Identification of Hydroxylation Enzymes and the Metabolic Analysis of Dihydromyricetin Synthesis in Ampelopsis grossedentata.

Ampelopsis grossedentata leaves are highly rich in dihydromyricetin. They have been used to make tea in China for centuries. Dihydromyricetin has many potential applications in foods and medicine. This are because it has five phenolic hydroxyl groups. However, the hydroxylases involving the biosynthesis of dihydromyricetin have not been identified and characterized. In this study, a series of hydroxylases genes, including flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), and cytochrome P450 reductase (CPR), were identified after RNA sequencing. The full-length CDSs of AgF3H, AgF3'H, AgF3'5'H, and AgCPR genes were amplified from the cDNA library of leaves. The aforementioned enzymes were expressed and verified in Saccharomyces cerevisiae. Through the substrate specificity assay, the functional AgF3'H, AgF3'5'H, and AgCPR in A. grossedentata were identified. The dihydromyricetin hydroxylation process in A. grossedentata was successfully identified. We found that substantial carbon flux occurred through the Naringenin (NAR)-Eriodictyol (ERI)-Dihydroquercetin (DHQ)-Dihydromyricetin (DHM) and NAR-Dihydrokaempferol (DHK)-DHQ-DHM pathways. This study provides some reference for the development and utilization of the germplasm resources and molecular breeding of A. grossedentata.

Exploring the genes involved in biosynthesis of dihydroquercetin and dihydromyricetin in Ampelopsis grossedentata.

Dihydroquercetin (DHQ), an extremely low content compound (less than 3%) in plants, is an important component of dietary supplements and used as functional food for its antioxidant activity. Moreover, as downstream metabolites of DHQ, an extremely high content of dihydromyricetin (DHM) is up to 38.5% in Ampelopsis grossedentata. However, the mechanisms involved in the biosynthesis and regulation from DHQ to DHM in A. grossedentata remain unclear. In this study, a comparative transcriptome analysis of A. grossedentata containing extreme amounts of DHM was performed on the Illumina HiSeq 2000 sequencing platform. A total of 167,415,597 high-quality clean reads were obtained and assembled into 100,584 unigenes having an N50 value of 1489. Among these contigs, 57,016 (56.68%) were successfully annotated in seven public protein databases. From the differentially expressed gene (DEG) analysis, 926 DEGs were identified between the B group (low DHM: 210.31 mg/g) and D group (high DHM: 359.12 mg/g) libraries, including 446 up-regulated genes and 480 down-regulated genes (B vs. D). Flavonoids (DHQ, DHM)-related DEGs of ten structural enzyme genes, three myeloblastosis transcription factors (MYB TFs), one basic helix-loop-helix (bHLH) TF, and one WD40 domain-containing protein were obtained. The enzyme genes comprised three PALs, two CLs, two CHSs, one F3'H, one F3'5'H (directly converts DHQ to DHM), and one ANS. The expression profiles of randomly selected genes were consistent with the RNA-seq results. Our findings thus provide comprehensive gene expression resources for revealing the molecular mechanism from DHQ to DHM in A. grossedentata. Importantly, this work will spur further genetic studies about A. grossedentata and may eventually lead to genetic improvements of the DHQ content in this plant.

Annotation of genes involved in high level of dihydromyricetin production in vine tea (Ampelopsis grossedentata) by transcriptome analysis.

