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Flavonoid glycosides are widespread in plants, and are of great interest owing to their diverse biological activities and effectiveness in preventing chronic diseases. Periploca forrestii, a renowned medicinal plant of the Apocynaceae family, contains diverse flavonoid glycosides and is clinically used to treat rheumatoid arthritis and traumatic injuries. However, the mechanisms underlying the biosynthesis of these flavonoid glycosides have not yet been elucidated. In this study, we used widely targeted metabolomics and full-length transcriptome sequencing to identify flavonoid diversity and biosynthetic genes in P. forrestii. A total of 120 flavonoid glycosides, including 21 C-, 96 O-, and 3 C/O-glycosides, were identified and annotated. Based on 24,123 full-length coding sequences, 99 uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) were identified and classified into 14 groups. Biochemical assays revealed that four UGTs exhibited O-glycosyltransferase activity toward apigenin and luteolin. Among them, PfUGT74B4 and PfUGT92A8 were highly promiscuous and exhibited multisite O-glycosylation or consecutive glycosylation activities toward various flavonoid aglycones. These four glycosyltransferases may significantly contribute to the diversity of flavonoid glycosides in P. forrestii. Our findings provide a valuable genetic resource for further studies on P. forrestii and insights into the metabolic engineering of bioactive flavonoid glycosides.

Abstract Horse chestnut (Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.

Abstract Background Karst-adapted plant, Lysionotus pauciflours accumulates special secondary metabolites with a wide range of pharmacological effects for surviving in drought and high salty areas, while researchers focused more on their environmental adaptations and evolutions. Nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone), the main active component in L. pauciflours, has unique bioactivity of such as anti-inflammatory, anti-tubercular, and anti-tumor or cancer. Complex decoration of nevadensin, such as hydroxylation and glycosylation of the flavone skeleton determines its diversity and biological activities. The lack of omics data limits the exploration of accumulation mode and biosynthetic pathway. Herein, we integrated transcriptomics, metabolomics, and microbial recombinant protein system to reveal hydroxylation and glycosylation involving nevadensin biosynthesis in L. pauciflours. Results Up to 275 flavonoids were found to exist in L. pauciflorus by UPLC-MS/MS based on widely targeted metabolome analysis. The special flavone nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone) is enriched in different tissues, as are its related glycosides. The flavonoid biosynthesis pathway was drawn based on differential transcripts analysis, including 9 PAL, 5 C4H, 8 4CL, 6 CHS, 3 CHI, 1 FNSII, and over 20 OMTs. Total 310 LpCYP450s were classified into 9 clans, 36 families, and 35 subfamilies, with 56% being A-type CYP450s by phylogenetic evolutionary analysis. According to the phylogenetic tree with AtUGTs, 187 LpUGTs clustered into 14 evolutionary groups (A-N), with 74% being E, A, D, G, and K groups. Two LpCYP82D members and LpUGT95 were functionally identified in Saccharomyces cerevisiae and Escherichia coli, respectively. CYP82D-8 and CYP82D-1 specially hydroxylate the 6- or 8-position of A ring in vivo and in vitro, dislike the function of F6H or F8H discovered in basil which functioned depending on A-ring substituted methoxy. These results refreshed the starting mode that apigenin can be firstly hydroxylated on A ring in nevadensin biosynthesis. Furthermore, LpUGT95 clustered into the 7-OGT family was verified to catalyze 7-O glucosylation of nevadensin accompanied with weak nevadensin 5-O glucosylation function, firstly revealed glycosylation modification of flavones with completely substituted A-ring. Conclusions Metabolomic and full-length transcriptomic association analysis unveiled the accumulation mode and biosynthetic pathway of the secondary metabolites in the karst-adapted plant L. pauciflorus. Moreover, functional identification of two LpCYP82D members and one LpUGT in microbe reconstructed the pathway of nevadensin biosynthesis.

