the appearance of new alleles in chronically irradiated samples of the B. inermis. Smooth brome seedlings were characterised by the content of anthocyanins, comparable with other types of cereals. In the gradient of chronic irradiation, the relative content of anthocyanins was not significantly changed. For the first time, the partial nucleotide sequences of the key genes of anthocyanin biosynthesis (Chi and F3h) in the brome were determined, these sequences were found to be 191 and 356 bp in length, respectively, and were cloned and sequenced. Three copies of the Chi gene were identified in the B. inermis genome. One copy (BiChi-1) clustered with the sequences of the Aegilops tauschii gene (D genome), and the other two copies (BiChi-2 and BiChi-3) formed a separate cluster in the Pooideae subfamily adjacent to Hordeum vulgare. In the copy of BiChi-1, a complete deletion of intron 1 was detected. For the F3h gene, one copy of the B. inermis gene was obtained, which forms a separate branch in the subfamily Pooideae.

Flavanone-3-Hydroxylase Plays an Important Role in the Biosynthesis of Spruce Phenolic Defenses Against Bark Beetles and Their Fungal Associates.

Conifer forests worldwide are becoming increasingly vulnerable to attacks by bark beetles and their fungal associates due to the effects of global warming. Attack by the bark beetle Ips typographus and the blue-stain fungus it vectors (Endoconidiophora polonica) on Norway spruce (Picea abies) is well known to induce increased production of terpene oleoresin and polyphenolic compounds. However, it is not clear whether specific compounds are important in resisting attack. In this study, we observed a significant increase in dihydroflavonol and flavan-3-ol content after inoculating Norway spruce with the bark beetle vectored fungus. A bioassay revealed that the dihydroflavonol taxifolin and the flavan-3-ol catechin negatively affected both I. typographus and E. polonica. The biosynthesis of flavan-3-ols is well studied in Norway spruce, but little is known about dihydroflavonol formation in this species. A flavanone-3-hydroxylase (F3H) was identified that catalyzed the conversion of eriodictyol to taxifolin and was highly expressed after E. polonica infection. Down-regulating F3H gene expression by RNA interference in transgenic Norway spruce resulted in significantly lower levels of both dihydroflavonols and flavan-3-ols. Therefore F3H plays a key role in the biosynthesis of defense compounds in Norway spruce that act against the bark beetle-fungus complex. This enzyme forms a defensive product, taxifolin, which is also a metabolic precursor of another defensive product, catechin, which in turn synergizes the toxicity of taxifolin to the bark beetle associated fungus.

Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.

The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.

The light-induced transcription factor FtMYB116 promotes accumulation of rutin in Fagopyrum tataricum.

Tartary buckwheat (Fagopyrum tataricum) not only provides a supplement to primary grain crops in China but also has high medicinal value, by virtue of its rich content of flavonoids possessing antioxidant, anti-inflammatory, and anticancer properties. Light is an important environmental factor that can regulate the synthesis of plant secondary metabolites. In this study, we treated tartary buckwheat seedlings with different wavelengths of light and found that red and blue light could increase the content of flavonoids and the expression of genes involved in flavonoid synthesis pathways. Through coexpression analysis, we identified a new MYB transcription factor (FtMYB116) that can be induced by red and blue light. Yeast one-hybrid assays and an electrophoretic mobility shift assay showed that FtMYB116 binds directly to the promoter region of flavonoid-3'-hydroxylase (F3'H), and a transient luciferase activity assay indicated that FtMYB116 can induce F3'H expression. After transforming FtMYB116 into the hairy roots of tartary buckwheat, we observed significant increases in the content of rutin and quercetin. Collectively, our results indicate that red and blue light promote an increase in flavonoid content in tartary buckwheat seedlings; we also identified a new MYB transcription factor, FtMYB116, that promotes the accumulation of rutin via direct activation of F3'H expression.