BACKGROUND: Leaves of the medicinal plant Ampelopsis grossedentata, which is commonly known as vine tea, are used widely in the traditional Chinese beverage in southwest China. The leaves contain a large amount of dihydromyricetin, a compound with various biological activities. However, the transcript profiles involved in its biosynthetic pathway in this plant are unknown. RESULTS: We conducted a transcriptome analysis of both young and old leaves of the vine tea plant using Illumina sequencing. Of the transcriptome datasets, a total of 52.47 million and 47.25 million clean reads were obtained from young and old leaves, respectively. Among 471,658 transcripts and 177,422 genes generated, 7768 differentially expressed genes were identified in leaves at these two stages of development. The phenylpropanoid biosynthetic pathway of vine tea was investigated according to the transcriptome profiling analysis. Most of the genes encoding phenylpropanoid biosynthesis enzymes were identified and found to be differentially expressed in different tissues and leaf stages of vine tea and also greatly contributed to the biosynthesis of dihydromyricetin in vine tea. CONCLUSIONS: To the best of our knowledge, this is the first formal study to explore the transcriptome of A. grossedentata. The study provides an insight into the expression patterns and differential distribution of genes related to dihydromyricetin biosynthesis in vine tea. The information may pave the way to metabolically engineering plants with higher flavonoid content.

Comparative Transcriptome Analysis of Ampelopsis megalophylla for Identifying Genes Involved in Flavonoid Biosynthesis and Accumulation during Different Seasons.

Ampelopsis megalophylla is an important species used in Chinese folk medicine. Flavonoids, the most important active components of plants, greatly determine the quality of A. megalophylla. However, biosynthesis of flavonoids at the molecular and genetic levels in A. megalophylla is not well understood. In this study, we performed chemical analysis and transcriptome analysis of A. megalophylla in different seasons (i.e., May, August, and October). Accumulation of flavonoids was higher in May than in the other two months. Genes involved in the flavonoid biosynthesis pathway, such as chalcone synthase, anthocyanidin synthase, flavanone 3-hydroxylase, flavonoid-3',5'-hydroxylase, caffeoyl-CoA O-methyltransferase, dihydroflavonol 4-reductase, 4-coumarate-CoA ligase, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, flavonoid 3'-monooxygenase, shikimate O-hydroxycinnamoyltransferase, and leucoanthocyanidin reductase, were identified based on transcriptome data. Fifty ATP binding cassette (ABC) transporter, nine SNARE, forty-nine GST, and eighty-four glycosyltransferases unigenes related to flavonoid transport and biomodification were also found. Moreover, seventy-eight cytochrome P450s and multiple transcription factors (five MYB, two bHLH, and three WD40 family genes) may be associated with the regulation of the flavonoid biosynthesis process. These results provide insights into the molecular processes of flavonoid biosynthesis in A. megalophylla and offer a significant resource for the application of genetic engineering in developing varieties with improved quality.

Evolution of the intercontinental disjunctions in six continents in the Ampelopsis clade of the grape family (Vitaceae).

BACKGROUND: The Ampelopsis clade (Ampelopsis and its close allies) of the grape family Vitaceae contains ca. 43 species disjunctly distributed in Asia, Europe, North America, South America, Africa, and Australia, and is a rare example to study both the Northern and the Southern Hemisphere intercontinental disjunctions. We reconstruct the temporal and spatial diversification of the Ampelopsis clade to explore the evolutionary processes that have resulted in their intercontinental disjunctions in six continents. RESULTS: The Bayesian molecular clock dating and the likelihood ancestral area analyses suggest that the Ampelopsis clade most likely originated in North America with its crown group dated at 41.2 Ma (95% HPD 23.4-61.0 Ma) in the middle Eocene. Two independent Laurasian migrations into Eurasia are inferred to have occurred in the early Miocene via the North Atlantic land bridges. The ancestor of the Southern Hemisphere lineage migrated from North America to South America in the early Oligocene. The Gondwanan-like pattern of intercontinental disjunction is best explained by two long-distance dispersals: once from South America to Africa estimated at 30.5 Ma (95% HPD 16.9-45.9 Ma), and the other from South America to Australia dated to 19.2 Ma (95% HPD 6.7-22.3 Ma). CONCLUSIONS: The global disjunctions in the Ampelopsis clade are best explained by a diversification model of North American origin, two Laurasian migrations, one migration into South America, and two post-Gondwanan long-distance dispersals. These findings highlight the importance of both vicariance and long distance dispersal in shaping intercontinental disjunctions of flowering plants.