Artemisia Linn. is a large genus within the family Asteraceae that includes several important medicinal plants. Because of their similar morphology and chemical composition, traditional identification methods often fail to distinguish them. Therefore, developing an effective identification method for Artemisia species is an urgent requirement. In this study, we analyzed 15 chloroplast (cp) genomes, including 12 newly sequenced genomes, from 5 Artemisia species. The cp genomes from the five Artemisia species had a typical quadripartite structure and were highly conserved across species. They had varying lengths of 151,132–151,178 bp, and their gene content and codon preferences were similar. Mutation hotspot analysis identified four highly variable regions, which can potentially be used as molecular markers to identify Artemisia species. Phylogenetic analysis showed that the five Artemisia species investigated in this study were sister branches to each other, and individuals of each species formed a monophyletic clade. This study shows that the cp genome can provide distinguishing features to help identify closely related Artemisia species and has the potential to serve as a universal super barcode for plant identification.

The seeds of Chinese horse chestnut are used as a source of starch and escin, whereas the potential use of whole plant has been ignored. The astringency and bitterness of tea produced from the leaves and flowers were found to be significantly better than those of green tea, suggesting that the enriched flavonoids maybe sensory determinates. During 47 flavonoids identified in leaves and flowers, seven flavonol glycosides in the top 10 including astragalin and isoquercitrin were significantly higher content in flowers than in leaves. The crude proteins of flowers could catalyze flavonol glucosides' formation, in which three glycosyltransferases contributed to the flavonol glucosylation were screened out by multi-dimensional integration of transcriptome, evolutionary analyses, recombinant enzymatic analysis and molecular docking. The deep exploration for flavonol profile and glycosylation provides theoretical and experimental basis for utilization of flowers and leaves of Aesculus chinensis as additives and dietary supplements.

Artemisinin is currently the most effective ingredient in the treatment of malaria, which is thus of great significance to study the genetic regulation of Artemisia annua. Alternative splicing (AS) is a regulatory process that increases the complexity of transcriptome and proteome. The most common mechanism of alternative splicing (AS) in plant is intron retention (IR). However, little is known about whether the IR isoforms produced by light play roles in regulating biosynthetic pathways. In this work we would explore how the level of AS in A. annua responds to light regulation. We obtained a new dataset of AS by analyzing full-length transcripts using both Illumina- and single molecule real-time (SMRT)-based RNA-seq as well as analyzing AS on various tissues. A total of 5,854 IR isoforms were identified, with IR accounting for the highest proportion (48.48%), affirming that IR is the most common mechanism of AS. We found that the number of up-regulated IR isoforms (1534/1378, blue and red light, respectively) was more than twice that of down-regulated (636/682) after treatment of blue or red light. In the artemisinin biosynthetic pathway, 10 genes produced 16 differentially expressed IR isoforms. This work demonstrated that the differential expression of IR isoforms induced by light has the potential to regulate sesquiterpenoid biosynthesis. This study also provides high accuracy full-length transcripts, which can be a valuable genetic resource for further research of A. annua, including areas of development, breeding, and biosynthesis of active compounds.

Ginseng, which contains ginsenosides as bioactive compounds, has been regarded as an important traditional medicine for several millennia. However, the genetic background of ginseng remains poorly understood, partly because of the plant's large and complex genome composition. We report the entire genome sequence of Panax ginseng using next-generation sequencing. The 3.5-Gb nucleotide sequence contains more than 60% repeats and encodes 42 006 predicted genes. Twenty-two transcriptome datasets and mass spectrometry images of ginseng roots were adopted to precisely quantify the functional genes. Thirty-one genes were identified to be involved in the mevalonic acid pathway. Eight of these genes were annotated as 3-hydroxy-3-methylglutaryl-CoA reductases, which displayed diverse structures and expression characteristics. A total of 225 UDP-glycosyltransferases (UGTs) were identified, and these UGTs accounted for one of the largest gene families of ginseng. Tandem repeats contributed to the duplication and divergence of UGTs. Molecular modeling of UGTs in the 71st, 74th, and 94th families revealed a regiospecific conserved motif located at the N-terminus. Molecular docking predicted that this motif captures ginsenoside precursors. The ginseng genome represents a valuable resource for understanding and improving the breeding, cultivation, and synthesis biology of this key herb. [ABSTRACT FROM AUTHOR]

Flavonoids, natural products abundant in the model legume Glycine max, confer benefits to plants and to animal health. Flavonoids are present in soybean mainly as glycoconjugates. However, the mechanisms of biosynthesis of flavonoid glycosides are largely unknown in G. max. In the present study, 212 putative UDP-glycosyltransferase (UGT) genes were identified in G. max by genome-wide searching. The GmUGT genes were distributed differentially among the 20 chromosomes, and they were expressed in various tissues with distinct expression profiles. We further analyzed the enzymatic activities of 11 GmUGTs that are potentially involved in flavonoid glycosylation, and found that six of them (UGT72X4, UGT72Z3, UGT73C20, UGT88A13, UGT88E19 and UGT92G4) exhibited activity toward flavonol, isoflavone, flavone and flavanol aglycones with different kinetic properties. Among them, UGT72X4, UGT72Z3 and UGT92G4 are flavonol-specific UGTs, and UGT73C20 and UGT88E19 exhibited activity toward both flavonol and isoflavone aglycones. In particular, UGT88A13 exhibited activity toward epicatechin, but not for the flavonol aglycones kaempferol and quercetin. Overexpression of these six GmUGT genes significantly increased the contents of isoflavone and flavonol glucosides in soybean hairy roots. In addition, overexpression of these six GmUGT genes also affected flavonol glycoside contents differently in seedlings and seeds of transgenic Arabidopsis thaliana. We provide valuable information on the identification of all UGT genes in soybean, and candidate GmUGT genes for potential metabolic engineering of flavonoid compounds in both Escherichia coli and plants. [ABSTRACT FROM AUTHOR]

Flavonols are one of the largest groups of flavonoids that confer benefits for the health of plants and animals. Flavonol glycosides are the predominant flavonoids present in the model legume Lotus japonicus. The molecular mechanisms underlying the biosynthesis of flavonol glycosides as yet remain unknown in L. japonicus. In the present study, we identified a total of 188 UDP-glycosyltransferases (UGTs) in L. japonicus by genome-wide searching. Notably, 12 UGTs from the UGT72 family were distributed widely among L. japonicus chromosomes, expressed in all tissues, and showed different docking scores in an in silico bioinformatics docking analysis. Further enzymatic assays showed that five recombinant UGTs (UGT72AD1, UGT72AF1, UGT72AH1, UGT72V3, and UGT72Z2) exhibit activity toward flavonol, flavone, and isoflavone aglycones. In particular, UGT72AD1, UGT72AH1, and UGT72Z2 are flavonol-specific UGTs with different kinetic properties. In addition, the overexpression of UGT72AD1 and UGT72Z2 led to increased accumulation of flavonol rhamnosides in L. japonicus and Arabidopsis thaliana. Moreover, the increase of kaempferol 3-O-rhamnoside-7-O-rhamnoside in transgenic A. thaliana inhibited root growth as compared with the wild-type control. These results highlight the significance of the UGT72 family in flavonol glycosylation and the role of flavonol rhamnosides in plant growth. [ABSTRACT FROM AUTHOR]

Galloylated catechins and flavonol 3-O-glycosides are characteristic astringent taste compounds in tea (Camellia sinensis). The mechanism involved in the formation of these metabolites remains unknown in tea plants. In this paper, 178 UGT genes (CsUGTs) were identified in C. sinensis based on an analysis of tea transcriptome data. Phylogenetic analysis revealed that 132 of these genes were clustered into 15 previously established phylogenetic groups (A to M, O and P) and a newly identified group R. Three of the 11 recombinant UGT proteins tested were found to be involved in the in vitro biosynthesis of β-glucogallin and glycosylated flavonols. CsUGT84A22 exhibited catalytic activity toward phenolic acids, in particular gallic acid, to produce β-glucogallin, which is the immediate precursor of galloylated catechin biosynthesis in tea plants. CsUGT78A14 and CsUGT78A15 were found to be responsible for the biosynthesis of flavonol 3-O-glucosides and flavonol 3-O-galactosides, respectively. Site-directed mutagenesis of the Q373H substitution for CsUGT78A14 indicated that the Q (Gln) residue played a catalytically crucial role for flavonoid 3-O-glucosyltransferase activity. The expression profiles of the CsUGT84A22, CsUGT78A14, and CsUGT78A15 genes were correlated with the accumulation patterns of β-glucogallin and the glycosylated flavonols which indicated that these three CsUGT genes were involved in the biosynthesis of astringent compounds in C. sinensis. [ABSTRACT FROM AUTHOR]

Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway. [ABSTRACT FROM AUTHOR]